Complete genome and comparative analysis of Xanthomonas oryzae pv. oryzae isolated from northern Thailand
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Rice (Oryza sativa L.) is a vital global crop with a predominant presence in Asia, including Thailand. However, it faces a significant threat from bacterial blight disease (BB), primarily caused by Xanthomonas oryzae pv. oryzae (Xoo). This research aims to provide an insights into the genetic virulence and variation of Xoo strains isolated in Thailand. Our phylogenetic analysis unveils that the 20 Thai strains align with the Asian strains, setting them apart from African and USA strains. Remarkably, the Average Nucleotide Identity (ANI) values, in comparison to the Xanthomonas oryzae type strain 35933 (XO35933), consistently exceed 99%. These strains can be classified into three assigned ribosomal sequence types (rST). Our investigation into the pangenome and the phylogenetic relationships of these 20 Xoo genomes reveals a diverse genetic landscape, with the pangenome comprising 11,872 orthologous gene clusters, of which roughly 30% form the core genome. Notably, all of these genomes exhibit the presence of a CRISPR-Cas I-C array, indicative of their adaptive immune mechanisms. All strains belonged to BXO1 type LPS cassette with high identity. Furthermore, our analysis identifies two distinct types of plasmids, namely, Xanthomonas oryzae pv. oryzicola strain GX01 plasmid pXOCgx01 (A46, A57, A83, A112, D, and E) and the Xanthomonas oryzae strain AH28 plasmid pAH28 (A97). This genomic resource will be valuable for advancing research on surveillance, prevention, management, and comparative studies of this critical pathogen in the future.
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Please provide more detail in your method section as detailed by the reviewers. It also seems that the articles structure is hard to follow, please make sure the narrative is clear. Please refer to specific comments made by the reviewers.
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Comments to Author
This study describes the genomes of 20 previously uncharacterised strains of Xanthomonas oryzae pv. Oryzae from Thailand through a variety of bioinformatic tools. They use both long and short read sequencing to generate the genome assemblies. They perform phylogenetic analyses comparing these strains to both African and USA strains, investigate the pangenome of these strains and identify the core genome. In addition, they identify the metabolic potential of these organisms through CAZyme and COG annotation as well as characterising plasmids, CRISPR sequences and virulence gene variations. Firstly, I think that the paper is clear and well structured, however I do have some editing suggestions below because I think that too much detail is given to explanation of tool use in the methods. The analysis …
Comments to Author
This study describes the genomes of 20 previously uncharacterised strains of Xanthomonas oryzae pv. Oryzae from Thailand through a variety of bioinformatic tools. They use both long and short read sequencing to generate the genome assemblies. They perform phylogenetic analyses comparing these strains to both African and USA strains, investigate the pangenome of these strains and identify the core genome. In addition, they identify the metabolic potential of these organisms through CAZyme and COG annotation as well as characterising plasmids, CRISPR sequences and virulence gene variations. Firstly, I think that the paper is clear and well structured, however I do have some editing suggestions below because I think that too much detail is given to explanation of tool use in the methods. The analysis methodology is sound however I think in a few places a couple of extra small details need to be added, as mentioned below. I think the paper would benefit from a few formatting changes including ensuring figure captions are at the foot of figures not on top. The authors present an informative set of analyses and the sequencing data, which will be publicly available, will be a good resource for the scientific community. In the methods (between line 107-163) there are a few sentences which I think have unnecessary detail I would remove: Line 107: This high throughput sequencing technology enables a comprehensive examination of the entire genome of these Xoo isolates, facilitating a detailed genomic analysis Line 117: Here is a breakdown of the bioinformatics workflow - though this could be a useful overflow figure. Line 118: This step helps remove any low-quality or irrelevant sequences from the data Line 124: They are tools for annotating bacterial genomes, identifying genes, and determining their functions - you already say this in the sentence before Line 163: Secretion systems play a crucial role in pathogenicity. My additional comments: Line 101: I would instead say that the extraction protocol was performed to manufacturer's guidelines Line 112: Which basecaller was used, dorado/guppy/other? Which version? Line 126: How was the comparison and alignment to the reference genome done? What tools? Line 143: I would shorten this to: The resulting phylogenetic trees were edited and visualized using FigTree v1.4.4 and Mega v11.0(22). Line 162: Are cumulative anotations in prokka and rast if they were identified in both or just all results from both together? Line 171: In the study, plasmid analysis and the characterization of antimicrobial resistance genes were conducted using specific tools and methods - this is quite vague and you then go on to list tools, so I would remove this and then update the next sentence. Line 172: Where is the plasmid database from? NCBI? Or is the fIDBAC the one you mean? Line 176-177: Too much detail on what resfinder does in the methods, just need what you used it for e.g. it was used to characterise the antimicrobial resistance genes. Line 197: Why not quote the range of genome sizes if they're quite uniform and mention table 1. Figure 1 contains no bootstrap values or distances on it, these are both important. Line 213: The rMLST isn't illustrated, the tree is coloured based on rMLST values. Figure 3: You have a legend that is above low abundance bars, but I would put this legend more to the side (not covering the AWZ COGs) Fig2B: indicate these four clusters, it's not very easy to tell. Fig 2B: looks like there are lots of genes absent (lots of yellow), might be worth removing these if they are absent in all of the genomes you annotated Reference 1 format is incorrect- non capitalise reference For the CAZyme annotation mention lack of AAs. Fig5 change table heading to make sure it's the strains in the top row How did you identify (lack of raxX genes), was this by aligning your reads to that region in the ref? Should give a bit more detail as to how you did this. Line 290: annotale - how many TALES do strains have on average and min and max of tales. For higher diversity of TALES, is this the total number or more than are unique?
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
General comments The study investigated 20 genomes of Xanthomonas oryzae pv. oryzae (Xoo) from Northern Thailand. Overall, the used methods are sound, however details are missing making it harder to reproduce results. The text needs a clearer structure, there is a mix between Methods, Results and Discussion. At times it is hard to follow the main story. Bigger focus needs to be placed on the value of the research for Xoo surveillance and management. See below specific comments. Introduction L56: Re-phrase the sentence, the 30 races were identified by researchers. L74-86: State the main aim of the research (mentioned in the next paragraph) but avoid giving too many details on the results. What is the knowledge gap? The last two paragraphs are hard to follow. Results of the study are presented in the …
Comments to Author
General comments The study investigated 20 genomes of Xanthomonas oryzae pv. oryzae (Xoo) from Northern Thailand. Overall, the used methods are sound, however details are missing making it harder to reproduce results. The text needs a clearer structure, there is a mix between Methods, Results and Discussion. At times it is hard to follow the main story. Bigger focus needs to be placed on the value of the research for Xoo surveillance and management. See below specific comments. Introduction L56: Re-phrase the sentence, the 30 races were identified by researchers. L74-86: State the main aim of the research (mentioned in the next paragraph) but avoid giving too many details on the results. What is the knowledge gap? The last two paragraphs are hard to follow. Results of the study are presented in the 3rd paragraph, then the reader is going back to the study objectives in the 4th paragraph. Material and Methods L99: Were extractions done from pure culture? Provide information on culturing conditions. L104: Remove: This step is crucial in guaranteeing the quality and quantity of the genetic material obtained. L105: Were fragments extracted from the agarose gel and cleaned? What library prep method was used? What sequencing reagents? Was it paired-end, 300 cycles? How many reads per sample was the target? L107: Remove: This high throughput sequencing technology enables a comprehensive examination of the entire genome of these Xoo isolates, facilitating a detailed genomic analysis. L111: Was any size selection done during the Nanopore library prep? L112: How long was the sequencing run? L113: Add version of the MinKnow software L117: Remove: as the platform. Here is a breakdown of the bioinformatics workflow L118: Remove: This step is crucial to ensure the reliability of the data and identify any potential issues L119: What quality scores were used for the trimming? L121: What software was used for the basecalling of the Nanopore reads? Did you remove any low-quality reads? Or filtering based on fragment size? L124: Remove: They are tools for annotating bacterial genomes, identifying genes, and determining their functions. L129: Remove: This submission ensures that the research findings and data are accessible to the broader scientific community. This comprehensive bioinformatics analysis provides valuable insights into the genomic characteristics of the Xoo isolates and allows for a better understanding of their genetic characterization and potential virulence factors. L171: Remove the first sentence. L172: Remove: then L173: What do you mean "manually"? L174: Remove: Antimicrobial Resistance gene characterization was studied to characterize antimicrobial resistance genes within the Xoo strains. L175: Which database was used for the AMR? L176: Remove simple explanations of popular tools L181 - 185: Not methods Results L194: Add: were successfully sequenced. L195: Give information on the number of reads (before and after trimming), quality scores, fragment size (N50) for the Nanopore reads. L196: Any information on the genome assembly: coverage, error rate, number of contigs etc. Table 1 - move to Supplementary materials. Just provide brief description in the main text. L205: This should be in the Methods, not Results: The phylogenetic tree was constructed using the neighbour-joining (NJ) method with a K-tuple length of 6. L255-264: Not Results. L302: Role of CRISPR-Cas system is not Results. Discussion Big part of Results contains Discussion. Both sections need to be re-organised. L342: The findings offer valuable insights for future investigations into genome virulence and pathogenesis - elaborate more on this. L490: Figure 1 or 2? Too many figures, move some to the Supplementary materials.
Please rate the manuscript for methodological rigour
Poor
Please rate the quality of the presentation and structure of the manuscript
Poor
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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