Viroid GYSVd1 Exhibited Different Regulations on the Qualities of Berries and Wines from 6 Grape Varieties

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript by Wu et al. addresses the study of the impact of Grapevine yellow speckle viroid 1 on the quality of grapes and wines in six grape varieties. The work is original, as there are no prior reports on this subject, and it is of significant importance to the industry.

However, upon reviewing the study, it appears that the experimental design is not adequate for this type of research. It seems that the study was conducted on sets of five infected plants and five healthy plants, with grapes collected from all plants and, ultimately, pseudo-replications being performed. True biological replicates do not appear to have been employed. Similarly, insufficient details are provided regarding the vinification process, leading the reviewer to assume that pseudo-replication may have also been used in that context.

No mention is made regarding the sanitary status of the plants with respect to other viruses or viroids. Was this sanitary status assessed? Which viruses were analyzed? Were all the plants exclusively infected by the viroid?

There are several determinations that are not correct. For example, to determine residual sugars in wine, a refractometer is used, whereas the appropriate method should involve the determination of reducing sugars, such as the Fehling method.

There are data inconsistencies throughout the paper. For example, Table 1 indicates that the TSS value for the healthy Chardonnay cultivar was 12.13 ºBx, whereas the sugar determination (Fig. 6) shows a value close to 300 mg/g.

The authors state that the gene expression of the viroid was determined, which is not correct since these organisms do not possess the transcriptional machinery required for gene expression. Nonetheless, they report values that do not make much sense, as the comparison between infected and healthy plants shows an expression value in the healthy plants, suggesting that they may not be entirely free of viroids. Was there positive qPCR amplification in these plants?

The variety names are not correct. The varieties "Riesling" and "Italian Riesling" may refer to either the same or different varieties according to the Vitis International Variety Catalogue. The appropriate prime name should be used.

 

Comments on the Quality of English Language

The quality of the English should be thoroughly reviewed by a native speaker

Author Response

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Reviewer 2 Report

Comments and Suggestions for Authors

Wu et al , present an interesting manuscript in which they show for the first time the effects of Grapevine yellow speckle viroid 1 (GYSVd1) over both the grapes and the wine quality, This study is really useful and provide new information about the relationship between the the viroid and the wine industry,  however there are some concerns that must be addressed before its publication

1. In the Y axis chart 1 the scale ( GYSvd1 detection rate % show maximum grade 25 , however there are several bar results representing in chart with higher values than 25 with a size represented values below 25%, please could you clarify this ?
2. The study present a very interesting results about the chemical modification of grapes and wines varieties , however in the discussion section this findings are not discussed deeply  for example : lines 157-158 the expression level of GYSvd1  infection are presented but this found is not discussed deeply , just a brief mention on lines 295-298 mention that some metabolic process in berries development could be inhibited but this is remain speculative because no one experimental data was added or cited.
3. other aspects like sugar, anthocyanine  and canine  content on grape berries and wine deserved a major discussion than presented 

Finally,  the manuscript is very interesting, the results are solid and unveiling , however in my opinion the authors could be do a better effort for improve the discussion in order to provide more robustness to the conclusion presented in the study

Author Response

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Reviewer 3 Report

Comments and Suggestions for Authors

Paper “Viroid GYSVd1 Exhibited Different Regulations on the Qualities of Berries and Wines from 6 Grape Varieties” provides important insights into the effect of a viroid on the quality of grapes and wine from affected grapevines. Currently, there are few reports on the effects of viroids on grapevines. Several viroids have been reported infecting grapevines worldwide, highlighting the importance of the presented knowledge. I believe the study should be published after the authors address some of the observations made. Observations and comments are in the text.

Comments for author File: Comments.pdf

Author Response

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Reviewer 4 Report

Comments and Suggestions for Authors

The paper “Viroid GYSVd1 Exhibited Different Regulations on the Qualities of Berries and Wines from 6 Grape Varieties” relates to a little-explored topic, but which affects one of the main crops on the planet. Therefore the purpose of the research is appreciable. The aims of the work are clear, with an updated and sufficient bibliography. The M&Ms are updated and adequate for the purpose of the research. The results are interesting and the discussion is not speculative. However, I have serious doubts about the validity of some data, such as those reported in figure 1 (see specific comments).

L12-13: The quotation marks must not be for a group, but for a single cultivar

L15-L18: Reporting that the quality of berries or wines is different between varieties does not make much sense, as each variety is different, with pros and cons, one cannot make rankings by saying that variety A is better than B. Therefore the sentence must be reformulated considering which variety suffers the greatest impact from the presence of the viroid on the berries or wine.

L78: reporting the cultivar is not sufficient for the vine. There are big differences between the different clones. It is therefore necessary to know if the vineyards were monoclonal for all varieties and which clones they are. Furthermore, there is no information on rootstocks. Also in this case it is necessary to know if, for each cultivar, the same rootstock was used and which one.

L152: what about transmission of GYSVd1? Some info should reported in Introduction or Discussion. I know about seed transmission of  GYSVd1, so why so high incidence?

Figure 1: I generally don't dispute the numbers reported by authors, but I notice an oddity. For three cultivars, exactly 20 out of 50 infected plants were found following a single monitoring. I wouldn't have been surprised if they had been three very similar but slightly different values ​​(e.g. 21, 19, 20, or even 20, 20, 19), but three identical values ​​for a viroid whose manner of transmission we don't know well is strange.

Figure 1: the figure is completely wrong. The reported incidence range is 0-25 (Y axis), but values ​​also reach 40%. This, together with the previous consideration, raises serious doubts about the validity of the data.

L288: the incidence is not only related to this, but is closely connected to the mode of transmission. It is necessary to add information on this aspect and correlate it with the results obtained.

L293-294: again I don’t agree with this approach. Maybe you can write that detrimental effect of the viroid is bigger in Merlot.

L266-269 and L341-343.  As mentioned in the abstract, it cannot be said that the berry of variety A is better than B. What can be said is that the impact of the viroid on variety A is less than that on B.

Author Response

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Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I would like to thank to the author for the consideration of my previous observations.

The manuscript requires significant revisions to address methodological inconsistencies, terminological inaccuracies, and statistical concerns. Specific issues are outlined below:

1. Lines 35-36: The authors mention the relevance of grapevine cultivation (e.g., higher yield, cultivated area, etc.). However, the comparative context is unclear: Is this relative to other fruit crops, general crops, or a specific regional benchmark? The cited reference does not directly support this claim and should be replaced with a pertinent source.

2. Line 41: The term "introduction of seedlings" is misleading. Grapevines are predominantly propagated via cuttings, whereas seedlings are primarily used in breeding programs. Revise terminology to reflect standard practices.

3. Line 73: Correct "alcohol fermentation" to alcoholic fermentation (standard technical term).

4. Line 43: The statement "there are 86 described species" is not upated. Update with the provided reference: [https://link.springer.com/article/10.1007/s42161-025-01871-9].

5. Lines 67-68: The categorization of quality parameters into "external" and "internal" appears to originate from studies on apples and is not widely recognized in viticulture. Either cite the original source for this framework or remove the distinction.

6. Line 77: A rise in pH is caused by a decrease in acidity, not an increase. Correct this biochemical inaccuracy.

7. Line 105: The authors report harvesting 10 kg of grapes for winemaking, yet the supplementary material describes winemaking trials using 500 g of grapes. Clarify this discrepancy. Additionally, microvinifications (500 g scale) are unconventional and poorly documented in the literature. Specify the number of replicates performed.

8. Line 134 (and subsequent sections): The term "phenol" is incorrect in the context of grapes; use "phenolic compounds". The methods for quantifying phenolics, anthocyanins, and tannins in wines are insufficiently detailed. Provide analytical protocols (e.g., HPLC, spectrophotometry).

9. Line 137: The phrase "gene accumulation" is invalid, as viroids lack genes. Replace with "viroid accumulation" or "viroid RNA levels".

10. Lines 200-220 & Table 1:

    • The reported pH difference for Chardonnay (3.92 ± 0.01 vs. 3.90 ± 0.18) is labeled as statistically significant, but the overlapping standard deviations suggest otherwise. Re-evaluate the statistical analysis.

    • Non-infected Welsch Riesling grapes had notably higher sugar content than Riesling Weiss, yet the latter produced wine with higher alcohol. Explain this contradiction.

11. Figure 4: Infected Cabernet Sauvignon grapes show higher anthocyanin levels, but the opposite is observed in wines. Justify this inconsistency.

12. Figure 5: Similarly, infected Riesling Weiss grapes exhibit higher tannins, but wines show the reverse trend. Clarify the mechanisms behind this disparity.

13. Line 258 (Section 3.7): Residual sugar levels in wines vary significantly across samples. For comparative validity, fermentations must be halted at a uniform residual sugar threshold (e.g., via physical/chemical parameters, not time). Describe the fermentation stopping criteria used.

14. Lines 329-332: The term "gene accumulation" reappears (see comment #9). Additionally, if qRT-PCR detected viroid RNA in non-infected plants, this implies contamination or false positives. Address this critical concern.

15. Lines 344-345: Berry morphology is emphasized, but this trait is far less critical for wine grapes than for table grapes. Support the claim about its relevance with citations from enology literature.

Comments on the Quality of English Language

The overal quality of the english should be improoved

Author Response

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Reviewer 2 Report

Comments and Suggestions for Authors

The Authors made a great effort to improve the article and response satisfactory to all my concerns and in my opinion the article is now in condition to be published 

Author Response

There is no problem with reviewer 2.