Patterns of Copper Bioaccumulation and Translocation in Grapevine Grafts Depending on Rootstocks
Round 1
Reviewer 1 Report
This manuscript analyses the capacity of 5BB and SO4 rootstocks to accumulate Cu, as well as its pattern of translocation in the grafts. After heat forcing (callusing), the grapevine grafts of Sauvignon Blanc on 5BB and SO4 rootstocks were grown in pots for six months in a glasshouse, and exposed to various Cu formulations (Cu-17 oxychloride, Cu-gluconate) and concentrations in peat (50, 150, 500, and 1000 mg Cu kg-1 of dry 18 weight (DW)). In addition to monitoring the shoot growth dynamics and analysis of copper content 19 in grafts organs, bioaccumulation (BAFs) and translocation factors (TFs) of Cu were calculated. The
This manuscript presents a study of Cu accumulation in grapevine grafts. A pot experiment was carried out using peat as a substrate, during six months. The variety Sauvignon Blanc has been grafted in two different rootstocks; two different Cu formulations and four different concentrations were studied. Shoot growth was analyzed two times; Cu concentrations were measured in roots, rootstocks trunk and canes. Bioaccumulation and translocation factor were calculated.
This work is well organized, allowing a thorough analysis of the effects of rootstock type and substrate Cu concentration on accumulation and translocation of Cu just after grafting. The methods used are in general adequately described. Conclusions are in line with objectives and supported by the obtained results.
Main Concern
My main concern is the extrapolation of results obtained on a substrate based on peat to soils contaminated with Cu. Please, consider to address this subject in the introduction.
Additional comments
The Material and Methods section is well organized. However, it should be improved at two points:
1) Calculation of biomass. This is because the statement in page 3, line 137 is different from the information given in Table 1 (page 7). So, In page it is stated that total biomass is calculated using a method different from method the method quoted in line 137. Please, be consistent.
2) Page 2. Lines 138-172. The description of Cu extraction by acid digestion is lengthy and should
The Conclusion should be tightened up. This is because they contain sentences, which are difficult to understand.
Page 2, Line 48. Please, indicate for which country or countries are mandatory the given concentrations.
Page 2, Line 50. Please, provide references regarding Cu toxicity for grapevine.
AN ANNOTATED PDF IS INCLUDED
Comments for author File: 
Comments.pdf
The English Language should be tightened up, before this manuscript can be accepted for publication. There are frequent redundancies, which should be avoided. Next I’m providing a list with several lines, where amelioration is recommended.
Page 1, Line 25. Here is an example of redundancy.
Page 2, Line 63. Are you meaning “in general” instead of “for the most part”?
Page 3, Line 102. “Due to equalized….” Is difficult to understand.
Page 3, Line 114. Please change “N 210 mg/L” by 210 mgN/L”, to be consistent with the criteria used for the other elements (P, K, etc.).
Page 3, Line 121. In Cu++, please ++ as a super index.
Page 3, Lines 129-130. To redundant.
Page 3, Lines 133-134, redundant.
Page 3, Line 138. Here it is stated “roots, cane and rootstock”. However, in general authors state “roots, rootstock and cane”, which seems more adequate. Please, be consistent and use “roots, rootstock and cane”, all over the text.
Page 3, Line 143. Please, delete “for further processing in vessels”. And again, please, simplify the description of total Cu determination. This is a routinely performed determination.
Page 4, Lines 158 – 162. Please rewrite the procedure used to assess growth rate. Also indicate explicitly if this parameter is given as a percent value.
Page 4, Line 173. Typo error. Kg-1 should include super index fo “-1”.
Page 11, Lines 407 – 09. This sentence is difficult to understand.
Page 12, Lines 413, 428, 430, etc. “we think” results to colloquial. Please, avoid its use.
AN ANNOTATED PDF IS INCLUDED
Author Response
Reviwer 1
This manuscript analyses the capacity of 5BB and SO4 rootstocks to accumulate Cu, as well as its pattern of translocation in the grafts. After heat forcing (callusing), the grapevine grafts of Sauvignon Blanc on 5BB and SO4 rootstocks were grown in pots for six months in a glasshouse, and exposed to various Cu formulations (Cu-17 oxychloride, Cu-gluconate) and concentrations in peat (50, 150, 500, and 1000 mg Cu kg-1 of dry 18 weight (DW)). In addition to monitoring the shoot growth dynamics and analysis of copper content 19 in grafts organs, bioaccumulation (BAFs) and translocation factors (TFs) of Cu were calculated. The
This manuscript presents a study of Cu accumulation in grapevine grafts. A pot experiment was carried out using peat as a substrate, during six months. The variety Sauvignon Blanc has been grafted in two different rootstocks; two different Cu formulations and four different concentrations were studied. Shoot growth was analyzed two times; Cu concentrations were measured in roots, rootstocks trunk and canes. Bioaccumulation and translocation factor were calculated.
This work is well organized, allowing a thorough analysis of the effects of rootstock type and substrate Cu concentration on accumulation and translocation of Cu just after grafting. The methods used are in general adequately described. Conclusions are in line with objectives and supported by the obtained results.
Dear reviewer, thanks a lot for your carefully revision again. We tried to do our best in order to check all the comments and incorporate the suggestions. We hope that now, the paper deserves to be accepted.
Dear reviewer, we are not a native English speaker. English was improved by Robert McKenzie Group again (Teachers College, Columbia University USA) Robert McKenzie is Senior Copywriter/Proof reader, and this he does, among other things also for 15 university faculties in Slovenia and Slovenian parliament and government.
Main Concern
My main concern is the extrapolation of results obtained on a substrate based on peat to soils contaminated with Cu. Please consider to address this subject in the introduction.
The concern is justified. In the introduction section, we added a comment about the
The research is a contribution to the set of information on the effects of copper on the development of grapevine grafts in the early developmental stages. We recognize that the results of pot experiments cannot be directly extrapolated to the conditions of the development of grafts in the soil in the vineyard.
Additional comments
The Material and Methods section is well organized. However, it should be improved at two points:
1) Calculation of biomass. This is because the statement in page 3, line 137 is different from the information given in Table 1 (page 7). So, In page it is stated that total biomass is calculated using a method different from method the method quoted in line 137. Please, be consistent.
We corrected for clarity and used the same definition as used in the table 1. *Biomass is the sum of DW of canes, roots, and annual growth of the rootstock trunks (difference between rootstock trunks weight at the start and the end of the trial).
For statistical evaluation, the biomass is defined as the sum of DW of canes, roots, and annual growth of the rootstock trunks (the difference between rootstock trunks' weight at the start and the end of the trial).
2) Page 2. Lines 138-172. The description of Cu extraction by acid digestion is lengthy and should
We shortened the description of the method.
The Conclusion should be tightened up. This is because they contain sentences, which are difficult to understand.
We tried to make the conclusions shorter, but other reviewers suggested some additional conclusions.
Page 2, Line 48. Please indicate for which country or countries are mandatory the given concentrations.
The Regulation EC No. 473/2002 is obligatory for EU countries.
The limit concentration of clearly detectable ecotoxic effects of Cu is between 60–90 mg Cu kg-1 of soils (Official Gazette RS no. 84/05). This is valid only for Republic of Slovenia. It was given only as example.
Page 2, Line 50. Please, provide references regarding Cu toxicity for grapevine.
We provided literature source regarding Cu toxicity to grapevine
- Toselli, M.; Baldi, E.; Marcolini, G.; Malaguti, D.; Quartieri, M.; Sorrenti, G.; Marangoni, B. Response of Potted Grapevines to Increasing Soil Copper Concentration. Aust. J. Grape Wine R. 2009, 15, 85–92, doi.org/10.1111/j.1755-0238.2008.00040.x.
AN ANNOTATED PDF IS INCLUDED
peer-review-31665604.v1.pdf
Comments on the Quality of English Language
The English Language should be tightened up, before this manuscript can be accepted for publication. There are frequent redundancies, which should be avoided. Next I’m providing a list with several lines, where amelioration is recommended.
Page 1, Line 25. Here is an example of redundancy. We corrected
Page 2, Line 63. Are you meaning “in general” instead of “for the most part”? we corrected
Page 3, Line 102. “Due to equalized….” Is difficult to understand. We made sentence more clear
Page 3, Line 114. Please change “N 210 mg/L” by 210 mgN/L”, to be consistent with the criteria used for the other elements (P, K, etc.).
Page 3, Line 121. In Cu++, please ++ as a super index.
Thank you for your correction, both of them were corrected (mgN/L and Cu++),
Page 3, Lines 129-130. To redundant. It was written because of clarity of presentation of experiment methodology
Page 3, Lines 133-134, redundant.
Page 3, Line 138. Here it is stated “roots, cane and rootstock”. However, in general authors state “roots, rootstock and cane”, which seems more adequate. Please, be consistent and use “roots, rootstock and cane”, all over the text.
Yes, we agree with you. We changed the sequence and use “roots, rootstock and cane”, all over the text. Thanks!
Page 3, Line 143. Please, delete “for further processing in vessels”. And again, please, simplify the description of total Cu determination. This is a routinely performed determination.
We shortened it significantly
Page 4, Lines 158 – 162. Please rewrite the procedure used to assess growth rate. Also indicate explicitly if this parameter is given as a percent value.
Our given definition
To analyze the growth rate of the shoots, the relative growth rate of shoots (%) was used, which is defined as the ratio between the length of shoots (in cm) of grafts developing in control pots without added copper and the length of graft shoots developing in pots with added copper (Cu-gluconate or Cu-oxychloride).
Growth rate was presented as relative growth rate to the control expressed in % of control - the unit of measurement is percentage if control 50 cm and treated grape is 25 cm - the relative growth rate is 50 % (this is given in the axis of Fig 1)
Page 4, Line 173. Typo error. Kg-1 should include super index fo “-1”.
Yes, it is true. We corrected that error.. Thanks
Page 11, Lines 407 – 09. This sentence is difficult to understand. We took into account suggestions
Page 12, Lines 413, 428, 430, etc. “we think” results to colloquial. Please, avoid its use. We replaced word we think
Reviewer 2 Report
Dear authors,
I found this article well -designed with the scientific sound and very actual topic. The content and accumulation of copper in the viticultural industry is always good and interesting. The methodology used in the article is good and up to date from Kurnik et al., 2012.
All used references are appropiate and new. Expecially I found important to emphasize the articles: 1) Mirleana et al., 2007. and mentioning the highest amount of Cu found in Brasilian soils.; 2) Juang et al., 2012. and the toxic effect of Cu residuals to plants and soils mentioned in lines 51-57. Also, the big trend in viticulture is organic production that has limited application of Cu on year level.
It is normal to get the highest concentrations in roots, so these was good observed also in this experiment. I can not judge the concentrations applied, this should comment the plant nutricionist.
I can only give two small objections:
1) It could be better if you used one more genotype for this research. Even Sauvignon Blanc is a widespread cultivar, it can be useful for viticultural industry.
2) In conclusion, based on your results you should suggest futher research activities.
Best regards,
Author Response
Reviewer 2
I found this article well -designed with the scientific sound and very actual topic. The content and accumulation of copper in the viticultural industry is always good and interesting. The methodology used in the article is good and up to date from Kurnik et al., 2012.
All used references are appropiate and new. Expecially I found important to emphasize the articles: 1) Mirleana et al., 2007. and mentioning the highest amount of Cu found in Brasilian soils.; 2) Juang et al., 2012. and the toxic effect of Cu residuals to plants and soils mentioned in lines 51-57. Also, the big trend in viticulture is organic production that has limited application of Cu on year level.
It is normal to get the highest concentrations in roots, so these was good observed also in this experiment. I can not judge the concentrations applied, this should comment the plant nutricionist.
I can only give two small objections:
1) It could be better if you used one more genotype for this research. Even Sauvignon Blanc is a widespread cultivar, it can be useful for viticultural industry.
2) In conclusion, based on your results you should suggest futher research activities.
Dear reviewer, thank you for your comments and suggestions. We were added in conclusion our suggestions for further research.
Also we are of the opinion that it is necessary to test more genotypes of varieties and rootstocks and to increase copper concentrations as reported from Bordeaux, Brazil, etc. We used the grafts in the most sensitive phase immediately after callus formation. It would make sense to include already rooted grafts in the research, which are mainly used for planting vineyards.
Thanks again.
Reviewer 3 Report
The publication discusses the results of the use of two forms of copper in the vase experiment at four levels of its use in the cultivation of grapevines on two rootstocks. In the methodology of the work, no number of grapevine plants was given, either in total or in one copper treatment. It is not known in which year the plants were grown. How many plant samples were taken for research on the copper content in plant organs? For the sake of clarity of the publication, the results of individual parameters should be given in the text and not only on the graph, which is difficult to read with this scale of units. The scientific work is suitable for publication after making the indicated corrections.
Comments for author File: Comments.pdf
Publication written correctly in English.
Author Response
Reviewer 3
Dear reviewer, thank you a lot for your corrections and sugestions.
The publication discusses the results of the use of two forms of copper in the vase experiment at four levels of its use in the cultivation of grapevines on two rootstocks. In the methodology of the work, no number of grapevine plants was given, either in total or in one copper treatment.
We were added in text of »Experimental settings« the number of plants per replicate three times as follows:
- The pot experiment was conducted in a greenhouse (four plants per replicate) with……
- .....the grafts were removed from the pots and sampled destructively by separating the plant organs (four plant per replicate). All plant samples of roots, canes, and the rootstock trunks
- How many plant samples were taken for research on the copper content in plant organs?
All plant included in experiment were analysed (see below)
The Cu content (mg kg−1) of dried tissues of roots, canes, and the rootstock trunk of all plants in each replicate (n =4) was determined from homogenised samples obtained by acid digestion in microwave heating using an atomic absorption spectropho-tometer (Varian AA 240FS; Agilent Technologies, California, USA) performing absorbance readings at 324.8 nm, as described by Kurnik et al. [39].
It is not known in which year the plants were grown.
Thank you for your warning; the year of experiment was added as follows
Experimental settings
Pot trials were carried out at the Faculty of Agriculture and Life Sciences in Maribor (Slovenia; 46°30′17.4″ N, 15°37′34.6″ E), in vegetation period (from May to October 2011).
For the sake of clarity of the publication, the results of individual parameters should be given in the text and not only on the graph, which is difficult to read with this scale of units. The scientific work is suitable for publication after making the indicated corrections.
More detailed comments of data presented in Fig 1 and Fig 2 are proposed.
In the existing text, we have several times inserted additional notes in parentheses regarding where in the graphs the information we are commenting on is visible. in lines 288 to 308 we explain the numerical data from Fig 2 quite precisely so we hope the level of clarity is OK.
Thank you again for your review and all welcome comments.
