208 Plant Disease / Vol. 93 No. 3
Research
A New ‘Candidatus Liberibacter’ Species Associated with Diseases
of Solanaceous Crops
Lia W. Liefting, Plant Health and Environment Laboratory, MAF Biosecurity New Zealand, P.O. Box 2095, Auck-
land 1140, New Zealand; Paul W. Sutherland, The Horticulture and Food Research Institute of New Zealand Ltd.,
Private Bag 92 169, Auckland, New Zealand; and Lisa I. Ward, Kerry L. Paice, Bevan S. Weir, and Gerard R. G.
Clover, Plant Health and Environment Laboratory, MAF Biosecurity New Zealand, P.O. Box 2095, Auckland 1140,
New Zealand
In January 2008, a disease of glasshouse
tomato (Solanum lycopersicum) crops was
observed in Auckland, New Zealand.
Symptoms included spiky, chlorotic apical
growth, general mottling of the leaves,
curling of the midveins, overall stunting of
the plants, and in some cultivars fruit de-
formation. Cultivars varied in severity of
symptoms and levels of disease incidence.
This disease problem has been reported
from three commercial glasshouse tomato
growers in Auckland, and losses of up to
NZ$1 million have been reported.
Extensive testing was done to determine
the etiology of this disease. No pathogenic
fungi or culturable bacteria were isolated.
Generic tests for viruses, including herba-
ceous indexing, transmission electron mi-
croscopy (leaf dip method), and dsRNA
purification, were all negative. Polymerase
chain reaction (PCR) tests for phytoplas-
mas, geminiviruses, tombusviruses, ilarvi-
ruses, potexviruses, Beet pseudo yellows
virus, Cucumber mosaic virus, Tomato
spotted wilt virus, Tobacco mosaic virus,
Tomato chlorosis virus, Tomato infectious
chlorosis virus, and pospiviridae were also
negative.
The tomato/potato psyllid, Bactericera
cockerelli (Homoptera: Psyllidae), was
reported to occur in all tomato glasshouses
where these disease symptoms were pre-
sent. B. cockerelli was first discovered in
an Auckland glasshouse tomato crop in
May 2006, and is now established
throughout the North Island and the top
part of the South Island of New Zealand.
Due to the similarity of symptoms with
those reported as “psyllid yellows” and the
absence of a positive result for other possi-
ble causal organisms, the symptoms were
thought to be those of psyllid yellows, first
reported by Richards (28), and thought to
be caused by a toxin associated with feed-
ing by the nymphal instars.
In April 2008, similar symptoms ap-
peared in a glasshouse pepper (Capsicum
annuum) crop on the same property as one
of infected tomato crops. Peppers are not
known to be susceptible to psyllid yellows;
therefore, further investigation was done.
Transmission electron microscopy of thin
sections of leaf tissue revealed the pres-
ence of bacterium-like organisms (BLOs)
in the phloem of symptomatic tomato and
pepper. There are no previous reports of
BLOs in tomato or pepper, and only a
small number of BLOs causing plant dis-
ease have been characterized at the mo-
lecular level. Of these, the three ‘Candida-
tus Liberibacter’ species associated with
huanglongbing of citrus are the best char-
acterized (13,35). Other BLOs with 16S
rRNA gene sequences available include
‘Candidatus Phlomobacter fragariae’ of
strawberry (38), yellow vine disease of
cucurbits (2), and syndrome “basses
richesses” of sugar beet (31). A difficulty
in the characterization of BLOs is the in-
ability to culture these organisms in vitro.
In this study, we report the initial dis-
covery of a BLO in tomato and pepper by
electron microscopy and the strategy used
to determine its identity. Phylogenetic
analysis of the 16S rRNA gene, the
16S/23S rRNA spacer region, and the
rplKAJL-rpoBC operon revealed that the
BLO is a new species of the ‘Candidatus
Liberibacter’ genus. A specific PCR primer
pair for its detection was developed.
MATERIALS AND METHODS
Plant and insect material. Sympto-
matic tomato and pepper plants were col-
lected from commercial glasshouses in the
Auckland region between January and
May 2008. Healthy tomato and pepper
were grown from seed in an insect-proof
glasshouse to use as negative controls. B.
cockerelli psyllids were collected from
glasshouses with symptomatic plants. Total
DNA from citrus infected with ‘Candida-
tus Liberibacter asiaticus’ and ‘Candidatus
Liberibacter americanus’ was kindly pro-
vided by Diva Teixeira (Fundecitrus, Ara-
raquara, Brazil) and Helvécio Coletta-Filho
(Centro APTA Citros Sylvio Moreira – In-
stituto Agronômico de Campinas, Brazil);
and total DNA from citrus infected with
‘Ca. L. africanus’ was provided by
Gerhard Pietersen (Citrus Research Inter-
ABSTRACT
Liefting, L. W., Sutherland, P. W., Ward, L. I., Paice, K. L., Weir, B. S., and Clover, G. R. G.
2009. A new ‘Candidatus Liberibacter’ species associated with diseases of solanaceous crops.
Plant Dis. 93:208-214.
A new disease of glasshouse-grown tomato and pepper in New Zealand has resulted in plant
decline and yield loss. Affected plants are characterized by spiky, chlorotic apical growth, curl-
ing or cupping of the leaves, and overall stunting. Transmission electron microscopy revealed the
presence of phloem-limited bacterium-like organisms in symptomatic plants. The strategy used
to identify the bacterium involved using specific prokaryote polymerase chain reaction (PCR)
primers in combination with universal 16S rRNA primers. Sequence analysis of the 16S rRNA
gene, the 16S/23S rRNA spacer region, and the rplKAJL-rpoBC operon revealed that the bacte-
rium shared high identity with ‘Candidatus Liberibacter’ species. Phylogenetic analysis showed
that the bacterium is distinct from the three citrus liberibacter species previously described and
has been named ‘Candidatus Liberibacter solanacearum’. This is the first report of a liberibacter
naturally infecting a host outside the Rutaceae family. A specific PCR primer pair was devel-
oped for its detection.

Corresponding author: Lia Liefting
E-mail: lia.liefting@maf.govt.nz
The nucleotide sequence data reported in this arti-
cle appear in the GenBank nucleotide sequence
database with the accession numbers EU834130
and EU834131.
Accepted for publication 21 November 2008.
doi:10.1094 / PDIS-93-3-0208
This article is in the public domain and not copy-
rightable. It may be freely reprinted with custom-
ary crediting of the source. The American Phyto-
pathological Society, 2009.

Plant Disease / March 2009 209
national, University of Pretoria, South
Africa).
Transmission electron microscopy.
Secondary leaf veins of symptomatic and
healthy plants were cut into 1 × 5 mm
pieces and fixed in 2% freshly prepared
paraformaldehyde and 2.5% glutaralde-
hyde in 0.1 M phosphate buffer at pH 7.2
under vacuum for 1 h. The samples were
then washed three times in buffer, post-
fixed in 1% osmium tetroxide for 1 h, de-
hydrated in an ethanol series, infiltrated,
and embedded in Spurr’s resin. Sections
120 nm thick were cut and placed on
Formvar coated grids and stained in 1%
vol/vol aqueous uranyl acetate and lead
citrate (29). Sections were viewed on a
JEOL JEM-1200EX II transmission elec-
tron microscope (Tokyo) operating at 80
kV.
DNA isolation. Plant DNA was ex-
tracted from leaf midribs and petioles of
symptomatic and healthy plants using the
DNeasy Plant Mini Kit (Qiagen, Valencia,
CA) according to the manufacturer’s in-
structions. Psyllid nymphs in batches of
approximately 20 were placed in 1.5-ml
microcentrifuge tubes with 180 µl of 0.01
M PBS (0.138 M NaCl, 0.0027 M KCl, pH
7.4; Sigma-Aldrich, St Louis, MO) and
homogenized using a disposable microtube
pestle. DNA was extracted from the ho-
mogenized psyllids using the DNeasy
Tissue Kit (Qiagen) according to the
manufacturer’s instructions.
PCR amplification. PCR primers used
in attempts to amplify a fragment of the
BLO were as follows: Fra 4/Fra 5 (38),
LSg2f/LSg2r (4), and 16S-23SF/16S-23SR
(24). The primer pairs Fra 4/Fra 5 and
LSg2f/LSg2r were designed to detect ‘Ca.
Phlomobacter fragariae’ and ‘Ca. L.
americanus’, respectively (4,38). The
primer pair 16S-23SF/16S-23SR was de-
signed by Martinati et al. (24) to amplify
the 16S/23S rRNA spacer region of Xylella
fastidiosa. These primers were selected
because BLASTn analysis determined that
they also match the 16S rRNA or 23S
rRNA gene of a wide range of bacteria.
The universal prokaryote 16S rRNA
primer pair fD2/rP1 (36) was used to ob-
tain PCR products for cloning. These
primers amplify the 16S rRNA gene from
bacteria and plants. The fD2/rP1 primers
were also used in combination with each
of the more specific primers described
above. Amplification was performed in 20-
µl reactions containing 1× PCR buffer
(Invitrogen, Carlsbad, CA), 1.5 mM
MgCl2, 0.2 mM dNTPs, 0.25 µM of each
primer, and 1 U of Platinum Taq poly-
merase (Invitrogen). The PCR conditions
were: an initial cycle at 94°C for 5 min,
then 40 cycles of 94°C for 30 s, 48°C for
30 s, and 72°C for 1 min, plus an addi-
tional cycle of 10 min at 72°C. All ampli-
fications were performed in a GeneAmp
PCR System 9700 (Applied Biosystems,
Foster City, CA). The amplified DNA was
analyzed by agarose gel electrophoresis,
and bands were visualized with SYBR
Safe (Invitrogen).
DNA cloning and sequencing. PCR
products were either gel purified using the
Quantum Prep Freeze ‘N Squeeze DNA
Gel Extraction Spin Columns (Bio-Rad,
Hercules, CA) or using the QIAquick PCR
Purification Kit (Qiagen). The purified
PCR products were either sequenced di-
rectly or cloned into the pCR 4-TOPO
vector (Invitrogen) followed by transfor-
mation into One Shot TOP10 chemically
competent Escherichia coli according to
the manufacturer’s instructions (Invitro-
gen). Plasmid DNA was purified using the
QIAprep Spin Miniprep Kit (Qiagen). The
DNA fragments were sequenced in both
directions using the M13 forward and re-
verse primers for cloned inserts or the
relevant PCR primer for direct sequencing
of PCR products. DNA was sequenced on
an ABI Avant 3100 Genetic analyzer using
BigDye 3.2 chemistry.
Sequence analysis. The sequences were
assembled using the SeqMan software of
the LaserGene package (DNASTAR,
Madison, WI). Searches of the GenBank
database for homologous sequences were
performed using the BLASTn network
service available at the National Center for
Biotechnology Information (Bethesda,
MD, USA). The software tRNAscan-SE
(23) was used to find transfer RNA genes.
Phylogenetic analysis. Multiple se-
quence alignments were performed using
Clustal X 2.0 (17). Modeltest 3.7 (27) was
used to determine the optimal analysis
method. The general time reversible model
with the proportion of invariant sites and
the gamma shape parameter (GTR+I+Γ)
was selected. Neighbor-joining (NJ) trees
were constructed using HKY85 parame-
ters, maximum likelihood (ML) trees were
constructed using the Modeltest parame-
ters, with 10 replicates, and both the NJ
and ML trees were analyzed in PAUP*
4.0b10 (32). Bayesian inference trees were
constructed with MrBayes 3.12 (12,30)
with a flat Dirichlet distribution, and run
for seven million generations with the
burnin set past the point of log probability
leveling off.
Primer design and PCR protocol for
specific detection of the BLO in tomato
and pepper. The forward primer OA2 (5′-
GCGCTTATTTTTAATAGGAGCGGCA-
3′) was designed from the 16S rRNA se-
quence of the tomato/pepper BLO. This
primer is located in the same region as OI1
and OA1, and when used in combination
with OI2c, a 1,160-bp product is expected
(14). The forward primers OI1 and OA1
were designed by Jagoueix et al. (14) to be
specific to ‘Ca. L. asiaticus’ and ‘Ca. L.
africanus’, respectively, while the reverse
primer OI2c matches both liberibacter
species. The sequences of OI1 and OA1
are identical except for a trinucleotide
located 12 bp from the 3′ end. PCR condi-
tions were the same as above except that
the annealing step was carried out at 65°C.
RESULTS
Symptomatology. Symptoms associ-
ated with the BLO in tomato vary in se-
verity and prevalence between cultivars.
New leaves develop interveinal chlorosis
(Fig. 1A), and the growing tips have a
spiky appearance and may show purpling
of the midveins depending on the cultivar
(Fig. 1B). Severely affected leaflets can
sometimes have a curled midvein forming
a cupped structure, with extended petioles
(Fig. 1C). The plants exhibit overall
stunting. Fruit may be misshapen with a
strawberry-like appearance and uneven
development of fruit locules (data not
shown). In some cases, there is no fruit
set at all.
Pepper plants infected with the BLO
have chlorotic or pale green leaves (Fig.
1D), shortened internodes, and overall
stunting. Other symptoms include leaf
cupping (Fig. 1E) and sharp tapering of the
leaf apex, resulting in a spiky appearance.
Apical meristem necrosis and flower abor-
tion may also occur.
Transmission electron microscopy.
Transmission electron microscopy revealed
the presence of numerous BLOs in the
phloem sieve tubes of symptomatic tomato
plants (Fig. 2) but not in healthy tomato
plants grown from seed (data not shown).
Fewer BLOs were observed in sympto-
matic pepper plants, which may reflect
uneven distribution in the plant rather than
a lower titer (data not shown). A total of 50
cross-sections and 20 longitudinal-sections
were measured, and the BLOs were deter-
mined to be approximately 0.2 µm in width
and 4 µm in length.
Identification of the BLO. Two strate-
gies were used to obtain sequence from the
16S rRNA gene of the BLO from sympto-
matic tomato plants. The first was that
used to isolate the 16S rRNA gene of ‘Ca.
L. africanus’ (13), ‘Ca. L. asiaticus’ (13),
‘Ca. L. americanus’ (35), and ‘Ca. Phlo-
mobacter fragariae’ (38). This method
involved amplifying the 16S rRNA gene
from symptomatic tomato plants with the
fD2 and rP1 primers (36), which are uni-
versal primers for amplification of the 16S
rRNA gene of prokaryotes as well as plant
organelles. The resulting PCR product was
cloned, and 56 transformants were ana-
lyzed by digestion with BclI and EcoRI.
The clones exhibited two different types of
restriction profiles (data not shown), and
database searches of the sequences of these
clones indicated that they were of plant
mitochondrial and chloroplast origin.
The second strategy was to use primers
specific for prokaryote 16S rRNA se-
quences in combination with the universal
fD2 and rP1 primers (36). Sémétey et al.
(31) used this strategy to amplify the 16S
rRNA gene from syndrome “basses
richesses” BLO of sugar beet. The primer