Metagenomic Sequencing for Identification of Xylella fastidiosa from Leaf Samples

ABSTRACT Xylella fastidiosa (Xf) is a globally distributed plant-pathogenic bacterium. The primary control strategy for Xf diseases is eradicating infected plants; therefore, timely and accurate detection is necessary to prevent crop losses and further pathogen dispersal. Conventional Xf diagnostics primarily relies on quantitative PCR (qPCR) assays. However, these methods do not consider new or emerging variants due to pathogen genetic recombination and sensitivity limitations. We developed and tested a metagenomics pipeline using in-house short-read sequencing as a complementary approach for affordable, fast, and highly accurate Xf detection. We used metagenomics to identify Xf to the strain level in single- and mixed-infected plant samples at concentrations as low as 1 pg of bacterial DNA per gram of tissue. We also tested naturally infected samples from various plant species originating from Europe and the United States. We identified Xf subspecies in samples previously considered inconclusive with real-time PCR (quantification cycle [Cq], >35). Overall, we showed the versatility of the pipeline by using different plant hosts and DNA extraction methods. Our pipeline provides taxonomic and functional information for Xf diagnostics without extensive knowledge of the disease. This pipeline demonstrates that metagenomics can be used for early detection of Xf and incorporated as a tool to inform disease management strategies. IMPORTANCE Destructive Xylella fastidiosa (Xf) outbreaks in Europe highlight this pathogen’s capacity to expand its host range and geographical distribution. The current disease diagnostic approaches are limited by a multiple-step process, biases to known sequences, and detection limits. We developed a low-cost, user-friendly metagenomic sequencing tool for Xf detection. In less than 3 days, we were able to identify Xf subspecies and strains in field-collected samples. Overall, our pipeline is a diagnostics tool that could be easily extended to other plant-pathogen interactions and implemented for emerging plant threat surveillance.

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The manuscript entitled "Metagenomic sequencing for rapid identification of Xylella fastidiosa from leaf 2 samples" reports a new strategy in the detection and contrast Xylella fastidiosa. The common strategies to identify the bacterium involved ELISA and qPCR methods, but, since their weak limit of detection, they are ineffective in early disease identification and in the host eradication strategies. The tool proposed by Roman-reyna et al., is very promising as demonstrated by experiments and bioinformatic analyses reported in this manuscript.
The main weakness of this strategy, in my opinion, is the lack of suitable equipment and specialized employees (bioinformaticians) in next-generation sequencing analyses at phytosanitary European institutes. I suggest the following revisions: INTRODUCTION: 1) Line 65: Reference 4 is not specific about tyloses. You can add, for example, Sabella et al., 2019 (Xylem cavitation susceptibility and refilling mechanisms in olive trees infected by Xylella fastidiosa) 2) Line 74-76. I partially disagree with the authors: The authors stated "fast and accurate detection" to prevent losses. Anyway, even with fast and accurate methods, we are not able to avoid the dispersion of the pathogen. The most important parameter in Xf detection (and host eradication) is the early detection. Methods with the lowest limit of detection should be developed. Since the method developed by the authors have a very low LOD, they should stress this point. 3) Line 87: please correct the typo "is". 4) Line 95: please remove the typo ",". RESULTS 5) Line 129-132: I didn't understand very well. Can you explain better? Thanks. 6) Line 137: MLST. Please define it. The acronym is explained only on page 21. 7) Line 187-189: Figure S2. It is very difficult to distinguish the 0.081 on the y-axis. Why did you consider the ratio of 60:40 as mixed infection and then 80:20 as not mixed infection? 8)Line 287 to 298. Congratulation on this result. This is a key point for the future development of Xf detection tools.
Reviewer #2 (Comments for the Author): Manuscript by Roman-Reyna et al., entitiled 'Metagenomic sequencing for rapid....' This study describes the set-up and use of a metagenomics pipeline using short reads via the iSeq in order to detect the plant pathogen Xylella fastidiosa. The detection system proved effective and very sensitive.

General comment
This pipeline is very powerful and allows sensitive detection and classification of X. fastidiosa from plant samples. It is described as an alternative to the current standard methodology of quantitative real time PCR (qPCR); its sensitivity and more in depth characterization provides clear advantages. It is however not a convenient, fast or cheap alternative since it requires NGS equipment and computational know-how. Detection methods of plant pathogens are advisable to be fast, cheap and requiring as little equipment and know as possible. This methodology has however many advantages resulting in a more in depth analysis of the plant samples. It can also be adapted to different pathogens or even pathobiome analysis.
Specific comments: 1. The analysis most likely resulted in the identification of many more microorganisms; authors have not presented or commented on this data. It would have been interesting to learn the significant presence of other microorganisms co-occurring with X. fastidiosa. 2. Explain more clearly in the discussion the advantages/disadvantages of using short reads. 3. Nanopore sequencing technology is evolving quickly and in the near future it will be a very cheap, quick and easily adaptable techniques. Possibly this technology can be better suited to pathogen detection since it is a more 'movable' field-like technology. It is encouraged to discuss this as a possible alternative NGS technology for this pipeline. 4. I recommend to remove the word 'rapid' from the title. Thank you for submitting your manuscript to mSystems. I have completed my evaluation of your revision, which did not require an additional round of external comments, and I am pleased to inform you that, in principle, we expect to accept it for publication in mSystems. However, acceptance will not be final until you have adequately these last, very minor, points: - Figure S2 does not seem to be cited in the main text. Shouldn't be "located" on or around paragraph 192-98, instead of Figure  -In a few instances you refer to "hope" regarding the application of the proposed method. I suggest a more "emotionally-detached" and result-based approach, something along the lines: this investigation (or our results) demonstrates that metagenomics can be efficiently integrated (or represent a valuable alternative) to conventional detection methods.
Else it is a well written manuscript proposing a timely and potential impactful use of metagenomicscongratulations! Sincerely yours,

Davide Bulgarelli
Thank you for the privilege of reviewing your work. Below you will find instructions from the mSystems editorial office and comments generated during the review.

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Prof. Jonathan M Jacobs Ohio State University Columbus
Re: mSystems00591-21R2 (Metagenomic sequencing for identification of Xylella fastidiosa from leaf samples) Dear Prof. Jonathan M Jacobs, I have no further comment and I'd like to take this occasion to congratulate with all co-authors for the very interesting and timely manuscript.
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