Review Reports

Round 1

Reviewer 1 Report

Please refer to the detailed comments 

The manuscript titled “Characterization of Viral Communities and Two Novel Viruses in Symptomatic Tea Plants via lncRNA Sequencing” by Tan et al identifies viral diversity in symptomatic tea leaves and characterizes two novel viruses, which could contribute valuable insights for disease management in tea cultivation. Overall, the manuscript is well-structured, with clear methods and informative figures. However, I have several critical suggestions to enhance clarity, scientific rigor, and depth. Addressing these points particularly methodological clarifications and deeper linkages to literature will strengthen it.

Please change title to “…via Ribodepleted Transcriptome Sequencing” “lncRNA sequencing” is incorrect and misleading.

From the abstract please delete “long non-coding RNA (lncRNA)” and also from everywhere as these are standard rRNA-depleted total RNA libraries.

Line 30: remove “lncRNA sequencing”, add “virome, metagenomics”.

Line 45-46: “prior to 2018, there were no documented viral infections” please change to “very few confirmed reports existed” (older serological reports do exist).

Line 69-79: I suggest adding two recent 2023-2024 tea virus papers (Tea plant virus 1 from Yunnan, TeaOLV2) would be nice.

Line 95: “systematic studies… are still lacking” this seems nice to me but I would suggest to cite a 2024 review that says the same to strengthen justification.

Line 98-110: delete “lncRNA” from all sentences.

Line 115-118: Pooling 6 cultivars destroys cultivar-level resolution. Add one sentence: “Pooling prevents assignment of viruses to individual cultivars; future RT-PCR validation on single plants is required.”

Line 120-124: “long non-coding RNA (lncRNA) libraries” please replace with “rRNA-depleted total RNA libraries”.

Line 129: ICTV 2016 is 9 years old. I would strongly suggest to use current ICTV Master Species List (2024) for novel virus taxonomy.

Line 132-140: GO enrichment on viral genes is inappropriate and produces artefacts. I strongly recommend removing the entire GO subsection or moving it to supplementary with clear caveat.

Line 147: ClustalX is outdated; MAFFT or MUSCLE is now standard, please justify choice or switch.

Line 158: Bootstrap ≥50 % is too low for taxonomic conclusions. Report only nodes ≥70 % as supported.

Line 182-187 in the Fig. 1 caption: I feel there is spelling inconsistency “Bizaoxiang” vs “Baizaoaxiang”, “Fudingdahao” vs “Fudingdahao” please check and unify throughout manuscript. Line 186: Red asterisks are helpful, but add: “Similar symptoms can also be caused by nutrient deficiency or phytoplasma.”

Line 188 (Table 1 captions): please change to “RNA-seq.

Figure 2 and 3 (A) are too blur, please replace it with HQ.

Line 207 & Fig. 2b: “locomotion”, “cellular locomotion” terms are clear database artefacts from animal rhabdoviruses. Delete or heavily caveat these GO results.

Line 228 & Fig. 3a: Taro bacilliform CH virus (badnavirus) dominant in Pool C is highly surprising in tea. I would suggest adding the following sentence in Results and Discussion: “Its presence requires independent confirmation because badnaviruses are usually monocot-specific and may represent contamination or integrated sequences.”

There are too many figures, I would suggest to move some into supplementary section

Discussion is poor and not validated by supported findings. For example, I have not seen any reference from Line 425-474. I must recommend to carefully revise with strong justification and appropriate literature. The authors have again discussed the results but without justification. I would suggest to carefully revise.

 

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

The authors, Rongrong Tan and coworkers presented a manuscript entitled "Characterization of Viral Communities and Two Novel Viruses in Symptomatic Tea Plants via lncRNA Sequencing".

The authors analyzed 18 varieties from the tea plant genetic resource collection with symptoms of leaf discoloration. Long non-coding RNA libraries were prepared from isolated RNA for high-throughput sequencing. The sequencing data revealed the presence of a number of viruses, with tea plant necrotic ring blotch virus predominating. The authors described and characterized two new viruses.

The authors describe in detail the harmfulness of viral infection for tea production.

The methodological approach to analyzing the health status of symptomatic tea plants was correctly chosen. The methods and procedures used are described in detail. The results are described and documented in great detail.

The description of two new tea viruses contributes to the knowledge of pathogens of this important agricultural crop and may thus contribute to improving the health status of tea plants and their yields.

Comments:

Please use the current virus taxonomy according to the ICTV website https://ictv.global/, which requires adjustments to all virus names throughout the article.

I am missing information on the number of reads detected for each virus and on the genome coverage of the detected viruses.

For the two new viruses, it is necessary to prove their presence in tea plants (e.g., using RT-PCR). They could be pathogens of other organisms associated with tea plants, such as endophytic fungi.

For the two new viruses, a schematic diagram of their genome organization needs to be provided.

According to Figure 5, the new virus tea plant associated ourmia-like virus 1 belongs to the genus Penoulivirus and not to the genus Botoulivirus, as stated in the article. Please check and correct.

Author Response

Please see the attachment.

Author Response File: Author Response.docx