International Journal of Systematic and Evolutionary Microbiology (2016), 66, 1212–1217
DOI 10.1099/ijsem.0.000858
Bacillus velezensis is not a later heterotypic
synonym of Bacillus amyloliquefaciens; Bacillus
methylotrophicus, Bacillus amyloliquefaciens
subsp. plantarum and ‘Bacillus oryzicola’ are later
heterotypic synonyms of Bacillus velezensis
based on phylogenomics
Christopher A. Dunlap,1 Soo-Jin Kim,1,2 Soon-Wo Kwon2 and
Alejandro P. Rooney1
Correspondence
Christopher A. Dunlap
[email protected]
1
Crop Bioprotection Research Unit, National Center for Agricultural Utilization Research,
Agricultural Research Service, United States Department of Agriculture, Peoria, IL, USA
2
Korean Agriculture Culture Collection (KACC), National Institute of Agricultural Science,
Rural Development Administration, Wanju-gun, Jeollabuk-do, Republic of Korea
Bacillus velezensis was previously reported to be a later heterotypic synonym of Bacillus
amyloliquefaciens, based primarily on DNA–DNA relatedness values. We have sequenced a
draft genome of B. velezensis NRRL B-41580T. Comparative genomics and DNA–DNA
relatedness calculations show that it is not a synonym of B. amyloliquefaciens. It was instead
synonymous with Bacillus methylotrophicus. ‘Bacillus oryzicola’ is a recently described species
that was isolated as an endophyte of rice (Oryza sativa). The strain was demonstrated to have
plant-pathogen antagonist activity in greenhouse assays, and the 16S rRNA gene was reported
to have 99.7 % sequence similarity with Bacillus siamensis and B. methylotrophicus, which are
both known for their plant pathogen antagonism. To better understand the phylogenetics of
these closely related strains, we sequenced the genome of ‘B. oryzicola’ KACC 18228.
Comparative genomic analysis showed only minor differences between this strain and the
genomes of B. velezensis NRRL B-41580T, B. methylotrophicus KACC 13015T and Bacillus
amyloliquefaciens subsp. plantarum FZB42T. The pairwise in silico DNA–DNA hybridization
values calculated in comparisons between the strains were all greater than 84 %, which is well
above the standard species threshold of 70 %. The results of morphological, physiological,
chemotaxonomic and phylogenetic analyses indicate that the strains share phenotype and
genotype coherence. Therefore, we propose that B. methylotrophicus KACC 13015T,
B. amyloliquefaciens subsp. plantarum FZB42T, and ‘B. oryzicola’ KACC 18228 should be
reclassified as later heterotypic synonyms of B. velezensis NRRL B-41580T, since the valid
publication date of B. velezensis precedes the other three strains.
Bacillus velezensis was originally described by Ruiz-Garcı́a
et al. (2005), after discovery in a screen of environmental
isolates for novel lipopeptides. In that study, B. velezensis
Abbreviations: ANI, Average Nucleotide Identity; DDH, DNA–DNA
hybridization; SNP, single nucleotide polymorphism.
The GenBank/EML/DDBJ accession numbers for the draft genome
sequences of Bacillus velezensis NRRL B-41580T and ‘Bacillus
oryzicola’ KACC 18228 are LLZC00000000 and LLZA00000000,
respectively.
Two supplementary figures and a supplementary table are available
with the online Supplementary Material.
1212
was shown to be closely related to Bacillus subtilis
and Bacillus amyloliquefaciens. However, it was subsequently declared a later heterotypic synonym of
B. amyloliquefaciens by Wang et al. (2008) based on the
results of DNA–DNA relatedness studies. Our laboratories
have recently been working on whole-genome sequencing
of type strains in the Bacillus subtilis group to resolve outstanding problems in their phylogenetic systematics
(Dunlap, 2015; Dunlap et al., 2015). Recently, we reported
that the names of two important biological control strains,
Bacillus methylotrophicus and Bacillus amyloliquefaciens
subsp. plantarum, were synonymous (Dunlap et al.,
2015). However, in a preliminary analysis of the draft
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000858 G 2015
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Phylogenomic analysis of Bacillus velezensis
genome of type strain of B. velezensis (Jeong et al., 2015),
we found that it was nearly identical to B. methylotrophicus.
The recent description of ‘Bacillus oryzicola’, a root endophyte of rice (Oryza sativa) (Chung et al., 2015), has
further complicated matters. ‘B. oryzicola’ YC7010 was
reported to be a plant growth promoter, a plant pathogen
antagonist, and an inducer of systemic resistance in rice
(Chung et al., 2015). B. methylotrophicus KACC 13015T
(5CBMB205T) was also isolated from the rice
rhizosphere (Korea) and has been shown to possess plant
growth promotion and antagonism towards plant pathogens (Ma et al., 2013; Madhaiyan et al., 2010).
Interestingly,‘B. oryzicola’ YC7010 was reported to have a
very high level of 16S rRNA gene sequence similarity to
B. methylotrophicus as well as B. siamensis (99.7 % for
both) (Chung et al., 2015). In addition, the strain was
reported to have a DNA G+C content of 50.5 mol%,
which is much higher than B. methylotrophicus with
46.4 mol% (Dunlap et al., 2015). When examined collectively, all of the above observations suggest that
‘B. oryzicola’ may be a later heterotypic synonym of
B. methylotrophicus.
The
above
situation
involving
B.
velezensis,
B. amyloliquefaciens subsp. plantarum, B. methylotrophicus
and ‘B. oryzicola’ creates a particularly confounding taxonomic problem (if not nightmare) for this species
group. In order to resolve this problem, we conducted a
phylogenomic study of the aforementioned taxa by using
complete genome sequence data of both type and representative strains in order to determine the extent to which
their genomes vary from one another and to reconstruct
their phylogenetic relationships.
‘B. oryzicola’ KACC 18228 (5YC7010) and B. methylotrophicus
KACC 13015T (5CBMB205T) were obtained from the Korean
Agriculture Culture Collection. B. velezensis NRRL B-41580T
(5CR-502T5BCRC 17467T5KCTC 13102T5LMG 22478T)
and B. amyloliquefaciens subsp. plantarum NRRL B-59544
(5FZB42T) were obtained from the Northern Regional
Research Laboratory in Peoria, IL, USA. Temperature
growth studies were conducted from 4 to 60 8C on tryptoneglucose-yeast extract agar and evaluated at 48 h. NaCl tolerance (%) was conducted in 2 % increments from 0 to 20 %
(w/v) in Luria–Bertani (LB) media at 30 8C and evaluated at
48 h. All strains were cultured overnight on Biolog universal
growth plates and prepared according to manufacturer’s
instructions for the GEN III MicroPlate test panel using protocol A (Biolog); the experiment was run in triplicate.
The previously published phenotypic data for B. velezensis
NRRL B-41580T, B. methylotrophicus KACC 13105T,
B. amyloliquefaciens subsp. plantarum FZB42T and ‘B oryzicola’ KACC 18228 are consistent with the strains belonging
to the same species (Borriss et al., 2011; Chung et al., 2015;
Madhaiyan et al., 2010; Ruiz-Garcı́a et al., 2005). The previously published fatty acid methyl ester data are slightly
variable, but the primary fatty acid components are the
same. In addition, we conducted a Biolog analysis of the
http://ijs.microbiologyresearch.org
four strains and found no notable differences (Fig. S1,
available in the online Supplementary Material). We also
conducted temperature and NaCl tolerance experiments
(data not shown), with no notable differences identified.
The only notable deviations found when comparing the
previously published phenotypic, physiological, or chemotaxonomic properties of these taxa are (1) DNA G+C
content and (2) 16S rRNA gene sequence similarity
(Chung et al., 2015; Madhaiyan et al., 2010).
The genomes of B. velezensis NRRL B-41580T and
‘B. oryzicola’ KACC 18228 were sequenced using a MiSeq
DNA sequencer (Illumina) and the MiSeq V3 26300
sequencing kit (Illumina) following the manufacturer’s
suggested protocols. The resulting reads were quality
trimmed to the Q30 confidence level. The draft genome
was assembled using CLCbio Genomics Workbench 8.0
(Qiagen) using default parameters. Genome comparisons
and alignments for phylogenetic trees were made using
BIGSdb software (Jolley & Maiden, 2010). The digital
DNA–DNA hybridizations (DDH) were determined
online at http://ggdc.dsmz.de/distcalc2.php using the
Genome-to-Genome Distance Calculation (GGDC) version 2.0 as described in Meier-Kolthoff et al. (2013). The
estimated DDH values were calculated using formula two
at the GGDC website, originally described in Auch et al.
(2010) and updated in Meier-Kolthoff et al. (2013). Average Nucleotide Identity (ANI) was performed as previously
described (Goris et al., 2007), with the following options;
minimum length 700 bp, minimum identity 70 %, minimum alignment 50 %, BLAST window size 1000 bp and
step size of 200 bp. Phylogenetic analyses were conducted
using MEGA software version 6.06 (Tamura et al., 2013).
Neighbour-joining trees were reconstructed using the
Tamura–Nei model (Tamura & Nei, 1993) with a
gamma correction (alpha value50.5); this model was
chosen on the basis of the likelihood test implemented in
MEGA 6.06. A total of 1500 bootstrap pseudoreplications
were performed in order to gauge support for internal
nodes.
The draft genome of B. velezensis NRRL B-41580T was
assembled and yielded 24 contigs with a total length of
4 034 335 bp at 796 coverage and 46.3 mol% G+C content. While the draft genome of ‘B. oryzicola’ KACC 18228
yielded 25 contigs with a total length of 3 928 423 bp at
406 coverage and 46.4 mol% G+C content. The draft
genomes were used to evaluate similarity of closely related
strains using an in silico DDH determination (Table 1) and
ANI determination (Table S1). The results show the type
strains of B. methylotrophicus, B. amyloliquefaciens
subsp. plantarum and ‘B. oryzicola’ all have pairwise
DDH values well above the recommended threshold of
70 % for species delineation (Wayne et al., 1987). The
results contradict the previously reported wet lab DDH
values for B. velezensis BCRC 17467T (Wang et al., 2008)
and ‘B. oryzicola’ KACC 18228 (Chung et al., 2015). The
ANI values produced a similar result with the type strains
of B. velezensis, B. methylotrophicus, B. amyloliquefaciens
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C. A. Dunlap and others
Table 1. Results of genome-to-genome distance comparisons of closely related type strains from the B. subtilis group
Strain
Genome-to-genome comparison (regression-based DNA–DNA hybridization, %) with:
1
B. velezensis NRRL-B-41580
‘B. oryzicola’ KACC 18228
B. methylotrophicus KACC 13105T
B. amyloliquefaciens FZB42T
B. amyloliquefaciens DSM 7T
B. siamensis KCTC-13613T
B. subtilis NRRL NRS-744T
84.9
84.5
85.8
55.5
56.7
20.6
2
3
4
5
6
7
84.9
84.5
84.1
85.8
89.7
85.1
55.5
55.2
55.2
56.2
54.7
56.5
56.5
56.8
54.7
20.6
20.7
20.6
20.9
20.6
20.7
84.1
89.7
55.2
56.5
20.7
85.1
55.2
56.5
20.6
subsp. plantarum and ‘B. oryzicola’ all having pairwise ANI
values greater 98 % (Table S1). The recommended cut-off
point of 70 % DDH for species delineation corresponds to
approximately 95 % ANI (Goris et al., 2007). Our draft
genome of B. velezensis NRRL B-41580T was essentially
clonal with the recently reported draft genome of
B. velezensis KCTC 13012T (Jeong et al., 2015).
The DNA G+C content for ‘B. oryzicola’ KACC 18228 was
originally reported at 50.5 mol% (Chung et al., 2015) and
45 mol% for B. methylotrophicus KACC 13015T (Madhaiyan et al., 2010) using wet lab methods for both.
54.7
20.9
20.7
While the sequenced genome data provide a G+C content
of 46.4 mol% for B. methylotrophicus KACC 13015T
(Dunlap et al., 2015) and 46.4 mol% for ‘B. oryzicola’
KACC 18228 in the current study. The discrepancy
between the original data generated using the conventional
(experimental) method and the whole genome data generated using next-generation sequencing techniques suggests
that the conventional method has some experimental error,
which is not surprising. This observation is supported by a
recent study comparing data from conventional methods
and genome-sequencing data, which showed G+C content
varies 3–5 mol% within species for conventional methods
B. subtilis subsp. spizizenii TU-B-10T (CP002905)
100
100
100
0.05
56.2
56.8
20.9
B. subtilis subsp. inaquosorum KCTC 13429T (AMXN01)
B. subtilis 168T (AL009126)
100
B. tequilensis KCTC 13622T (AYTO01)
100
B. vallismortis DV1-F-3T (AFSH01)
100
B. mojavensis KCTC 3706T (AFSI01)
B. atrophaeus 1942 (CP002207)
100
B. siamensis KCTC 13613T (AJVF01)
95
B. amyloliquefaciens DSM 7T (FN597644)
B. methylotrophicus KACC 13105T (JTKJ01)
100
100
100
100
100
100
B. velezensis NRRL B-41580T (LLZC01)
B. amyloliquefaciens subsp plantarum FZB42T (CP000560)
‘B. oryzicola’ KACC 18228 (LLZA01)
conspecific
1.
2.
3.
4.
5.
6.
7.
T
B. lichenformis DSM 13T (CP000002)
B. paralichenformis KJ-16T (LBMN01)
B. glycinifermentans GO-13T (LECW01)
100
B. sonorensis KCTC 13918T (AYTN01)
100
100
B. xiamenensis HYC-10T (AMSH01)
B. altitudinis 41KF2bT (ASJC01)
100
100
B. safenesis F0-36bT (ASJD01)
B. pumilus SAFR-032 (CP000813)
Fig. 1. Phylogeny of the Bacillus subtilis species group reconstructed from a neighbour-joining analysis of core-genome
sequence data (799 genes). Bootstrap values .50 %, based on 1500 pseudoreplicates are indicated at branch points.
Bar, 0.05 nt substitutions per site.
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Phylogenomic analysis of Bacillus velezensis
B. methylotrophicus OB9 (LGAU01)
B. methylotrophicus B26 (LGAT01)
0.05
B. amyloliquefaciens KHG19 (CP007242)
B. methylotrophicus UCMB 5113 (HG328253)
B. amyloliquefaciens G341 (CP011686)
B. methylotrophicus UCMB 5033 (HG328253)
Bacillus sp. 916 (AFSU01)
B. methylotrophicus EBL11 (JCOC01)
B. amyloliquefaciens LPL-K103 (JXAT01)
Bacillus sp. UNC69MF (JQKM01)
‘B. oryzicola’ KACC 18228 (LLZA01)
B. methylotrophicus AS 43.3 (CP003838)
B. methylotrophicus TrigoCor 1448 (CP007244)
B. methylotrophicus AP183 (JXAM01)
B. amyloliquefaciens 629 (LGYP01)
B. amyloliquefaciens subsp. plantarum FZB42T (CP000560)
B. methylotrophicus CC178 (CP006845)
B. amyloliquefaciens Bs006 (LJAU01)
B. amyloliquefaciens XK-4-1 (LJDI01)
Bacillus sp. Pc3 (CP010406)
B. methylotrophicus UCMB 5036 (HF563562)
B. amyloliquefaciens TF28 (JUDU01)
B. amyloliquefaciens HB-26 (AUWK01)
B. methylotrophicus SOR9 (CP006890)
B. methylotrphicus YJ11-1-4 (CP011347)
B. velezensis
B. subtilis ATCC 19217 (CP009749)
B. subtilis GB03 (AYTJ01)
Bacillus sp. 5B6 (AHST01)
B. methylotrophicus GR4-5 (GYGH01)
B. amyloliquefaciens Lx-11 (AUNG01)
B. methylotrophicus KACC 13105T (JTKJ01)
B. amyloliquefaciens X1 (JQNZ01)
B. methylotrophicus SK19.001 (AOFO01)
B. amyloliquefaciens DC-12 (AMQI01)
B. subtilis SPZ1 (AQGM01)
B. amyloliquefaciens EGD-AQ12 (AVQH01)
B. methylotrophicus JS25R (CP009679)
B. methylotrophicus NAU-B3 (HG514499)
B. methylotrophicus B9601-Y2 (HE774679)
B. subtilis NKYL29 (JPYY01)
B. velezensis NRRL B-4257 (LLZB01)
B. velezensis NRRL B-41580T (LLZC01)
B. velezensis KCTC 13012T (LHCC01)
B. methylotrophicus M27 (AMPK01)
B. methylotrophicus B1895 (JMEG01)
B. methylotrophicus AH159-1 (JFBZ01)
B. methylotrophicus IT-45 (CP004065)
B. amyloliquefaciens UASWSBA1 (AWQY01)
B. methylotrophicus LFB112 (CP006952)
B. methylotrophicus NJN-6 (CP007165)
B. methylotrophicus B946 (HE617159)
B. methylotrophicus JJ-D34 (CP0011346)
B. subtilis B-1 (CP009684)
B. amyloliquefaciens L-H15 (CP010556)
B. amyloliquefaciens L-S60 (CP011278)
B. amyloliquefaciens CMW1 (BBLH01)
B. amyloliquefaciens DSM 7T (FN597644)
B. amyloliquefaciens LL3 (CP002634)
B. amyloliquefaciens
B. amyloliquefaciens TA208 (CP002627)
B. amyloliquefaciens XHY (CP002927)
B. siamensis JJC33M (JTJG01)
B. siamensis XY18 (LAGT01)
B. siamensis
B. siamensis KCTC 13613T (AJVF01)
Fig. 2. Phylogeny of selected strains of species from the Bacillus velezensis, Bacillus amyloliquefaciens and Bacillus
siamensis clades reconstructed from a neighbour-joining analysis of core-genome sequence data (2740 genes). Bar, 0.05 nt
substitutions per site.
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1215
C. A. Dunlap and others
and within 1 % for whole-genome sequencing data (MeierKolthoff et al., 2014).
After genome sequencing of both B. velezensis NRRL
B-41580T and ‘B. oryzicola’ KACC 18228, we extracted the
16S rRNA gene sequence and used the EzTaxon-e database
(Kim et al., 2012) to identify the closest relatives. The analysis identified the nearest neighbour for both strains was
B. methylotrophicus (JTKJ01000077) with 100 % sequence
similarity. To understand the difference between our findings and the previous report of 99.7 % sequence similarity
for ‘B. oryzicola’ KACC 18228 (Chung et al., 2015), we did
an alignment of 16S rRNA gene sequences (Fig. S2). The
only difference observed between the ‘B. oryzicola’ KACC
18228 16S rRNA gene sequence generated in Chung et al.
(2015) and our study was a C to T single nucleotide polymorphism (SNP) at position 146. This difference was not
enough to explain the observed differences in reported
sequence similarity. After review, the differences arise from
different 16S rRNA gene sequences reported for the type
strain of B. methylotrophicus at the EzTaxon-e database.
In the current study, the 16S rRNA gene sequence of
B. methylotrophicus KACC 13105T obtained at the
EzTaxon-e database is from GenBank accession number
JTKJ01000077, based on whole genome data released 23
March 2015 (Dunlap et al., 2015). Prior to the EzTaxon-e
database being updated, the 16S rRNA gene of the type
strain of B. methylotrophicus would have been from GenBank
accession number EU194897 (Madhaiyan et al., 2010).
There are 8 nt differences between these two 16S rRNA
gene sequences, which arise from three N’s and three deletions in the first 15 nt, an insert at position 624 and two deletions at positions 1476–1477 of GenBank accession number
EU194897. We have recently reported similar discrepancies
in other strains from the Bacillus subtilis group 16S rRNA
gene sequence data obtained from capillary electrophoresis
as well as whole-genome sequence data, which also resulted
in part to another synonym being described as a new species
(Dunlap, 2015). It is notable that of the 16S rRNA gene
sequences extracted from the genomes of the four conspecific type strains, only two SNPs were observed.
The phylogenomic tree based on the core genome (799
genes) of the strains of the Bacillus subtilis group showed
that B. velezensis NRRL B-41580T, B. methylotrophicus
KACC 13105T, B. amyloliquefaciens subsp. plantarum
FZB42T and ‘B oryzicola’ KACC 18228 were closely related
(Fig. 1). The results are consistent with B. velezensis NRRL
B-41580T, B. methylotrophicus KACC 13105T, ‘B. oryzicola’
KACC 18228 and B. amyloliquefaciens subsp. plantarum
FZB42T being conspecific. To further resolve the confusion
surrounding these closely related strains, we downloaded
and analysed all of the genomes of species in the Bacillus
subtilis group present in the GenBank database. Strains
belonging to the clade containing B. velezensis and its nearest neighbours B. amyloliquefaciens and B. siamensis were
used to reconstruct a tree based on the sequences of
genes in their core genomes (Fig. 2). The results highlight
the confusion that has arisen regarding the nomenclature
1216
and taxonomy of these strains commonly isolated as
plant pathogen antagonists and developed as biological
control agents (see Dunlap et al., 2015). Because the valid
publication of B. velezensis (Ruiz-Garcı́a et al., 2005) predates the publication of B. methylotrophicus (Madhaiyan
et al., 2010), B. amyloliquefaciens subsp. plantarum
(Borriss et al., 2011) and ‘B. oryzicola’ (Chung et al.,
2015), we propose that B. methylotrophicus, B. amyloliquefaciens subsp. plantarum, and ‘B. oryzicola’ be reclassified as
later heterotypic synonyms of B. velezensis.
Acknowledgements
This study was supported by the ‘Research Program for Agricultural
Science & Technology Development (project no. PJ011248)’ of the
National Institute of Agricultural Science, Rural Development
Administration, Republic of Korea. The authors would like to
thank Heather Walker of the National Center for Agricultural Utilization Research, Peoria, IL, USA for expert technical assistance. Any
opinions, findings, conclusions, or recommendations expressed in
this publication are those of the author(s) and do not necessarily
reflect the view of the US Department of Agriculture. The mention
of firm names or trade products does not imply that they are
endorsed or recommended by the USDA over other firms or similar
products not mentioned. USDA is an equal opportunity provider and
employer.
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