# Letter to the Editor: The Ralstonia research community rejects the proposal to classify phylotype I Ralstonia into the new species Ralstonia nicotianae

Tiffany Lowe-Power, Parul Sharma, Poliane Alfenas-Zerbini, Belén Álvarez, Mohammad Arif, Caroline Baroukh, Elena Biosca, Ana Maria Bocsanczy, Jose Castillo, Gilles Cellier, et al.

► To cite this version:

Tiffany Lowe-Power, Parul Sharma, Poliane Alfenas-Zerbini, Belén Álvarez, Mohammad Arif, et al. Letter to the Editor: The Ralstonia research community rejects the proposal to classify phylotype I Ralstonia into the new species Ralstonia nicotianae. PhytoFrontiers, 2023, 10.1094/PHYTOFR-06-23-0071-LE. hal-04188467

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## Q1 Letter To The Editor

### Q2 The *Ralstonia* Research Community Rejects the Proposal to Classify Phylotype I *Ralstonia* into the New Species *Ralstonia nicotianae*

Tiffany Lowe-Power<fnref n="1" />,† ID | Parul Sharma<fnref n="2" /> | Poliane Alfenas-Zerbini<fnref n="3" /> ID | Belén Álvarez<fnref n="4" /> | Mohammad Arif<fnref n="5" /> ID |
Caroline Baroukh<fnref n="6" /> | Ana Maria Bocsanczy<fnref n="7" /> ID | Elena G. Biosca<fnref n="8" /> | José A. Castillo<fnref n="9" /> | Gilles Cellier<fnref n="10" /> ID | Teresa Coutinho<fnref n="11" /> ID |
André Drenth<fnref n="12" /> | Ville-Petri Friman<fnref n="13" /> | Stephane Genin<fnref n="6" /> | Alice Guidot<fnref n="6" /> | Yasufumi Hikichi<fnref n="14" /> ID | Qi Huang<fnref n="15" /> ID |
Anjali Iyer-Pascuzzi<fnref n="16" /> ID | Kenji Kai<fnref n="17" /> | Yann Pecrix<fnref n="18" /> | Stephane Poussier<fnref n="19" /> ID | Jane Ray<fnref n="20" /> | Maurício Rossato<fnref n="21" /> ID |
Rebecca Schomer<fnref n="22" /> | Maria Inés Siri<fnref n="23" /> ID | Boris Vinatzer<fnref n="2" />,† ID | Caitilyn Allen<fnref n="24" />,† |

Q4

Q5

## Abstract

The *Ralstonia solanacearum* species complex is a group of globally important plant pathogens. Bacteria in this very large and genetically diverse group all colonize the xylem elements of angiosperm plants and cause high-impact wilting diseases of many crops. Because they threaten economic and food security, several *R. solanacearum* species complex subgroups are strictly regulated as quarantine pests. Biologically meaningful and consistent nomenclature is essential for organisms that have major economic and regulatory importance, such as plant-pathogenic *Ralstonia*. There are currently three species of *Ralstonia* wilt pathogens: *R. pseudosolanacearum* (corresponding to two phylogenetic groups that are described in the literature as phylotypes I and III), *R. solanacearum* (phylotypes IIA, IIB, and IIC), and *R. syzygii* (phylotype IV, containing three subspecies: subsp. *syzygii*, subsp. *celebensis*, and subsp. *indonesiensis*). A recent paper proposed reclassifying phylotype I as a new species named "*Ralstonia nicotianae*." The purpose of this commentary is to register our objection to the taxon "*Ralstonia nicotianae*."

## Keywords:

The *Ralstonia solanacearum* species complex (RSSC) is a group of globally important plant pathogens. Bacteria in this very large and genetically diverse group all colonize the xylem elements of angiosperm plants and cause high-impact wilting diseases of many crops. Because they threaten economic and food security, several RSSC subgroups are strictly regulated as quarantine pests (see “Regulation” section before the references). Biologically meaningful and consistent nomenclature is essential for organisms that have major economic and regulatory importance, such as plant-pathogenic *Ralstonia*. There are currently three species of *Ralstonia* wilt pathogens: *R. pseudosolanacearum* (corresponding to two phylogenetic groups that are described in the literature as phylotypes I and III), *R. solanacearum* (phylotypes IIA, IIB, and IIC), and *R. syzygii* (phylotype IV, containing three subspecies: subsp. *syzygii*, subsp. *celebensis*, and subsp. *indonesiensis*). A recent paper proposed reclassifying phylotype I as a new species named “*Ralstonia nicotianae*” (Liu et al. 2023). The purpose of this commentary is to register our objection to the taxon “*Ralstonia nicotianae*.”

## ALTHOUGH CHANGING BACTERIAL TAXONOMY IS SOMETIMES NECESSARY, THE RALSTONIA NICOTIANAE PROPOSAL IS NOT JUSTIFIED

Changing the taxonomy of any group of organisms can be disruptive to both scientists and regulators, so it should not be proposed for trivial reasons, as explained in the

<figure><img src="image_30.png" /></figure>

<footnote n="1">¹ University of California, Davis, U.S.A.</footnote>

<footnote n="2">² Virginia Polytechnic Institute and State University, U.S.A.</footnote>

<footnote n="3">³ Department of Microbiology, Universidade Federal de Viçosa, Viçosa, MG, Brazil</footnote>

<footnote n="4">⁴ Plant Protection Laboratory, IMIDRA, Community of Madrid, Spain</footnote>

<footnote n="5">⁵ Department of Plant and Environmental Protection Sciences, University of Hawaii at Manoa, Honolulu, HI, U.S.A.</footnote>

<footnote n="6">⁶ LIPME, Université de Toulouse, INRAE, CNRS, Castanet-Tolosan, France</footnote>

<footnote n="7">⁷ University of Florida, U.S.A.</footnote>

<footnote n="8">⁸ Universitat de València, Spain</footnote>

<footnote n="9">⁹ Yachay Tech University, Ecuador</footnote>

<footnote n="10">¹⁰ Anses Laboratoire de la sante des vegetaux, Saint-Pierre, Reunion Island, France</footnote>

<footnote n="11">¹¹ Department of Biochemistry, Genetics and Microbiology, Centre for Microbial Ecology and Genomics/Forestry and Agricultural Biotechnology Institute, University of Pretoria, Pretoria 0002, South Africa</footnote>

<footnote n="12">¹² University of Queensland, Australia</footnote>

<footnote n="13">¹³ Department of Microbiology, University of Helsinki, Finland</footnote>

<footnote n="14">¹⁴ Faculty of Agriculture and Marine Science, Kochi University, Japan</footnote>

<footnote n="15">¹⁵ Floral and Nursery Plants Research Unit, US National Arboretum, USDA/ARS, Beltsville, MD, U.S.A.</footnote>

<footnote n="16">¹⁶ Department of Botany and Plant Pathology, Purdue University, U.S.A.</footnote>

<footnote n="17">¹⁷ Osaka Metropolitan University, Japan</footnote>

<footnote n="18">¹⁸ CIRAD, UMR PVBMT, F-97410 Saint-Pierre, Réunion Island, France</footnote>

<footnote n="19">¹⁹ Université de la Réunion, France</footnote>

<footnote n="20">²⁰ Northern Territory Government, Australia</footnote>

<footnote n="21">²¹ Universidade de Brasilia, Brazil</footnote>

<footnote n="22">²² School of Plant Sciences, University of Arizona, U.S.A.</footnote>

<footnote n="23">²³ Molecular Microbiology Laboratory, Biosciences Department, School of Chemistry, Universidad de la República, Montevideo, Uruguay</footnote>

<footnote n="24">²⁴ Department of Plant Pathology, University of Wisconsin-Madison USA</footnote>

<footnote>† Corresponding authors: T. Lowe-Power; Tlowepower@ucdavis.edu, B. Vinatzer; vinatzer@vt.edu, and C. Allen; caitilyn.allen@wisc.edu</footnote>

<footnote>Accepted for publication 13 June 2023.</footnote>

<footnote>T. Lowe-Power and P. Sharma are co-first authors.</footnote>

<footnote>The author(s) declare no conflict of interest.</footnote>
code of the International Committee on Systematics of Prokary-
otes (Oren et al. 2023). There are two main reasons to propose
new bacterial species. First, the isolation and discovery of novel
bacteria that do not belong to any named species justifies the
naming of a new species. Second, better data from technolog-
ical and analytical advances can change our understanding of
the diversity and evolution of bacterial lineages. With sufficient
evidence, these advances can justify taxonomic revisions so that
the newly named species better reflect evolutionary relationships.
However, the *R. nicotianae* proposal is not based on discovery of
a new lineage, nor does it reflect novel insight into the evolution-
ary relationships within the RSSC. As demonstrated below, the
*R. nicotianae* proposal ignores natural phylogenetic gaps among
the existing three species. Moreover, it is based on inappropriately
selective use of molecular analyses.

During their decades-long careers, Drs. Philippe Prior and
Mark Fegan collected and studied the diversity of RSSC plant
pathogens from around the world. Both research group leaders
concurred that the RSSC is properly divided into three species
(Fegan and Prior 2005; Prior et al. 2016; Remenant et al. 2010,
2011; Safni et al. 2014). Specifically, extensive genomic and bio-
logical analyses of phylotype I and III strains led these and other
experts to conclude that phylotype I and III should not be di-
vided into distinct species (Prior et al. 2016; Sharma et al. 2022;
Truchon et al. 2023). As a result, three RSSC species were validly
published in 2014 as *R. solanacearum*, *R. pseudosolanacearum*,
and *R. syzygii* (Safni et al. 2014). These names were subse-
quently validated by the list editors of the *International Journal of
Systematic and Evolutionary Microbiology* (IJSEM). Figure 1
shows the overall relationships among subgroups of the RSSC
in a phylogenetic tree constructed using the core genome of the
species complex. *R. pseudosolanacearum* is composed of two
major subgroups (phylotype I and III). *R. solanacearum* is com-
posed of three major subgroups (phylotype IIA, IIB, and IIC).

NATURAL GAPS IN GENETIC DIVERSITY SEPARATE THE THREE
RSSC SPECIES

Average nucleotide identity (ANI) is now an accepted way to
use whole-genome sequences to measure relationships between
strains and propose species delineations (Oren et al. 2023). The
ANI between any pair of genomes can be calculated based on
different algorithms, such as BLAST comparisons (“ANIB”) or
the MUMMER index (“ANIm”). We used pyani (Pritchard et al.
2016), a Python-based ANIb software, to calculate pairwise ANIb
values for 300 RSSC genomes, including genomes of 11 phylo-
type III strains and 148 phylotype I strains. When the resulting
90,000 ANIb values are hierarchically clustered and visualized
as a heatmap, three obvious clusters correspond to the three ac-
cepted RSSC species (Fig. 2A).

AN ANI THRESHOLD OF 96% IS NOT THE APPROPRIATE
CUTOFF FOR DELINEATING SPECIES IN THE RSSC

Depending on the taxon, bacterial species borders can be drawn
using ANI threshold values of 95 to 96% (Chun et al. 2018; Oren
et al. 2023). However, an ANI <95% is the most widely used
cutoff for dividing species. This threshold has been applied across
the bacterial domain in the Genome Taxonomy Database (Parks
et al. 2020). We investigated the distributions of 90,000 ANIb
comparisons among 300 RSSC genomes to determine if there is
a biologically relevant cutoff that separates RSSC species.

The *R. nicotianae* proposal applied an 96% ANI species thresh-
old value. However, our analysis of 300 RSSC genomes sug-
gests that 95% is the appropriate threshold for delineating species

within the RSSC (Fig. 2). Visualizing the distribution of ANI
values reveals an obvious natural gap in ANIb values: No pair-
wise comparison yields an ANI value between 92.57 and 95.06%
(Fig. 2B). Applying an ANI cutoff of 96% (indicated by the red
lines in Fig. 2 graphs) would interrupt a continuous distribution
of genetic distances within the RSSC as a whole (Fig. 2B), within
*R. solanacearum* (Fig. 2C), and within *R. pseudosolanacearum*
(Fig. 2D). In contrast, a 95% ANI cutoff (indicated by the blue
lines) separates the RSSC into three species with clear gaps that
suggest that these groups have distinct evolutionary histories
(Fig. 2E), and the existing three-species nomenclature may thus
represent their natural phylogenetic order.

THE DATA PRESENTED IN THE R. NICOTIANAE PROPOSAL DO NOT SUPPORT A DIVISION OF PHYLTYPE I INTO A NEW SPECIES

This section provides a detailed dissection of ANI data to high-
light the methodological problems in the *R. nicotianae* proposal.

The *R. nicotianae* proposal was based on limited analyses that
compared genomes of a single phylotype I and a single phylotype
III genome against other *R. pseudosolanacearum* genomes. This
approach significantly biased the statistics and phylogenetic anal-
yses, as it does not reflect the diversity of a representative popula-
tion of isolates. The focal strains were the established type strain
of *R. pseudosolanacearum* (phylotype III strain LMG9673T) and
a phylotype I strain (RS) that was proposed as a type strain for
the novel species. Hereafter, we refer to this strain as RS*proposed_T*.

The *R. nicotianae* proposal calculated ANI with three meth-
ods: FastANI using the Genome Taxonomy Database website
interface, ANIb using the JSpeciesWS website interface, and
MUMMER-based ANI (ANIm) using the JSpeciesWS website
interface. The authors then carried out 434 FastANI compar-
isons (LMG9673T and RS<sup>proposed_T</sup> vs. 204 phylotype I and 11
phylotype III strains), 24 ANIb comparisons (LMG9673T and
RS<sup>proposed_T</sup> vs. 1 phylotype I and 11 phylotype III strains), and
24 ANIm comparisons (LMG9673T and RS<sup>proposed_T</sup> vs. 1 phy-
lyotype I and 11 phylotype III strains).

Comparing RS<sup>proposed_T</sup> to the 11 phylotype III genomes yielded FastANI values from 95.85 to 96.06%, ANIm values from 96.12 to 96.26%, and ANIb values from 94.95 to 95.33% as described in the *R. nicotianae* proposal. We also computed ANIb values, but we used the Python-based pyani tool over a larger sample size of phylotype I and III genomes (Fig. 2). In the subset of comparisons that overlap between our analysis and that of the *R. nicotianae* proposal, pyani yielded ANIb values from 95.77 to 96.02%. An overview of these data is presented in Figure 3A, which compares the ANI values obtained for each of the comparisons and methods.

For taxonomic classification, the most important ANI compar-
isons are between type strains. In the *R. nicotianae* proposal, com-
parisons between RS<sup>proposed_T</sup> and the *R. pseudosolanacearum*
type strain LMG9673<sup>T</sup> yielded values of 95.97 to 96.02%
(FastANI), 96.14 to 96.15% (ANIm), and 95.23 to 95.30%
(ANIb). Our pyani calculation of ANIb yielded a narrow range
of values from 95.81 to 95.82%.

Before genome sequences were readily available, the gold
standard for classifying bacterial strains into species was a wet-
lab technique called DNA-DNA hybridization (DDH). A 70%
DDH threshold was used to delineate bacterial species. The *R.
nicotianae* proposal used three digital DDH calculations (dDDH)
to estimate DDH between RS<sup>proposed_T</sup> and LMG9673<sup>T</sup>. Two
dDDH calculations yielded values above the standard 70%
threshold (74.9 and 75.8%), and a third dDDH calculation yielded
a value of 66.2%. If averaged, the three calculations yield 72.3%,
<figure><img src="image_31.png" /><figcaption>FIGURE 1</figcaption></figure>

Core genome phylogenetic tree demarcating the three *Ralstonia solanacearum* species complex species and their major subdivisions: *R. pseudosolanacearum* (phylotype I and III subdivisions), *R. solanacearum* (phylotype IIA, IIB, and IIC subdivisions (Sharma et al. 2022), and *R. syzygii*. The tree was built using IQtree (Minh et al. 2020) using the core-genome alignments obtained with PIRATE (Bayliss et al. 2019) as input.
A

<figure><img src="image_32.png" /></figure>

B

<figure><img src="image_33.png" /></figure>

C

Comparison: Within
*R. pseudosolanacearum*
(phylotype I and III)

<figure><img src="image_34.png" /></figure>

D

Comparison:
Within *R. solanacearum*
(phylotype IIA, IIB and IIC)

<figure><img src="image_35.png" /></figure>

E

Comparison between
true species

<figure><img src="image_36.png" /><figcaption>FIGURE 2</figcaption></figure>

The biologically relevant average nucleotide identity (ANI) threshold for delineating *Ralstonia solanacearum* species complex (RSSC) species is 95%. A, Robust ANI analysis of 300 RSSC genomes reveals three species clusters corresponding to *R. pseudosolanacearum*, *R. solanacearum*, and *R. syzygii*. Pairwise comparisons are shown in an ANI heatmap calculated with the BLAST-based ANIb method using pyani (Pritchard et al. 2016). B, The distribution of pairwise ANIb values between 300 RSSC strains reveals a natural gap between pairs sharing 92.57 and 95.06% ANIb. ANIb was calculated with pyani (Pritchard et al. 2016). C, Comparison of ANI values within the *R. pseudosolanacearum* species and its two major subdivisions. D, Comparison of ANI values within the *R. solanacearum* species and its three major subdivisions. E, Comparison of ANI values between the three validated RSSC species. Blue lines show the biologically relevant ANI threshold of 95%, and red lines show the biologically inappropriate threshold of 96%.
above the 70% species cutoff. Figure 3B shows the full distribu-
tion of dDDH scores from the *R. nicotianae* proposal. However,
the text of the *R. nicotianae* proposal emphasized only the lowest
of these three DDH values.

The careful assessment above reveals that the conclusions in
the *R. nicotianae* proposal were based on the sole DDH analy-
sis and the sole ANI analysis where comparisons of type strains
yielded a value less than the 70% DDH threshold and an ANI
value in the gray zone of 95 to 96% ANI. This ignored the molec-
ular phylogenomic analysis results that suggested that phylotype
I should remain within the *R. pseudosolanacearum* species. Se-
lecting among obtained results to present only the subset of re-
sults that support a preferred narrative is not consistent with good
scientific practice (Casadevall and Fang 2016).

EVEN IF THERE WAS GENOMIC AND BIOLOGICAL
JUSTIFICATION FOR THE SEPARATION OF PHYTYPE I INTO A
NOVEL SPECIES, NICOTIANAE WOULD BE A MISLEADING
SPECIES EPITHET FOR PHYTYPE I

The epithet *nicotianae* was suggested because pathogenic bac-
teria are sometimes named for their host of isolation, usually the
primary host, and the proposed Type strain RS<sup>proposed_T</sup> was iso-
lated from an experimental tobacco plot. However, this name
would be misleading because infecting tobacco is not a distin-
guishing trait of phylotype I. RSSC strains from each of the four
phylotypes have been isolated from tobacco (Lowe-Power et al.
2020). Furthermore, phylotype I strains have the broadest host
range within the RSSC; Phylotype I strains have been isolated
from 95 plant species in 79 genera in 46 families (Lowe-Power
et al. 2020). In comparison, the other three phylotypes combined
have been isolated from only 69 plant species in 40 genera in 28
families (Lowe-Power et al. 2020).

Proposing new names without careful consideration can cre-
ate confusion in the research community and potentially in the

published literature. For example, the widely used NCBI genome
database transiently adopted the *R. nicotianae* proposal. Within
two weeks of the publishing of the *R. nicotianae* proposal in
*Frontiers in Microbiology*, we noticed that NCBI had renamed
the genome of the much studied model *R. pseudosolanacearum*
GMI1000 as “*Ralstonia nicotianae*.” This occurred be-
fore the IJSEM list editors had the opportunity to consider this
proposal and issue a decision about publishing the new name.
Although GMI1000 is a phylotype I *R. pseudosolanacearum*
strain, the GMI1000 genome was still labeled in NCBI as
“*Ralstonia solanacearum*” for historical reasons: The genome
was sequenced and deposited 14 years before the RSSC was for-
mally divided into three species (Salanoubat et al. 2002). Impor-
tantly, this error was promptly corrected when it was brought to
the attention of NCBI.

SUMMARY

Adopting “*R. nicotianae*” as a newly named species cor-
responding to phylotype I and reducing the validly published
species *R. pseudosolanacearum* to include only phylotype III is
not justified based on either genomic similarity or evolutionary
relationships. On the contrary, the comparative genomics analy-
ses presented in the *R. nicotianae* proposal are consistent with the
conclusion that phylotype I and phylotype III are two subgroups
of the same species, *R. pseudosolanacearum*. Furthermore, ac-
cepting a division of phylotype I and III into separate species
would complicate and disrupt scientific and regulatory communi-
cation about strains and genomes of plant-pathogenic *Ralstonia*.
Changing the name of a taxon that has been established and vali-
dated through multiple rigorous studies would create unnecessary
confusion. This proposal violates three of the four essential ele-
ments of Principle 1 of the International Code of Nomenclature
of Prokaryotes, which states that nomenclature should “1) Aim
at stability of names; 2) Avoid or reject names that create er-

<figure><img src="image_37.png" /><figcaption>FIGURE 3</figcaption></figure>

The *R. nicotianae* proposal focused on outlier average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) calculations that supported a new species. A, Comparison of ANI values from the 12 pairs of genomes that were shared between the *R. nicotianae* proposal and our larger-scale analysis (Figs. 1 and 2). The *R. nicotianae* proposal analyzed ANI between six phylotype III genomes to two strains: the *R. pseudosolanacearum* type strain (LMG9673T) and the phylotype I strain proposed as a new type strain (RS<sup>proposed_T</sup>). B, Comparison of dDDH calculations from the *R. nicotianae* proposal. Lines connect the same strain pairings that were analyzed using three different dDDH tools. ANI and DDH comparisons of *R. pseudosolanacearum* type strain LMG9673T and RS<sup>proposed_T</sup> are shown in red. Arrows indicate the outlier results favored in the *R. nicotianae* proposal.
ror or confusions; and 3) Avoid the useless creation of names” (Oren et al. 2023). Finally, the chosen species name would be misleading regarding the host range of the strains that belong to it and to the related strains in other species within the RSSC and is thus in conflict with International Code of Nomenclature of Prokaryotes Recommendation 12(c) 2: “Avoid [epithets] that express a character common to all, or nearly all, the species of a genus” (Oren et al. 2023). These reasons, together with the analyses presented in this letter, establish that “*Ralstonia nicotianae*” Liu et al. 2023 is at most a junior heterotypic synonym of *Ralstonia pseudosolanacearum* Safni et al. 2014.

Therefore, we strongly encourage our fellow scientists in the RSSC community not to adopt *R. nicotianae* in publications and scientific communication in general. We further respectfully request that the IJSEM list editors review the evidence presented here when considering whether *R. nicotianae* should be validly published.

## Regulation

**European Union: Regulation (EU) 2016/2031 of the European Parliament of the Council of 26 October 2016 on protective measures against pests of plants, amending Regulations (EU) No 228/2013, (EU) No 652/2014 and (EU) No 1143/2014 of the European Parliament and of the Council and repealing Council Directives 69/464/EEC, 74/647/EEC, 93/85/EEC, 98/57/EC, 2000/29/EC, 2006/91/EC and 2007/33/EC https://eur-lex.europa.eu/eli/reg/2016/2031/oj**

**United Kingdom: https://planthealthportal.defra.gov.uk/pests-and-diseases/pest-and-disease-factsheets/notifiable-diseases/**

**Canada: https://inspection.canada.ca/plant-health/invasive-species/regulated-pests/eng/1363317115207/1363317187811**

**United States: https://www.aphis.usda.gov/aphis/ourfocus/planthealth/import-information/rppl/rppl-table**

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