Genome sequences of pathogenic and non-pathogenic Pantoea ananatis strains in maize (Zea mays L.)
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We performed genome sequencing and comparative analysis of Pantoea ananatis strains isolated from corn leaves expressing typical bacterial leaf streak (BLS) and maize white spot (MWS) symptoms to confirm bacterial identity and to understand the relationship among these strains and P. ananatis strains isolated from different plant hosts in Brazil. In pathogenicity tests, strains 4.2 and 13.3 isolated from symptomatic BLS leaves were non-pathogenic on corn. In contrast, strain B13 isolated from MWS diseased leaf tissue caused symptoms typical of MWS. Our comparative analysis revealed that all three strains are very genetically similar. The G+C (%) content of the strains 4.2 and 13.3 was 53.5%, while B13 was 53.7%. Average nucleotide identity (ANI) analysis showed that strains B13 and 13.3, B13 and 4.2, and 4.2 and 13.3 shared ANIs of 99.17%, 99.15%, and 99.99%, respectively. Strains 13.3, B13 and 4.2 shared ~99% ANI with P. ananatis type strain LMG 2665. To our knowledge, these are the first genome sequences of P. ananatis strains isolated from corn in Brazil.
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Thank you for submitting your paper to Access Microbiology. The revisions undertaken in line with the reviewers' reports have been met satisfactorily and I confirmed that the genome data is accessible. However, in the revised version, the summary section (methods section, data summary, etc) still refers to the older version of texts and softwares such as Roary (which was replaced by a new software in the revised draft). This may cause confusion and I would encourage you to make the necessary correction
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This is a study that would be of interest to the field and community. The reviewers have highlighted major concerns with the work presented. Please ensure that you address their comments. The language used is poor, which can cause ambiguity at times. Please carefully rewrite it. We offer a discounted translation service, Editage (https://www.editage.com/; see https://www.microbiologyresearch.org/prepare-an-article#13 for more information). The reviewers raise concerns regarding the scientific rigour and experimental design of the work.
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Comments to Author
Reviewer summary: The study introduces the diverse ecological niches of Pantoea ananatis, a bacterium found in various environments such as water, soil, insects, plants, and humans. The bacterium can have different associations with plants, acting as endo- or epiphytes, biocontrol agents, plant-growth promoters, or true pathogens. Specifically, P. ananatis is known to cause economic losses in agricultural crop plants, including maize white spot (MWS) in Brazil. The research involves genome sequencing and comparative genomic analysis of three P. ananatis genomes from Brazil. The pathogenicity tests reveal that while one strain (B13) causes symptoms of MWS, two other strains (13.3 and 4.2) may not induce any disease symptoms in corn plants. Only the draft genome assemblies of these strains are deposited …
Comments to Author
Reviewer summary: The study introduces the diverse ecological niches of Pantoea ananatis, a bacterium found in various environments such as water, soil, insects, plants, and humans. The bacterium can have different associations with plants, acting as endo- or epiphytes, biocontrol agents, plant-growth promoters, or true pathogens. Specifically, P. ananatis is known to cause economic losses in agricultural crop plants, including maize white spot (MWS) in Brazil. The research involves genome sequencing and comparative genomic analysis of three P. ananatis genomes from Brazil. The pathogenicity tests reveal that while one strain (B13) causes symptoms of MWS, two other strains (13.3 and 4.2) may not induce any disease symptoms in corn plants. Only the draft genome assemblies of these strains are deposited in the GenBank database, and the authors suggest conservation in the core gene content across these strains. Suggested major revisions: I appreciate the effort and achievements so far. However, there's ample room for further exploration. It's worth noting that several opportunities have been missed to present the most comprehensive comparison and scientific output of these genomes. For instance, the authors mentioned a phylogeny in the introduction (page 3; line 61), which has yet to be addressed in the manuscript. I strongly encourage a more comprehensive and inclusive approach in the following ways: 1. Kindly provide explicit details on how the initial species identification was conducted. While the authors mention that "Silva and Tebaldi (2018) identified and characterized B13 strain based on cultural, biochemical and molecular analysis", it remains unclear how the initial species identification was performed for strains 13.3 and 4.2. 2. Can you provide information on the quality control measures undertaken beyond FastQC? How did the authors ensure accurate taxonomic profiling and in silico identification as P. ananatis? Seeing as only the assembled genomes are publicly available, I have verified that 13.3 and 4.2 are P. ananatis using a GTDB-tk analysis. Parks DH, Chuvochina M, Waite DW, Rinke C, Skarshewski A et al. A standardized bacterial taxonomy based on genome phylogeny substantially revises the tree of life. Nat Biotechnol 2018; 36:996-1004 Chaumeil P-A, Mussig AJ, Hugenholtz P, Parks DH. GTDB-Tk v2: memory friendly classification with the genome taxonomy database. Bioinformatics 2022; 38:5315-5316 3. Have the authors considered a BLASTn analysis to show the presence/absence of similar regions between the genomes 13.3, 4.2, and B13 relative to a reference sequence? E.g., BRIG: Alikhan N-F, Petty NK, Ben Zakour NL, Beatson SA. BLAST Ring Image Generator (BRIG): simple prokaryote genome comparisons. BMC Genomics 2011; 12:402 4. For the stated goal of comparing corn P. ananatis strains from Brazil and determining their phylogenetic relationships with other strains of the same species, it is noted that sequence data for other P. ananatis is available in the NCBI SRA. Could you clarify why this existing data was not downloaded and used for phylogenetic analysis as mentioned in the introduction (page 3; line 61)? By not including the phylogeny, important information has been overlooked, including: (i) the common ancestry shared between 13.3 and 4.2; (ii) the shared common ancestor between 13.3, 4.2, and strain PNA 200-7 (United States; collected in the year 2000; SRA: SRR7130690); and (iii) the genomic distinctiveness of B13 in comparison to 13.3 and 4.2 (see Figure 1). Figure 1. Maximum-likelihood phylogeny of Pantoea ananatis. The phylogeny was inferred from 104,310 core-genome single-nucleotide variants (SNVs) from 32 genomes. SNVs were derived from a core-genome alignment of 3,707,830 bp and are called against the reference chromosome of P. ananatis strain PA13 (GenBank: CP003085). The scale bar represents nucleotide substitutions per site. The pink taxon represents the strain sequenced in this study. For details on the methodology used to construct this phylogeny, please refer to: White RT, Taylor W, Klukowski N, Vaughan-Higgins R, Williams E, Petrovski S, Rose JJA, Sarker S. A discovery down under: decoding the draft genome sequence of Pantoea stewartii from Australia's Critically Endangered western ground parrot/kyloring (Pezoporus flaviventris). Microbial Genomics 2023;9:001101 Additionally, considering Illumina short-read sequence data are available in the public domain (SRA) for only 28 P. ananatis genomes (meeting my quality control criteria), it would greatly benefit the scientific community if the raw Illumina short-reads were deposited in the SRA (linked to the same BioSample ID) rather than solely making the assembled genomes publicly available. Suggested minor revisions: Firstly, it would be useful to have subheadings to break up the sections. Introduction, methods, results, Discussion, etc Page 2; line 34: "P. ananatis" should be italicised Page 3; line 62: should "no pathogenic" be non-pathogenic? Page 4; line 79: "under 60 rpm shaken conditions". The information provided could benefit from increased clarity. Using xg (times gravity) as a measurement would standardise the expression of force during shaking or centrifugation, offering a more standardised, quantitative, and easily comparable measure for the centrifugal force applied in experiments. This approach would enhance the clarity and reproducibility. Page 4; line 83: "after emergency." Please clarify; it's unclear to me. Page 4; line 91: "at 200 rpm". Again, please consider changing to xg (see above). Page 4; line 99: The authors used TrimGalor! for trimming raw sequence reads. However, the specific parameters used are not provided. Please include detailed methods to ensure the clarity and reproducibility of the experiment. Page 4 line 101: The term "clean reads" might be misleading. Are you referring to trimmed reads instead? Page 5; line 102: "…De novo assembly using SPAdes incorporate in the Uniclycler pipeline (Wick et al., 2017)". Intriguingly, the authors opted for a hybrid assembly pipeline for short and long reads when working with short-read-only data. Although Unicycler provides a short-read-only method, it primarily operates as a SPAdes optimiser in this context. The authors have yet to state their use of the short-read-only method explicitly and need to provide sufficient information, such as parameters, for others to reproduce this experiment. Also, the version of SPAdes (and proper citation for SPAdes) should be added here. Page 5; line 105: "and the other Pantoea ananatis strains…". What other strains? Page 5; line 107: "specie: should be species. Page 6; line 126: The results section unexpectedly mentions "The Prokka annotation (Seemann 2014)" without prior mention in the methods. This information should be included in the methods section, specifying the Prokka version used and the parameters applied. Page 6; line 132: I downloaded the three genomes from NCBI. I noticed an error in Table 1 regarding the number of contigs for genome B13. It appears there are actually 41 contigs, not 34. Upon investigation, it seems that the lower threshold for contig length at the default (--min-contig 500 bp) in QUAST might be the reason for the discrepancy (I verified this). Page 7; line 143: "Based on the output file generated from Roary (Page et al., 2015)". The authors have not mentioned this until now and it is in the same sentence as the results. To enhance clarity and reproducibility, please provide details on the Roary version used and the specific parameters applied in the methods section. Page 9; line 186 and 187: "Our comparative analysis revealed that the genomes of the three P. ananatis strains, isolated from corn in Brazil, are highly similar". This statement is incorrect. Based on a core-genome alignment of 3,707,830 bp (80.8% of the 4,586,378 bp reference genome PA13), 13.3 and 4.2 are separated by 3 pairwise SNVs, meanwhile 4.2 and B13 are separated by 22,994 pairwise SNVs (see Figure 1). 13.3 and 4.2 are more genomically similar (~325 pairwise SNV distance) to strain PNA 200-7 (United States; collected in the year 2000; SRA: SRR7130690). Page 9; line 189: "This suggested a low core genome diversity among the three strains" - No, it doesn't imply that; rather, it suggests conservation in the core gene content across these strains. Figure 1 was derived from 104,310 core-genome SNVs based on a core-genome alignment of 3,707,830 bp, indicating a substantial diversity within the core genome.
Please rate the manuscript for methodological rigour
Poor
Please rate the quality of the presentation and structure of the manuscript
Very poor
To what extent are the conclusions supported by the data?
Not at all
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
In this study, authors analyzed the draft genome sequences of pathogenic and non-pathogenic P. ananatis strains in corn in Brazil. They compared the sequence data of their isolates with reference strains. I like this idea and also think this is a good study, but I will recommend some revisions for the authors to improve the manuscript. The comments about the manuscript are as follows: - The manuscript is written in poor English. Authors should improve English of their manuscript. - This is only recommendation. please, change title like this "Draft genome sequences of pathogenic and non-pathogenic strains of Pantoea ananatis in maize (Zea mays L.)" - Line 16-21 Please, rephrase this part. - Line 20 They are non-pathogenic isolates for corn. - Line 23-26 These isolates are almost the same. …
Comments to Author
In this study, authors analyzed the draft genome sequences of pathogenic and non-pathogenic P. ananatis strains in corn in Brazil. They compared the sequence data of their isolates with reference strains. I like this idea and also think this is a good study, but I will recommend some revisions for the authors to improve the manuscript. The comments about the manuscript are as follows: - The manuscript is written in poor English. Authors should improve English of their manuscript. - This is only recommendation. please, change title like this "Draft genome sequences of pathogenic and non-pathogenic strains of Pantoea ananatis in maize (Zea mays L.)" - Line 16-21 Please, rephrase this part. - Line 20 They are non-pathogenic isolates for corn. - Line 23-26 These isolates are almost the same. Maybe I overlooked it. Are there any big differences at a genomic and gene level between pathogenic and non-pathogenic isolates? - Line 23-26 These isolates are almost the same. Maybe I overlooked it. Are there any big differences at a genomic and gene level between pathogenic and non-pathogenic isolates? - Line 40-41 please, rephrase. - Line 42-44 please, rephrase this part. - Line 44-46 I think that the terms maize and corn are generally used to refer to the same plant. please, use the same term throughout the text. - Line 46-49 please, rephrase. - Line 50-53 please, rephrase. - Line 57-59 Sorry, I do not understand why you have insisted on this disease (BLS). Please, either you explain the background of the association between these diseases (BLS and MWS) or delete parts about this disease (BLS) in the text. You mentioned first part of the manuscript, P. ananatis could be isolated from any ecological niches. P. ananatis in this study, has been isolated from maize leaves infected with Xvv. I think this is possible. also, I see these isolates from also non-pathogenic. Maybe, these isolates (non-pathogenic) could be saprophytic. In my opinion, You aim to perform genome sequencing of pathogenic and non-pathogenic isolates of P. ananatis in this study. - Line 63- 64 please rephrase. Sorry, MWS strain and P. ananatis are same, arn't they? - Line 67 I think it could be saprophytic strains. - line 85 Please, add concentration information of bacterial suspension. Also, in each of these wound spots was inoculated with bacterial suspension (For example 100 µl). - Please, edit these parts. -The strains were grown in TSB (Tryptic Soy Broth) medium for 12 h at 30 °C under 60 rpm shaken conditions. (line 79) - The strains were grown in nutrient broth (NB) at 200 rpm and 28 ºC for 16 h. (line 90-91) - Line 106-111 do you have any idea to the pathogenic reaction to these reference strains on corn plants. if you have, please share with us. - Line 112-114 please rephrase. - Line 144-145 please, rephrase. - Line 147 Are there any endophytic strains in this study? could I have overlooked them? - Line 165-173 I agree with this idea. non-pathogenic isolates of P. ananatis could have potential traits to biocontrol of BLS. but this is the subject of another study. - Line 174-178 This is not the correct comment. all these strains are not pathogenic, so they do not give typical symptoms on the same host. - Line 239-240 please, check. not see in text.
Please rate the manuscript for methodological rigour
Satisfactory
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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