Effects of ‘Candidatus’ Liberibacter Asiaticus on the Root System of Poncirus trifoliata Hybrids as a Rootstock for ‘Valencia’ Scion

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Huanglongbing (HLB), which caused by the bacterium Candidatus Liberibacter asiaticus (CLas) is currently the most destructive disease of citrus, responsible for huge economic losses in the world's major citrus production areas. In this study, the impact of CLas-infected 'Valencia' scion on 2 kinds of rootstock, citrandarin H-222 (a hybrid of Poncirus trifoliata and Sunki mandarin) and Swingle citrumelo are investigated. The results show that CLas in the scion has a lower impact on the roots of H-222 than that of Swingle citrumelo. The research work is original, data are sufficient, analytical method is correct, the conclusion is reliable, and the references are up to date. I suggest the consideration for publication after minor revision.
Comments:

1.    Abstract, L20, it is described that “At 360 days post-inoculation”, but in Materials and Methods, L113, it is “Samples were collected 13 months after inoculation”. Which one is correct? Please check.

2.     Abstract, L15, “Citrus genera” is not correct, because Poncirus trifoliate is just a close relative with Citrus species.

3.    Introduction, the purpose of the interstock (Treatment 3 and Treatment 4) is not described. I also wonder how to insert the interstock between the rootstock and the scion?

4.    L173, title of table 1 is not appropriate. I think “Impact of 'Candidatus Liberibacter asiaticus' infected 'Valencia' orange scion on the root system” may be better.  

5.    Table 1, the data, standard error is necessary after the mean.

6.    Please check the statistical analysis result. I think Column No.3, “B” should be “A”; Column No.5, “B” should be “A”, “C” should be “B”; Column No.8, “b a c a” should be “a b c b”.

7.    Add scientific name to all citrus and rootstock species on first occurrence.

     8. Check the format of references: upper and lower case in the title.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Huanglongbing (HLB) caused by the bacterium Candidatus Liberibacter asiaticus (CLas) is currently the most devasting disease of citrus plants. There is an urgent need to develop effective methods and measures to prevent and control the disease. Thanks to the innate resistance/tolerance to CLas infection of certain citrus varieties, grafting technique and hybridization technique, the conventional horticultural techniques, are glowing with new hopes in mitigating HLB. Previously, the authors obtained a series of Citrus hybrids, some of which showed more tolerance to HLB disease and low titers of CLas, but little is known about the effects of CLas on the root system of these species. In this manuscript, the authors present some new findings about the effects of multiple grafting using H-222 (a tolerant hybrid) as rootstock and interstock. The results showed that H-222 rootstock was less affected by the CLas-infected scion compared to the Swingle rootstock, mainly manifesting that the damage to the root system of H-222 was significantly less severe compared to the Swingle rootstock. The manuscript elucidated the effects and the contributions of rootstocks in disease tolerances, providing some new references in rootstock selection in citrus graft breeding for the effective prevention and control of HLB disease.

 

The manuscript is not well organized and written.

 

Questions and Suggestions:

1.       The amount of data in the manuscript is relatively limited. It is suggested that the manuscript published as a Note.

2.       In addition to the visible phenotypic variations, it is worth emphasizing the changes of microstructure and chemical transportation in the rootstock, interstock and scion, especially the graft interfaces and intersections of the grafted trees.

3.       What is the difference between H-222 and H222? If they are the same hybrid, please unify naming, also with the original references.

4.       How many biological repeats in one treatment? Why there is no standard deviation or standard error for the data in Table 1?

5.       Please carefully check the data and the letters for differences in Table 1.

It is difficult to figure out the associations of the numbers with the differential letters.

Why the differential letter is all “B” in column “Root system volume/infected”?

How can we get the results, “31.33a”, “37.39b”, and “27.88c” in column “CT values/Roots”?

6.       The writing quality of the manuscript needs to be improved.

7.       Please also pay attention to typos and grammar mistakes.

8.       Line 31, beeing?

9.       Line 58, Jhonson?

10.    Line 103, and30.

11.    Line 141, leess

12.    Line 165, Jhonson?

13.    Line 229, as weel as?

Comments on the Quality of English Language

1.       The writing quality of the manuscript needs to be improved.

2.       Please also pay attention to typos and grammar mistakes.

3.       Line 31, beeing?

4.       Line 58, Jhonson?

5.       Line 103, and30.

6.       Line 141, leess

7.       Line 165, Jhonson?

8.       Line 229, as weel as?

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The paper entitled “Effects of 'Candidatus' Liberibacter asiciatus on root system of Poncirus trifoliata hybrids as a rootstock for ‘Valencia’ scion” is an interesting work on an hot topic of plant pathology. The topic is clearly stated and the literature cited is sufficient and up-to-date. The materials and methods are correct and updated. The results are consistent with the analyzes and discussed appropriately. The conclusions are not speculative. The article is well written and there are few changes to suggest. In particular, I appreciate the agronomic and concrete approach of the experiment, as I believe it can provide information that can be immediately used by farmers.

 

Here my few comments:

Abstract and M&M: The Ct is not the best parameter for estimating the concentration of a pathogen, as the values ​​can vary depending on many factors (e.g. machinery, operator, reagents). Therefore it is a parameter that is not very useful for making comparisons between different experiments with respect to an estimate of the quantity of bacteria. I therefore ask you to use a more representative parameter or to clearly justify the exclusive use of the Ct. Even the use of decimals (even two in several cases) is not very useful as they are easily subject to internal variability.

L110. No details are reported on the bacterium, i.e. where it was obtained from, how it was characterized, any information on the virulence of the strain. It would also be important to understand if the budsticks had a similar bacterial load and, more generally, how they were chosen and how they were applied. There is not enough information to be able to understand, in practice, how the inoculation was done in such a way that it is reproducible by other researchers.

L113. 13 months is a long time and it is not clear how/where the plants were maintained and what was done to avoid natural infections by CLa or other pathogens.

L114. “pieces of fibrous roots” is not very informative. Please add data about quantities, sampling method, etc.

L158. "absence" is too rigid a term, as it is not possible to exclude the presence of the bacterium below the detection threshold of the analysis.

 

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

This is for the revised version of MS3038690. The authors revised the manuscript carefully. All my concerns and suggestions have been well addressed. The revised manuscript has been improved.

 

There are still some inappropriate contents in Table 1 and Figure 2.

1.       In pair-wise comparison, asterisk “*” is used for indicating differences between two samplings at 5% probability. It is suggested to used asterisk replace the capital letters in Table 1. Normally, the capital letters are used for indicating differences in the Multiple Comparison.

2.       In Figure 2, the direction of the dendrogram is not common, which resulted in a visual discomfort for capturing the information in each branch. It is suggested to rearrange the dendrogram by rotating 90 degrees counterclockwise and horizontally placing the branches, phrases and the scale.

Author Response

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Author Response File: Author Response.pdf