Abstract
The pathogen that causes stem gall in Loropetalum chinense was first identified in Florida and Alabama in 2018 and named Pseudomonas amygdali pv. loropetali. We report the genome sequence of the pathotype strain of this pathogen, Pseudomonas amygdali pv. loropetali DSM105780 PT.
Announcement
Pseudomonas amygdali is a member of the Pseudomonas syringae complex and was initially identified as the cause of hyperplastic bacterial canker of almond (Prunus communis Arc) [1]. A novel pathovar, Pseudomonas amygdali pv. loropetali pv. nov, reported to cause stem galls on Loropetalum chinense, was isolated in Florida and Alabama [2]. L. chinensis is a hardwood species often used as firewood in mountainous part of southern China, whose individual flowers are clustered, covering the branches blooming like fireworks [3]. The strain of Pseudomonas amygdali pv. loropetali pv. nov, PDC13-208, isolated from Florida in 2012, was designated as the pathotype strain and deposited in DSM culture collection as DSM 105780PT. Harmon et al. (2018) identified this organism as a pathovar within P. amygdali based on multi-locus sequence analysis (MLSA) [2]. In order to corroborate the MLSA analysis on placement within this species and to understand its relatedness with other members of the Pseudomonas syringae complex, we applied next-generation sequencing to clarify the whole-genome sequence of the pathotype strain.
The bacterial strain was grown for 24 h at 28 °C in nutrient broth (BBL; Becton, Dickinson and Co., Franklin Lakes, NJ) and DNA was extracted with a Wizard genomic DNA purification kit (Promega, Chicago, IL). The DNA was sent to Microbial Genome Sequencing Center (Pittsburgh, PA) for library preparation and genome sequencing. A NextSeq 550 system (Illumina, San Diego, CA) was used to produce 151 bp end reads. The paired-end reads were assembled using SPAdes v. 3.15.3 and ‘--—careful’ switch [4]. Contigs smaller than 500 bp and with less than 2.0 of K-mer coverage were filtered using script filter-spades.py 1 . Trim Galore! 2 v. 0.6.5 with default parameters was used to remove the adaptors from the paired-end reads and generate validated read files. Alignment of filtered contigs and remaining steps were performed as described by Fulton et al. (2020) [5].
Sequencing and genome statistics of the strain were calculated using Python and R scripts. R packages used were ape [6] and adegenet [7]. The draft genome comprises 314 contigs, with a G+C content of 58.068 %, N50 length of 73 733, sequencing genome coverage of 83.62×, and genome size of 6.35 Mb.
Average nucleotide identity (ANI) has been described as one of the best alternatives to DNA–DNA hybridization for prokaryotic species circumscriptions to the gold standard in which an ANI greater than 95–96 % can be used as a cut-off for considering a new prokaryotic species [8]. Pairwise ANIs of the strains and members of P. syringae complex were calculated (Table 1) and visualized (Fig. 1) using Python3 module Pyani 0.2.11 3 . Pairwise ANIs of the studied strain ( P. amygdali pv. loropetali DSM 105780 PT) were at least 98.1996 % within P. amygdali pathovars, including the P. amygdali type strain (ICMP 3198, ATCC 33050, DSM7298, NCPPB 3033) and 87.21 % with the type strain of P. syringae (DSM10604 T) and the pathotype strain of P. syringae pv. tomato (ICMP 2844). Pairwise ANI of the strain DSM 105780PT was at least 98.5 % with type strains of P. savastanoi pv. fraxini and P. savastanoi pv. savastanoi, and at least 97.4 % with the type strain of P. ficuserectae (ICMP7848).
Table 1.
Comparison of percent average nucleotide identities of Pseudomonas amygdali pv. loropetali strain DSM 105780 PT and members of P. amygadali and P. syringae complex
|
P. viridiflava DSM6694T |
ICMP7848 T |
P. caricapapayae ICMP2855 T |
P. amygdali pv. loropetali DSM 105780 PT |
P. tremae ICMP9151 T |
P. amygdali ICMPP3918 T |
P. amygdali pv. ciccaronei ICMP5710 PT |
P. avellanae JCM11937 T |
P. cannabina ICMP2823 T |
P. amygdali pv. ulmi CFBP1407 PT |
P. savastanoi pv. fraxini CFBP 5062 PT |
P. syringae pv. tomato ICMP2844 PT |
P. savastanoi pv. savastanoi ICMP4352 PT |
P. syringae DSM10604 T |
|
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|
P. viridiflava DSM6694 T |
100.000 % |
82.765 % |
82.598 % |
82.915 % |
82.431 % |
82.849 % |
82.915 % |
83.223 % |
83.054 % |
82.930 % |
82.854 % |
83.070 % |
82.840 % |
82.893 % |
|
P. ficuserectae ICMP7848 T |
82.941 % |
100.000 % |
89.044 % |
97.417 % |
85.447 % |
97.367 % |
97.458 % |
87.297 % |
85.667 % |
97.430 % |
97.453 % |
87.132 % |
97.438 % |
88.623 % |
|
P. caricapapayae ICMP2855 T |
82.606 % |
89.020 % |
100.000 % |
89.094 % |
84.743 % |
89.073 % |
89.149 % |
86.382 % |
85.136 % |
89.232 % |
89.154 % |
86.215 % |
89.135 % |
87.947 % |
|
P. amygdali pv. loropetali DSM 105780 PT |
82.831 % |
97.385 % |
89.151 % |
100.000 % |
85.629 % |
98.510 % |
98.572 % |
87.483 % |
86.026 % |
98.200 % |
98.549 % |
87.255 % |
98.566 % |
88.725 % |
|
P. tremae ICMP9151 T |
82.362 % |
86.036 % |
84.979 % |
86.405 % |
100.000 % |
86.187 % |
86.354 % |
86.150 % |
85.667 % |
86.402 % |
86.390 % |
86.008 % |
86.308 % |
85.142 % |
|
P. amygdali ICMPP3918 T |
82.867 % |
97.433 % |
89.143 % |
98.668 % |
85.462 % |
100.000 % |
98.773 % |
87.393 % |
85.776 % |
98.312 % |
98.741 % |
87.129 % |
98.752 % |
88.723 % |
|
P. amygdali pv. ciccaronei ICMP5710 PT |
82.911 % |
97.396 % |
89.185 % |
98.555 % |
85.717 % |
98.627 % |
100.000 % |
87.571 % |
86.324 % |
98.234 % |
99.459 % |
87.237 % |
99.585 % |
88.696 % |
|
P. avellanae JCM11937 T |
83.259 % |
87.293 % |
86.454 % |
87.608 % |
86.247 % |
87.363 % |
87.463 % |
100.000 % |
87.028 % |
87.773 % |
87.624 % |
95.511 % |
87.453 % |
86.318 % |
|
P. cannabina ICMP2823 T |
83.171 % |
85.764 % |
85.318 % |
86.420 % |
86.269 % |
85.991 % |
86.190 % |
87.155 % |
100.000 % |
86.433 % |
86.418 % |
86.892 % |
86.070 % |
85.469 % |
|
P. amygdali pv. ulmi CFBP1407 PT |
82.975 % |
97.427 % |
89.240 % |
98.223 % |
85.668 % |
98.157 % |
98.281 % |
87.620 % |
86.248 % |
100.000 % |
98.289 % |
87.191 % |
98.326 % |
88.780 % |
|
P. savastanoi pv. fraxini CFBP5062 PT |
82.835 % |
97.348 % |
89.160 % |
98.494 % |
85.672 % |
98.492 % |
99.423 % |
87.566 % |
86.148 % |
98.237 % |
100.000 % |
87.148 % |
99.663 % |
88.631 % |
|
P. syringae pv. tomato ICMP2844 PT |
83.133 % |
87.009 % |
86.223 % |
87.210 % |
86.023 % |
87.087 % |
87.238 % |
95.485 % |
86.719 % |
87.250 % |
87.173 % |
100.000 % |
87.210 % |
86.299 % |
|
P. savastanoi pv. savastanoi ICMP4352 PT |
82.883 % |
97.431 % |
89.206 % |
98.668 % |
85.516 % |
98.659 % |
99.608 % |
87.395 % |
85.858 % |
98.358 % |
99.774 % |
87.206 % |
100.000 % |
88.621 % |
|
P. syringae DSM10604 T |
82.787 % |
88.582 % |
87.860 % |
88.804 % |
84.898 % |
88.608 % |
88.767 % |
86.098 % |
85.420 % |
88.874 % |
88.717 % |
86.080 % |
88.720 % |
100.000 % |
PT: pathotype strain.
T: type strain.
Fig. 1.
Average nucleotide identity (ANI) among members of Pseudomonas syringae complex. The ANI was calculated using the pyani program after blastn alignment. Only regions present in all genomes were used in the ANI calculation.
Previously published reports [2, 9] place the pathogen closely in two different species: P. amygdali and P. savastanoi, respectively. The highest pairwise ANI of the P. amygdali pv. loropetali DSM 105780PT was found with P. amygdali pv. ciccaronei ICMP 5710. However, we cannot deny the closeness of the strain in study with the pathovars of P. savastanoi . Previous discussions considering the high ANIs among P. savastanoi, P. ficuserectae and P. amygdali support the claim that P. savastanoi and P. ficuserectae are synonyms of P. amygdali [10]. Given that P. savastanoi is a later synonym of P. amygdali , and the relatedness based on ANI, we believe that the evidence is sufficient that the pathogen should continue to reside in Pseudomonas amygdali as pathovar loropetali.
Funding information
This work received no specific grant from any funding agency.
Conflicts of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Funding information
This work received no specific grant from any funding agency.
Abbreviations: ANI, average nucleotide identity; ATCC, American Type Culture Collection; BBL, The Baltimore Biological Laboratory; CA, California; CFBP, French collection of plant pathogenic bacteria; DNA, deoxyribose nucleic acid; DSM, German Collection of Microorganisms; GCA, GenBank assemblies; ICMP, International Collection of Microorganisms from Plants; IL, Illinois; MLSA, multi locus sequence analysis; NJ, New Jersey; nov, novel; PDC, Plant Diagnostics Center; PT, pathotype; pv, pathovar; SPAdes, St. Petersburg genome assembler.
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