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‘Candidatus Liberibacter americanus’, associated
with citrus huanglongbing (greening disease) in
Sa
˜
o Paulo State, Brazil
Diva do Carmo Teixeira,
1
,
3
Colette Saillard,
2
Sandrine Eveillard,
2
Jean Luc Danet,
2
Paulo Ina
´
cio da Costa,
3
Antonio Juliano Ayres
1
and Joseph Bove
´
2
Correspondence
Sandrine Eveillard
jagoueix@bordeaux.inra.fr
1
Fundecitrus, Av. Dr. Adhemar Pereira de Barros, 201, CEP 14807-040, Araraquara, SP, Brazil
2
UMR 1090, Institut National de la Recherche Agronomique et Universite
´
Victor Segalen
Bordeaux 2, BP 81, 33883 Villenave d’Ornon cedex, France
3
Instituto de Quı´mica – UNESP, R. Francisco Degni, s/n, CEP 14800-900, Araraquara, SP, Brazil
Symptoms of huanglongbing (HLB) were reported in Sa
˜
o Paulo State (SPS), Brazil, in March 2004.
In Asia, HLB is caused by ‘Candidatus Liberibacter asiaticus’ and in Africa by ‘Candidatus Liberibacter
africanus’. Detection of the liberibacters is based on PCR amplification of their 16S rRNA gene
with specific primers. Leaves with blotchy mottle symptoms characteristic of HLB were sampled in
several farms of SPS and tested for the presence of liberibacters. ‘Ca. L. asiaticus’ was detected in a
small number of samples but most samples gave negative PCR results. Therefore, a new HLB
pathogen was suspected. Evidence for an SPS-HLB bacterium in symptomatic leaves was obtained
by PCR amplification with universal primers for prokaryotic 16S rRNA gene sequences. The amplified
16S rRNA gene was cloned and sequenced. Sequence analysis and phylogeny studies showed
that the 16S rRNA gene possessed the oligonucleotide signatures and the secondary loop structure
characteristic of the a-Proteobacteria, including the liberibacters. The 16S rRNA gene sequence
phylogenetic tree showed that the SPS-HLB bacterium clustered within the a-Proteobacteria,the
liberibacters being its closest relatives. For these reasons, the SPS-HLB bacterium is considered a
member of the genus ‘Ca. Liberibacter’. However, while the 16S rRNA gene sequences of ‘Ca.L.
asiaticus’ and ‘Ca.L.africanus’had98?4 % similarity, the 16S rRNA gene sequence of the SPS-HLB
liberibacter had only 96?0 % similarity with the 16S rRNA gene sequences of ‘Ca.L.asiaticus’or‘Ca.L.
africanus’. This lower similarity was reflected in the phylogenetic tree, where the SPS-HLB liberibacter
did not cluster within the ‘Ca.Lasiaticus’/‘Ca. L. africanus group’, but as a separate branch.
Within the genus ‘Candidatus Liberibacter’ and for a given species, the 16S/23S intergenic region
does not vary greatly. The intergenic regions of three strains of ‘Ca. L. asiaticus’, from India, the
People’s Republic of China and Japan, were found to have identical or almost identical sequences.
In contrast, the intergenic regions of the SPS-HLB liberibacter, ‘Ca.L.asiaticus’and‘Ca.L.africanus’
had quite different sequences, with similarity between 66?0and79?5 %. These results confirm
that the SPS-HLB liberibacter is a novel species for which the name ‘Candidatus Liberibacter
americanus’ is proposed. Like the African and the Asian liberibacters, the ‘American’ liberibacter is
restricted to the sieve tubes of the citrus host. The liberibacter could also be detected by PCR
amplification of the 16S rRNA gene in Diaphorina citri, the psyllid vector of ‘Ca.L.asiaticus’,
suggesting that this psyllid is also a vector of ‘Ca.L.americanus’inSPS.‘Ca. L. americanus’ was
detected in 216 of 218 symptomatic leaf samples from 47 farms in 35 municipalities, while ‘Ca.
L. asiaticus’ was detected in only 4 of the 218 samples, indicating that ‘Ca. L. americanus’ is the
major cause of HLB in SPS.
Abbreviations: HLB, huanglongbing; RIR, ribosomal intergenic region; SPS, Sa˜o Paulo State.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and the ribosomal 16S/23S intergenic region gene sequence of
‘Candidatus Liberibacter americanus’ isolate SPSA3 are AY742824 and AY859542, respectively.
Comparisons between the gene sequences of the ribosomal 16S/23S intergenic region for liberibacter isolates are available as supplementary data in
IJSEM Online.
63677
G
2005 IUMS Printed in Great Britain 1857
International Journal of Systematic and Evolutionary Microbiology (2005), 55, 1857–1862 DOI 10.1099/ijs.0.63677-0
In March 2004, symptoms of citrus huanglongbing (HLB),
formerly called greening disease, were recognized on sweet
orange trees near the city of Araraquara in Sa
˜
o Paulo State
(SPS), Brazil (Coletta-Filho et al., 2004; Teixeira et al.,
2005a, b). This was the first report of HLB on the American
continent. HLB is caused by two sieve-tube-restricted
species of the
a-Proteobacteria,‘Candidatus Liberibacter
africanus’ in Africa and ‘Candidatus Liberibacter asiaticus’
in Asia (Jagoueix et al., 1994). Neither of the two species has
been cultured. The HLB liberibacters were among the first
bacteria to receive ‘Candidatus’ designations according to
the rules established for uncultured organisms (Murray &
Schleifer, 1994). Phylogenetically, the closest relatives of
the liberibacters are members of the alpha-2 subgroup of
the Proteobacteria. However, the liberibacters do not belong
to the alpha-2 subgroup because their 16S rRNA gene
sequence has only one of the seven or more oligonucleotide
signatures characteristic of the alpha-2 subgroup and also
has signatures characteristic of the alpha-1 and alpha-3
subgroups (Jagoueix et al., 1994; Woese et al., 1984).
The HLB liberibacters are transmitted by two psyllid insects,
Trioza erytreae (Del Guercio) in Africa and Diaphorina citri
(Kuwayama) in Asia. The two insects are responsible for
the large geographical distribution of HLB in these areas.
The Mediterranean region and Australia are free of both
HLB and HLB psyllid vectors. However, Diaphorina citri, the
Asian psyllid vector, is established in South, Central and
North America (Florida and Texas) and the Caribbean
basin. D. citri has been present in Brazil for more than
50 years and for less than 10 years in Florida and Texas.
Even though symptoms of HLB were recognized in SPS less
than a year ago, the disease has been present in some
orchards for much longer, probably for 10 years.
Due to their devastating effects on citrus trees and their
efficient transmission by the psyllid vectors, ‘Ca. L. afri-
canus’ and ‘Ca. L. asiaticus’ have been classified by the US
Government as ‘select agents’ with potential for bioterror-
ism. Working with such agents in the USA requires per-
mission from the US Government. The two liberibacters are
quarantine organisms in Europe.
Detection and identification of the liberibacters in citrus
leaves showing blotchy mottle, the most characteristic
symptom of HLB (McClean & Schwarz, 1970), is based on
specific PCR amplification of their 16S rRNA gene sequence
with primer pair OA1/OI2c for ‘Ca. L. africanus’ and OI1/
OI2c for ‘Ca. L. asiaticus’ (Jagoueix et al., 1996; Teixeira
et al., 2005b). When, in a given region, the liberibacter
involved has not yet been identified, both forward primers
are used in the same reaction mixture to favour amplifica-
tion of either one of the two liberibacters. In all previous
cases, amplicons of 1160 bp have been obtained. In order
to identify the liberibacter causing HLB in SPS, 43 samples
of blotchy mottle-affected leaves were collected in seven
citrus farms in the Araraquara region and 16S rRNA gene
amplifications with both forward primers OA1 and OI1
and reverse primer OI2c were carried out. Surprisingly, all
samples gave negative PCR results under conditions where
symptomatic control leaves infected with ‘Ca. L. asiaticus’ or
‘Ca. L. africanus’ gave positive reactions (Teixeira et al.,
2005b). These negative results were unexpected because, in
the many African and Asian countries previously studied
(Jagoueix et al., 1996; Garnier & Bove
´
, 1996; Garnier et al.,
1996; Bove
´
et al., 2000), the PCR tests were always positive
when symptomatic leaves from HLB-affected trees were
used. In SPS, many negative PCR tests were also observed by
other investigators using the same PCR method as described
above, even though a few samples (2 of 10) tested positive
for ‘Ca. L. asiaticus’ (Coletta-Filho et al., 2004). In view of
the many negative PCR tests witnessed by us and others with
leaves showing characteristic HLB symptoms, the presence
of a new bacterial pathogen in the symptomatic HLB-
affected leaves from SPS was suspected.
Evidence for an ‘SPS-HLB’ bacterium responsible for HLB
in SPS was obtained by PCR amplification of its 16S rRNA
gene with universal primers fD1/rP1 for prokaryotic 16S
rRNA gene sequences (Weisburg et al., 1991). The method
was essentially that of Jagoueix et al. (1994) and is based on
the fact that BclI cuts plant mitochondria 16S rRNA genes,
but not bacterial 16S rRNA genes, and EcoRI has no effect on
chloroplast 16S rRNA genes, but cuts bacterial 16S rRNA
genes into two fragments of ~650 and ~850 bp. DNAs
from five symptomatic Valencia sweet orange leaf samples
that tested negative for ‘Ca. L. africanus’ and ‘Ca. L. asia-
ticus’ were digested with BclI to cut the plant mitochondria
16S rRNA gene and prevent it from being amplified in the
ensuing step (Zreik et al., 1998). The remaining chloroplast
16S rRNA gene and the putative SPS-HLB 16S rRNA gene
were amplified by PCR using universal primers fD1/rP1 and
submitted to EcoRI treatment. Under these conditions, the
bacterial 16S rRNA gene is revealed by the presence of the
~650 and ~850 bp fragments. Fig. 1 shows that, indeed,
two fragments of the expected size were obtained, not only
with control leaves infected with ‘Ca. L. asiaticus’ (lane 9) or
‘Ca. Phytoplasma aurantifolia’ (lane 8), but also with the five
leaf samples with HLB symptoms from SPS (lanes 2–6).
With DNA amplified from healthy leaves (lanes 1 and 7),
only the ~1500 bp chloroplast DNA band was seen on the
gel. These results gave evidence for the presence of an SPS-
HLB bacterium in HLB-affected leaves from SPS, even
though such leaves tested negative for ‘Ca. L. asiaticus’ and
‘Ca. L. africanus’. Characterization of the novel bacterium
required cloning and sequencing of its 16S rRNA gene as
follows.
DNA from Natal sweet orange leaves with HLB symptoms
(sample A3 from Luis Antonio municipality) was purified
according to Jagoueix et al. (1996). It was found to test
negative for ‘Ca. L. africanus’ and ‘Ca. L. asiaticus’ with
primers OA1 and OI1/OI2c. The DNA was digested with
BclI and amplified by PCR with universal primers fD1/rP1 as
described above. The ~1500 bp amplified DNA (16S rRNA
gene from SPS-HLB bacterium and chloroplast 16S rRNA
gene) was cloned in Escherichia coli using the TOPO TA
1858 International Journal of Systematic and Evolutionary Microbiology 55
D. C. Teixeira and others
cloning kit (Invitrogen) according to the manufacturer’s
instructions. In total, 46 clones were obtained, of which two
had plasmid inserts resistant to BclI digestion, but sensitive
to EcoRI digestion, characteristic of bacterial 16S rRNA
genes. These two clones were selected and their inserts were
sequenced. The two sequences were identical. The unique
sequence was 1447 bp. Search for homologies in general
databases (http://www.ncbi.nlm.nih.gov/blast) were carried
out using the
BLAST program (Altschul et al ., 1997).
Sequence analysis was also performed using Infobiogen
programs (http://www.infobiogen.fr/index.html). Multiple
sequence alignments were performed using
MULTALIN
(Corpet, 1988) (http://www.toulouse.inra.fr/Igc/multalin/
multalin.html) and
CLUSTAL W (Thompson et al., 1994)
software.
A
BLAST search of the GenBank database applied to the
1447 bp sequence revealed that it was the 16S rRNA gene
sequence of a bacterium, with liberibacters as the closest
relatives. The sequence was aligned with the 16S rRNA gene
sequences of ‘Ca. L. asiaticus’ isolate Poona and ‘Ca.L.
africanus’ isolate Nelspruit, with which it had 96?1 and
95?9 % similarity, respectively.
The following experiments were undertaken to demonstrate
that the bacterial 1447 bp 16S rRNA gene sequence did
represent the PCR amplification product of the SPS-HLB
bacterium involved in HLB in SPS and not that of a
contaminating bacterium. Forward primer GB1 and reverse
primer GB3 were designed from the bacterial 16S rRNA gene
sequence (Teixeira et al., 2005b). Two hundred and fourteen
leaf samples from SPS with characteristic HLB symptoms
and that tested negative for ‘Ca. L. africanus’ and ‘Ca.L.
asiaticus’ were used for PCR amplification with primers
GB1/GB3. All 214 samples gave positive PCR results and the
amplicons obtained were of the expected size (1027 bp).
Healthy leaves, or leaves infected with ‘Ca . L. africanus’ or
‘Ca. L. asiaticus’, gave negative PCR results (Teixeira et al.,
2005b). The 1027 bp amplicon, obtained with primers GB1/
GB3, specific for 16S rRNA gene amplification, is thus
characteristic of HLB leaves from SPS and represents the 16S
rRNA gene sequence amplified from the SPS-HLB bacter-
ium present in these leaves. Two amplicons, one from leaf
sample A5 (Luis Antonio municipality) and one from AA9
(Boa Esperanc¸a do Sul municipality) were cloned and
sequenced. The two sequences were identical and they had
100 % similarity with the corresponding sequence of the
1447 bp 16S rRNA gene. Finally, the ~850 EcoRI fragment
shown in Fig. 1, (lane 5) was also cloned and sequenced.
The fragment was 833 bp and its sequence showed 100 %
similarity to the corresponding region of the 1447 bp 16S
rRNA gene sequence. Therefore, the 833 bp fragment, the
1027 bp 16S rDNA amplicon and the 1447 bp 16S rRNA
amplicon, obtained from leaf samples of different geogra-
phical origins, were all 16S rRNA gene amplification pro-
ducts from the SPS-HLB bacterium and not from a
contaminating bacterium. The 1447 bp 16S rRNA gene
sequence, hereafter referred to as the SPS-HLB 16S rRNA
gene sequence, could thus be safely used to characterize
the novel SPS-HLB bacterium.
Phylogenetic and molecular evolutionary analyses were
conducted using
MEGA version 2.1 (Kumar et al., 2001). The
bacterial 16S rRNA gene sequences used for comparisons
were obtained from the GenBank database and were those
of ‘Ca. L. asiaticus’ isolate Poona, ‘Ca. L. endosymbiont’
from Diaphorina citri,‘Ca. L. asiaticus’ isolate Okinawa, ‘Ca.
L. asiaticus’ isolate Citrus grandis,‘Ca . L. africanus’ isolate
Nelspruit, ‘Ca. L. africanus subsp. capensis’, Brucella abortus
ATCC 23448
T
, Bartonella henselae ATCC 49882
T
, Afipia felis
ATCC 53690
T
,‘Candidatus Phlomobacter fragariae’ and
E. coli (MRE 600
T
).
The phylogenetic analysis (Fig. 2) indicated that the SPS-
HLB bacterium, like the liberibacters, clustered in the
a-subclass of the Proteobacteria and that the liberibacters
were its closest relatives. In addition, the SPS-HLB 16S
rRNA gene sequence had oligonucleotide signatures
(Woese, 1987; Zeff & Geliebter, 1987) very similar to
those of the liberibacters (Jagoueix et al., 1997). Also, the
secondary loop structure characteristic of the
a-Proteo-
bacteria (Woese, 1987) was shared by the liberibacters and
the SPS-HLB bacterium. For all these reasons, the SPS-HLB
bacterium is a member of the genus ‘Candi datus Liberi-
bacter’. The following results indicate that it represents a
novel ‘Candidatus Liberibacter’ species.
While the percentage similarity between the 16S rRNA gene
sequences of two species of liberibacters as different as ‘Ca.
L. africanus’ isolate Nelspruit and ‘Ca. L. asiaticus’ isolate
Poona was 98?4 % (gap-free sequences), the percentage
similarity in the case of the SPS-HLB liberibacter and ‘Ca.L.
Fig. 1. Electrophoresis on 1 % agarose gel of DNA treated
with EcoRI after amplification with primers fD1/rP1 from BclI-
treated DNA extracted from healthy Valencia sweet orange
leaves (lanes 1 and 7), control leaves infected with ‘Ca. L. asia-
ticus’ (lane 9) or ‘Ca. Phytoplasma aurantifolia’ (lane 8) and
Valencia sweet orange leaves from SPS with blotchy mottle
symptoms characteristic of HLB (lanes 2–6). The upper, middle
and lower arrows point to DNA bands of approximately 1500,
850 and 650 bp. Lane M, 1 kb Plus DNA ladder.
http://ijs.sgmjournals.org 1859
‘Candidatus Liberibacter americanus’
asiaticus’ was lower, 95?4 %, indicating that the latter two
liberibacters had to represent different species. This was also
indicated by the phylogenetic tree (Fig. 2), where the SPS-
HLB liberibacter did not cluster within the ‘Ca. L. asiaticus’/
‘Ca. L. africanus’ group, but formed a separate branch with a
bootstrap percentage of 100. In contrast, the four isolates of
‘Ca. L. asiaticus’ clustered together within the liberibacter
group. One of these strains, ‘Ca. L. endosymbiont’, was des-
cribed as an endosymbiont of Diaphorina citri, the psyllid
vector of the Asian liberibacter. Evidence for this liberi-
bacter was obtained by PCR amplification of the 16S rRNA
gene sequence from total D. citri DNA. Therefore, the D. citri
liberibacter should not be considered as an endosymbiont,
but as the pathogen transmitted by the psyllid, namely ‘Ca.
L. asiaticus’. The phylogenetic analysis (Fig. 2) clearly
showed that the so-called ‘Ca. L. endosymbiont’ clustered
with the isolates of ‘Ca. L. asiaticus’ and is an isolate of this
species. However, the SPS-HLB liberibacter did not cluster
in this region and is a novel species. This conclusion was
confirmed by analysis of the 16S/23S ribosomal intergenic
region (RIR) gene sequence.
The RIR of the SPS-HLB liberibacter was obtained by PCR
amplification of DNA extracted from HLB-affected leaf
sample AA9 from Boa Esperanca do Sul municipality which
was also used for the production, by PCR amplification, of
the 1027 bp SPS-HLB 16S rRNA gene sequence (see above).
The primers used were forward primer GB3c, complemen-
tary to primer GB3 and located in the 39 half of the SPS-HLB
16S rRNA gene sequence, and reverse primer 23S1, located
at the very start of the 23S rRNA gene (Jagoueix et al., 1997).
The leaf DNA was amplified for 35 cycles with the follow-
ing program: 92
u
C for 40 s, 64
u
C for 40 s and 72
u
C for 60 s.
The amplified DNA was cloned and sequenced and was
1026 bp long. As expected, the RIR was preceded upstream
by a 16S rRNA gene sequence, 425 bp, of which the 393 bp
sequence at the 59 end was identical to the 39 terminal region
of the SPS-HLB 16S rRNA gene. The remaining 32 bp
sequence was absent from this 16S rRNA gene sequence
because of the position of the reverse universal primer rP1
used for its amplification (see above). Downstream, the RIR
was followed by the first 18 bp of the 23S rRNA gene and
these 18 bp represented the 23S1 reverse primer used for
amplification of the RIR region. The fact that the 16S rRNA
gene sequence in the cloned 1026 bp RIR was identical to the
equivalent region of the SPS-HLB 16S rRNA gene sequence
demonstrated that the cloned 1026 bp RIR was indeed that
of the SPS-HLB liberibacter. The bacterial RIR sequences
used for comparisons were obtained from the GenBank
database and were those of ‘Ca. L. asiaticus’ isolates Poona
(U61359) and Okinawa (AB019793) and ‘Ca. L. africanus’
isolate Nelspruit (U61360). ‘Ca. L. asiaticus’ isolate Fuzhou
was from Jagoueix et al. (1997). The RIR of the SPS-HLB
liberibacter was 582 bp long, compared with 595 and 498 bp
for the RIRs of ‘Ca. L. asiaticus’ isolate Poona and ‘Ca .L.
africanus’ isolate Nelspruit, respectively (Jagoueix et al.,
1997). The RIR contained the sequences for two tRNAs:
tRNA
Ile
and tRNA
Ala
. The RIR of ‘Ca. L. asiaticus’ con-
tained two similar tRNA sequences, while only the tRNA
Ala
sequence was present in the ‘Ca. L. africanus’ RIR (Jagoueix
et al., 1997).
RIR sequence similarity comparisons between liberibacter
species or isolates are available as Supplementary Table S1
in IJSEM Online. In summary, when ‘Ca. L. asiaticus’ iso-
late Poona was compared with another isolate of the
same species, isolate Fuzhou or isolate Okinawa, the RIR
sequences were either identical, with 100 % similarity
(isolate Fuzhou) or very similar, 99?2 % similarity (isolate
Okinawa). Thus, within in a given liberibacter species, the
RIR sequence does not vary much, as already described
(Jagoueix et al., 1997). However, when the RIRs of two
different species, such as ‘Ca. L. asiaticus’ isolate Poona
and ‘Ca. L. africanus’ isolate Nelspruit were compared, the
similarity value for the total intergenic region was as low as
79?5 %. The figures were even lower when the RIR of the
SPS-HLB liberibacter was compared with that of ‘Ca.L.
asiaticus’, 77?8 % for isolate Poona, 77?7 % for isolate
Okinawa, or with ‘Ca. L. africanus’, 66?0 %. These results
confirm those based on 16S rRNA gene sequence compar-
isons and indicate that the SPS-HLB liberibacter is a novel
species, for which the name ‘Candidatus Liberibacter
americanus’ is proposed.
Additional properties of the SPS-HLB liberibacter fit those
of the other liberibacters. Transmission of the SPS-HLB
liberibacter to healthy sweet orange seedlings has been
achieved by graft-inoculations under greenhouse conditions
Fig. 2. Phylogenetic tree constructed from
16S rRNA gene sequences obtained from
GenBank (accession nos in parentheses)
using
MEGA version 2.1.
1860 International Journal of Systematic and Evolutionary Microbiology 55
D. C. Teixeira and others
in Bordeaux, France, as well as Araraquara, Brazil. The
inoculated seedlings showed foliar symptoms of blotchy
mottle within 4 months and the SPS-HLB liberibacter could
be detected by PCR in the symptomatic leaves. The liberi-
bacters have never been grown in culture and the same is
true for the novel species. Liberibacters are restricted to
the sieve tubes of the phloem tissue and possess a charac-
teristic double-membrane cell envelope (Garnier et al .,
1984). Electron microscopy has shown that this is also
present in the SPS-HLB liberibacter (Teixeira et al., 2005a;
Tanaka et al., 2004). The natural vector of ‘Ca. L. asiaticus’
in Asia is the psyllid D. citri, which has been present on
citrus in Brazil for more than 50 years. The SPS-HLB
liberibacter could be detected in D. citri by PCR amplifica-
tion of 16S rRNA gene sequence with the specific primer
pair GB1/GB3, strongly suggesting that the Asian psyllid is
also a vector of the SPS-HLB liberibacter in SPS (Teixeira
et al., 2005b).
In an extensive survey for HLB, 218 samples of leaves
showing symptoms of blotchy mottle were collected from
47 citrus farms in 35 municipalities of SPS. Each sample
was tested for liberibacters with two pairs of primers; the
OA1+OI1/OI2c pair, specific for ‘Ca. L. africanus’ and ‘Ca.
L. asiaticus’ (Jagoueix et al., 1996), and the GB1/GB3 pair,
specific for the SPS-HLB liberibacter (Teixeira et al., 2005b).
The SPS-HLB liberibacter could be detected in 214 samples,
‘Ca. L. asiaticus’ in two and ‘Ca. L. africanus’ in none. Two
samples were infected with both the SPS-HLB liberibacter
and ‘Ca. L. asiaticus’. The proportion of ‘Ca. L. asiaticus’ to
the SPS-HLB liberibacter is thus 4 : 216 or 1?9 %. These
figures indicate that the major cause of HLB in SPS is the
SPS-HLB liberibacter and not ‘Ca . L. asiaticus’, as stated
before the characterization of the SPS-HLB liberibacter
(Coletta-Filho et al., 2004).
It has been suggested previously that ‘Ca . L. africanus’
probably originated in Africa and ‘Ca. L. asiaticus’ probably
originated in Asia (Garnier & Bove
´
, 1996). Similarly, ‘Ca.L.
americanus’ is likely to be of American origin and will be
spread by D. citri to further American countries. Therefore,
in America, the new liberibacter might be a more serious
‘select agent’ than ‘Ca. L. africanus’ or ‘Ca. L. asiaticus’.
Finally, we propose to eliminate the taxon ‘Ca. L. endosym-
biont’ and merge it into ‘Ca. L. asiaticus’.
Description of ‘Candidatus Liberibacter
americanus’
In view of the genomic properties of the liberibacter
associated with citrus huanglongbing in Sa
˜
o Paulo State,
Brazil, it is proposed that this SPS-HLB bacterium be
assigned species status with the following designation
according to Murray & Schleifer (1994) and Murray &
Stackebrandt (1995):
‘Candidatus Liberibacter americanus’ (a.me.ri.ca9nus. N.L.
masc. adj. americanus American, referring to the fact that
the first detection and occurrence of the organism was on
the American continent).
Reference isolate is SPSA3 (from Natal sweet orange leaf
sample A3, Sa
˜
o Jose
´
farm, Luis Antonio municipality, SPS,
Brazil).
[(
a-Proteobacteria) NC; G2; F; NAS (GenBank accession
no. AY742824 for 16S rRNA gene and AY859542 for ribo-
somal 16S/23S intergenic region), oligonucleotide sequence
complementary to unique region of 16S rRNA 59-CTA-
TATTTGCCATCATTAAGTTGG-39,S(Citrus, phloem;
Diaphorina citri (Psyllidae), haemolymph, salivary glands);
M]. Teixeira et al., this study.
Acknowledgements
With deep emotion, we dedicate this publication to the memory of
Dr Monique Garnier (1949–2003). We would like to thank Dr Pascal
Sirand-Pugnet, Universite
´
Victor Segalen Bordeaux 2, for fruitful
discussions concerning phylogeny.
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