Is the Removal of Nettles along Ditches Effective in Controlling Bois Noir in Vineyards?

Round 1

Reviewer 1 Report

The presented manuscript provides interesting data on management strategies concerning Urtica dioica in the context of stolbur phytoplasma diseases, which is always a current topic. However, I have some concerns, particularly regarding the nomenclature of tuf types/genotypes throughout the manuscript, especially concerning the interpretation of the tuf-b1 type found in grapevines. I urge you to consider the paper by Aryan et al. (2014) in addressing my comments. Overall, the experimental setup appears sound, and the provided results are informative within the context of the BN control

Line 28 - Incorporating additional references beyond (or instead of some of) the noted one, such as Quaglino et al. (2013) (reference 5), would be more appropriate given that the cited papers predominantly focus on Italy, it would be beneficial to include references that encompass a broader scope concerning the Euro-Mediterranean region.

Line 30-34 - To enhance clarity, I would recommend to refine the sentence to specify precisely which plants serve as H. obsoletus host plants, avoiding imprecise terms like "living preferentially." The identified host plants include Urtica dioica, Convolvulus arvensis, Vitex agnus-castus, and Crepis foetida, as nymphs were observed on the roots of all four plants. Furthermore, the inclusion of Artemisia verlotiorum in this context would be ok also, as noted by Cargnus et al. (2012, reference 13), who documented the presence of nymphs and adults in the field and subsequent reared them in laboratory conditions. Although Artemisia vulgaris has been previously reported to host adults, especially in Italy. This would be an opportunity to give more precise information on the association of H. obsoletus and Artemisia spp.

Line 35 – ‘’tuf gene’’ instead of ‘’tufB gene’’

Lines 37-39 - I would suggest clarifying that tuf-a and tuf-b are types of the tuf gene, whereas tuf-b1 is more accurately described as a genotype. Additionally, it seems that tuf-b2 is omitted in this context, despite its description by Aryan et al. in 2014 (reference 18), where both tuf-b1 and tuf-b2 are delineated and referred mainly to as genotypes, sequences, or sequence types throughout the manuscript. Therefore, I would suggest the use of the term "genotype" when discussing both tuf-b1 and tuf-b2, as their differentiation necessitates sequencing after the initial RFLP analysis.

Line 39 – “ ‘Ca. P. solani’-positive” instead of “PCR-positive”

Line 40 – If the sentence pertains specifically to Italy, please ensure that this is explicitly stated. However, if the information is presented in a broader context, such as Europe-wide, I would advise specifying that the tuf-b1 genotype and the newly described tuf-d genotype were found infecting H. obsoletus ex C. arvensis, whereas genotypes tuf-a and tuf-b2 were detected infecting H. obsoletus from U. dioica, adding appropriate references.

Line 49 – Reference 15 (Kosovac et al., 2018, PlosOne) is not suitable, as the authors concluded in this study that populations of H. obsoletus from C. arvensis and U. dioica form a homogenous genetic group on pan-European level, rather than undergoing cryptic speciation.

Line 62 – ‘’ H. obsoletus is attracted by soil without herbaceous vegetation’’ – Any reference on this?

Line 98 – Please delete ‘’tuf-types’’

Line 117 – Please change double apostrophe with a single apostrophe.

Lines 120-121 – Please further explain ‘’36% for vineyard A and 42% for vineyard B’’. Are those samples from the both, ‘’weeding sector’’ and ‘’control sector’’? How were they selected? Randomly?

Line 122 – Citing seems somewhat odd, would recommend only to cite reference 36, or if preferring to leave more detailed explanation than cite also Doyle & Doyle 1990 (Doyle, J. & Doyle, J. (1990) Isolation of plant dNA from fresh tissue. Focus 12, 13–15).

Line 134- Please change double apostrophe with a single apostrophe.

Line 136 – Why is Fialova et al. 2009 cited?

Original name of the primers as described in Langer & Maixner 2004 is rTufAY and fTufAY

Line 140-142 – I suggest stating that the thermal protocol underwent modifications in comparison to the original description by Langer & Maixner and that these modifications correspond specifically to the durations of the corresponding steps, while temperatures were the same as in the original protocol.

Line 143 – Calculated in R as further described? Or in some other software, please specify.

Lines 221-222 – Please specify in the Materials and Methods section how the detection of FD was carried out and at which stage of the experiment it was conducted.

Line 224 – It is not possible to conclude that the stolbur strains of the tuf-b type detected via RFLP HpaII analysis belong exclusively to the tuf-b1 genotype. Subsequent sequencing has revealed that they may instead belong to the tuf-b2 genotype associated with Urtica dioica (Aryan et al., 2014).

’’For a clear discrimination between tuf type b1 and tuf type b2 or tuf a, however, a sequence analysis of the tuf gene is necessary. ’’ (Aryan et al. 2014).

According to the research of Aryan et al. 2014 (tuf-b2) and Curcic et al. 2021 (tuf-d), that both have showed that there is hidden diversity within the RFLP HpaII patterns that can be revealed by sequencing, (or with additional RFLP analysis with TaiI enzyme in case of the tuf-d), I would suggest using the term ‘type’ to describe the results of the RFLP HpaII analysis- tuf-a and tuf-b (or tuf-c when found). Asserting that a specific sample carries the tuf-b1 genotype solely on the HpaII RFLP analysis should be validated through sequencing, particularly within the context of research focused on Urtica dioica as a reservoir plant.

I would advise the authors to conduct sequencing of 'tuf-b' strains identified in grapevines to verify whether the detected 'tuf-b1 types' might actually be the tuf-b2 genotypes.

Line 226-227 – Same comment as previously

Lines 544-545 – I recommend including this data in the paper and possibly conducting sequencing on a subset of tuf-b type stolbur samples found in H.obsoletus from Convolvulus arvensis to verify the presence of the tuf-d genotype, if possible.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

The article describes the impact of chemical weeding of nettle on the spatial distribution of H. obsoletus populations and newly BN symptomatic grapevines in two vineyards of north-eastern Italy over several years. 

The introduction is well written and correctly documented. 

In the results section, the number of figures should be reduced. For example, Figures 1 and 2 could be merged as Figure 1A and 1B. Same for Figures 3 and 4: I would suggest to remove Table 3 or add it as supplementary material.

What is the difference between Figure 5 (% New symptomatic grapevine over the years) and Figure 9B and 10B (Newly symptomatic over the years)? Isn’t it the same? If yes, the % numbers do not correspond. 

The experimental design is not optimal. The 2 vineyards have different situations (distance from the ditch, length of the ditch). The authors state this in the discussion. This leads to discrepancies in the results from the 2 vineyards.

It would be of high importance to have vineyards with the same situation regarding the ditch to be able to compare the results. 

In my opinion, the main conclusion “a significant relationship between the size of H. obsoletus populations and the occurrence of newly symptomatic grapevines was demonstrated for the first time” (lines 557-558) is not supported by the results.

For example, in Vineyard B (Figure 5), there is a drastic decrease in newly symptomatic grapevines in 2017 but the captures of H. obsoletus the year before (2016) are still high.

The novelty/originality brought by this study is poor.

None

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Dear authors,

Thank you for providing answers.

In Table 3, the significance is true (barely, P=0.05) only for Vineyard A and not for Vineyard B (P=0.11). It would be of interest to pool the data from the 2 vineyards to do the regression analysis. Doing so would allow to draw the conclusion on the complete set of your data.

What are the data you used to feed the ANOVA and regression tests?

I still do not agree with this sentence in your conclusion:

"Furthermore, a significant relationship between the size of H. obsoletus populations and the occurrence of newly symptomatic grapevines was demonstrated for the first time. In particular, the appearance of symptoms is mainly associated with the planthopper populations observed in the previous year."

Best regards.

Author Response

Answer:

• In Table 3 we added the regression analysis based on both vineyards together. Pooling the years together, new results of regression analysis confirm the previous results increasing the statistical significance of the relationship.
• For the regressions, we used the total annual cumulative captures of obsoletus within vineyards and the percentage of newly symptomatic grapevines. Unfortunately, when transferring the manuscript into the template we had not copied the sentence from the materials and methods relating to the regression. We added in the manuscript the sentence: “For each vineyard and for the two vineyards together, linear regressions between the cumulative annual captures of H. obsoletus within vineyard and the percentage of grapevines that showed first symptoms in the same and the subsequent year were calculated.” (L414-416)
• Upon re-evaluation of the data using the analysis suggested by the reviewer, we have found that the new results strengthen our statement. Specifically, regression analyisis, that considers the data of the two vineyards together, revealed even greater significance of the relationship between the percentage of newly symptomatic grapevines and the vector captures recorded in the previous year. However, in consideration of the reviewer’s concern, we have opted to reflect a more cautious interpretation.

" Furthermore, a significant relationship between the size of H. obsoletus populations and the occurrence of newly symptomatic grapevines was observed here for the first time. This suggests that the symptoms mostly appear in the year after infection.” (L1531-1533)

Reviewer 3 Report

Comments for author File: Comments.pdf

Author Response

We apologize for not including the answer in the previous version. We have carefully considered both the differences between the two vineyards and the presence of the strain associated with Convolvolus arvensis. However, we believe that these factors do not invalidate our results.

In the vineyard where nettle weeding along a portion of the ditches did not differentiate the captures of Hyalesthes obsoletus between the two sectors of the vineyard, no effect was observed on the newly symptomatic grapevines (vineyard A). Conversely, in the vineyard where a differentiated effect on Hyalesthes obsoletus captures between the two sectors was observed, the incidence of new symptomatic vines was influenced.

Regarding the influence of the strain from Convolvulus, the percentage of grapevines with tuf-tb1 is lower (20% vs 80%). Moreover, the fact that the differences between the two sectors are significant despite the presence of the strain associated with bindweed reinforces the effectiveness of nettle weeding. For major clarity, we added this statement in the discussion section “In vineyard B, despite the presence of 20% of grapevines infected with the tuf-1b, associated with C. arvensis, the effectiveness of nettle weeding emerges.” (L1525-1526).

We hope that this explanation convincingly demonstrates the robustness of our results and addresses any concerns raised by the reviewer.