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Citrus huanglongbing in Sao Paulo State, Brazil: PCR detection of the
‘Candidatus’ Liberibacter species associated with the disease
Diva do Carmo Teixeiraa, Jean Luc Danetb, Sandrine Eveillardb, Elaine Cristina Martinsa,Waldir Cintra de Jesus Juniora, Pedro Takao Yamamotoa, Silvio Aparecido Lopesa,
Renato Beozzo Bassanezia, Antonio Juliano Ayresa, Colette Saillardb, Joseph Marie Boveb,*
aFundecitrus, Av. Dr. Adhemar Pereira de Barros, 201, CEP 14807-040 Araraquara, SP, BrazilbIBVM, Centre INRA, Institut National de la Recherche Agronomique and Universite de Bordeaux 2, Laboratoire de Biologie cellulaire et moleculaire,
71, Avenue Edouard Bordeaux, B.P. 81, 33883 Villenave d’Ornon Cedex, France
Received 16 September 2004; accepted for publication 12 November 2004
Abstract
Symptoms of huanglongbing (HLB), one of the most serious diseases of citrus in Asia and Africa, have been noticed in March 2004 in the
Araraquara region of Sao Paulo State, Brazil. HLB has not been reported previously from America. The causal HLB bacteria, Candidatus
Liberibacter africanus in Africa and Candidatus Liberibacter asiaticus in Asia, can be detected in symptomatic citrus leaves by PCR
amplification of their 16S rDNA with previously described primers. When this technique was applied to 43 symptomatic leaf samples from
the Araraquara region, all PCR reactions were negative. This suggested that a new pathogen, not detected by the above primers, could be
involved in HLB in the State of Sao Paulo. Indeed, by using universal primers for amplification of bacterial 16S rDNA, a new liberibacter
species, Candidatus Liberibacter americanus, has recently been identified. Specific primers for PCR amplification of the 16S rDNA of Ca. L.
americanus have been selected. Using these primers, the new liberibacter could be detected in 214 symptomatic leaf samples tested. The
leaves of two additional samples were infected with Candidatus Liberibacter asiaticus, and two further samples contained both Ca. L.
americanus and Ca. L. asiaticus. The samples came from 47 farms in 35 municipalities. The psyllid vector of Ca. L. asiaticus, Diaphorina
citri, is established in South, Central, and North America (Florida and Texas). Ca. L. americanus could be detected by PCR in several batches
of D. citri psyllids collected on symptomatic sweet orange trees infected with Ca. L. americanus, strongly suggesting that D. citri is the vector
of Ca. L. americanus. The results reported here confirm the presence of HLB in the State of Sao Paulo. Ca. L. americanus is the most widely
distributed pathogen.
q 2004 Elsevier Ltd. All rights reserved.
Keywords: Citrus; Huanglongbing; Greening; Liberibacter; 16S rDNA; Brazil
1. Introduction
Huanglongbing (HLB), previously called greening, is
one of the most serious diseases of citrus. The causal agent
is a non-cultured, sieve tube-restricted member of the a-
subdivision of the Proteobacteria: Candidatus Liberibacter
africanus in Africa and Candidatus Liberibacter asiaticus
in Asia [1]. HLB has not been reported previously from
America. However, the Asian psyllid vector of Ca. L.
0890-8508/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.mcp.2004.11.002
* Corresponding author. Tel.: C33 670 774 883; fax: C33 557 122 369.
E-mail address: [email protected] (J.M. Bove).
asiaticus, Diaphorina citri, is established in South, Central,
and North America (Florida and Texas). The insect reached
Brazil 60 years ago, but entered Texas only in 2001. In
March 2004, leaf and fruit symptoms resembling those of
HLB were observed in several sweet orange (Citrus sinensis
(L.) Osbeck) orchards in the Araraquara area of Sao Paulo
State. Leaf mottling or ‘blotchy mottle’ [2], a characteristic
feature of HLB, was the major foliar symptom. Fruits were
small and lopsided, and contained many aborted seeds [3].
A PCR method has been described previously, and
permits the detection of the two liberibacters in citrus leaves
by amplification of an 1160 bp fragment of their 16S rDNA
[4]. With both liberibacter species, the size of the amplicon is
Molecular and Cellular Probes 19 (2005) 173–179
www.elsevier.com/locate/ymcpr
D. do Carmo Teixeira et al. / Molecular and Cellular Probes 19 (2005) 173–179174
the same (1160 bp). However, the Ca. L asiaticus amplicon
contains one Xba1 restriction site, and yields two fragments
(640 and 520 bp) upon restriction, while Ca. L. africanus has
two such sites, and yields three fragments (520, 506 and
130 bp). It is thus easy to identify the liberibacter species
involved [4]. The PCR method has been assayed in many
Asian and African countries for the detection of the two HLB
liberibacters [see for instance 5–7]. Whenever leaves with
the classic blotchy mottle symptoms were used, positive PCR
reactions were always obtained, and yielded the character-
istic 1160 bp amplicon. In April 2004, it was decided to use
the PCR technique to confirm the presence of HLB in Sao
Paulo State, and identify the liberibacter involved: Ca. L.
asiaticus or Ca. L. africanus. Unexpectedly, none of the
symptomatic leaf samples from 43 affected trees, of which
many had severe fruit symptoms, yielded a positive PCR test,
under conditions where symptomatic control leaves infected
with Ca. L. asiaticus or Ca. L. africanus from the HLB
collection in Bordeaux, gave positive PCR reactions.
However, at the same time, and using the same PCR
technique, Ca. L. asiaticus was detected in two of 22 leaf
samples (M.A. Machado, Cordeiropolis, S.P., Brazil,
personal communication). In view of the many negative
PCR reactions witnessed, the presence of a new bacterial
pathogen in the symptomatic, blotchy mottle leaves from Sao
Paulo State was suspected and investigated. This work has
resulted in the discovery of a third liberibacter species, for
which we have proposed the name Candidatus Liberibacter
americanus [3]. The complete characterization of the new
liberibacter is in progress. In the work reported here, primers
for PCR amplification of the 16S rDNA of Ca. L. americanus
have been used to detect the new liberibacter in citrus leaves,
and evaluate its distribution, in comparison with that of
Ca. L. asiaticus and Ca. L. africanus, within the huanglongb-
ing-affected region of Sao Paulo State. Detection of the new
liberibacter in D. citri psyllids was also attempted.
2. Materials and methods
2.1. Plant material
Two-year-old ‘Hamlin’ sweet orange seedlings infected
with Ca. L. africanus or Ca. L. asiaticus were from the HLB
collection in Bordeaux, France, and served as positive
control material. Healthy and infected seedlings were
maintained in a greenhouse as previously described [4].
Symptomless citrus leaves and leaves with characteristic
blotchy mottle symptoms were collected in April, June and
August 2004, on trees from 47 citrus farms within the 35
municipalities of Sao Paulo State where symptoms of HLB
had been detected. Each leaf sample came from a single
tree. When symptomatic leaves were sampled on trees
bearing fruit, fruit symptoms of HLB always accompanied
the leaf symptoms. Leaves with symptoms other than HLB
blotchy mottle were also sampled. Each leaf sample
contained 10–20 leaves. Leaves were kept in plastic bags
at 4 8C before they were used for DNA extraction within
48 h [8]. In total, 271 leaf samples were collected. Most
symptomatic leaf samples came from sweet orange trees,
the major citrus cultivar grown in Sao Paulo State, but some
were also collected on affected Ponkan mandarin, Cravo
mandarin, and Murcott tangor trees.
2.2. Capture of D. citri psyllids
D. citri psyllids were captured with a motor-driven
aspirator on three fully infected, symptomatic ‘Pera’ sweet
orange trees, after PCR assays had shown the trees to be
infected with Ca. L. americanus only.
2.3. PCR detection of Ca. L. americanus
PCR primers specific for amplification of the 16S rDNA
of the new liberibacter were selected from the 16S rDNA
sequence (GenBank accession number: AY742824). Fig. 1
shows the sequence of forward primer (GB1) and reverse
primer (GB3) in comparison with corresponding sequences
of Ca. L. asiaticus and Ca. L. africanus 16S rDNAs. The use
of these primers leads to a 1027 bp amplicon. The PCR
reaction was performed in 40 ml of reaction mixture
containing 1 mM of each of the primers, 200 mM of each
of the four dNTP, 2 mM MgCl2, 20 mM Tris–HCl pH 8.4,
50 mM KCl, 1.5 U of Taq polymerase (Promega), and 1 ml
of DNA preparation. A mastercycle gradient thermocycler
(Eppendorf) with the following program was used for DNA
amplification: 35 cycles each at 94 8C for 45 s, 64 8C for
45 s, and 72 8C for 60 s. Following amplification, 10 ml
aliquots of each reaction mixture were analysed by
electrophoresis on 1.2% agarose gels.
2.4. PCR detection of Ca. L. africanus and Ca. L. asiaticus
PCR was performed according to [4] with 35 cycles each
at 92 8C for 40 s, and 72 8C for 90 s (annealing and primer
extension at same temperature). The reaction mixture was
the same than that for Ca. L. americanus. As shown on
Fig. 1, the sequence of reverse primer OI2c is the same for
both liberibacters. The sequences of forward primer OA1 for
Ca. L. africanus and OI1 for Ca. L. asiaticus are identical
except that GCA in OI1 is replaced by TTT in OA1. Both
forward primers were used in the reaction mixture to favor
amplification of either one of the two liberibacters [4].
Following amplification, the reaction mixture was analysed
on 1.2% agarose gels. The amplified DNA has a size of
1160 bp for both liberibacters. However, the 1160 bp
amplicon from Ca. L. asiaticus has one Xba1 restriction
site and yields two fragments upon digestion (640 and
520 bp), while Ca. L. africanus has two such sites, and gives
three fragments (520, 506, and 130 bp) [4]. The Xba1 test
was used to identify the liberibacter involved.
Fig. 1. Sequences of PCR primers for amplification of liberibacter 16S rDNA. GB1 and GB3: respectively, forward and reverse primers for Ca. L. americanus
16S rDNA amplification. OI1 and OI2c: respectively, forward and reverse primers for Ca. L. asiaticus 16S rDNA amplification. OA1 and OI2c: respectively,
forward and reverse primers for Ca. L. africanus 16S rDNA amplification. The ! indicates a mismatche between primers for Ca. L. americanus, and primers for
Ca. L. asiaticus and Ca. L. africanus.
D. do Carmo Teixeira et al. / Molecular and Cellular Probes 19 (2005) 173–179 175
3. Results
3.1. Attempts to detect Ca. L. africanus and Ca. L. asiaticus
by PCR in symptomatic leaf samples collected
in April and June, 2004
In April and June, 2004, soon after HLB had been
reported in Sao Paulo State, at a time when Ca. L.
americanus had not yet been discovered, 43 symptomatic
sweet orange leaf samples were collected in seven citrus
farms in the Araraquara region (Fig. 2). All samples gave
negative PCR reactions with the primers specific of Ca. L.
africanus and Ca. L.asiaticus 16S rDNA (Fig. 3) under
conditions where symptomatic control leaves infected with
Fig. 2. Map of Sao Paulo State showing the citrus area in green, and, in red, the 35 m
found. Position of Sao Paulo city and Araraquara city is indicated by a black dot
Ca. L. asiaticus (Fig. 3, AS) or Ca. L. africanus (Fig. 3, AF)
gave positive reactions. Fig. 3 represents the results from
only 31 of the 43 samples. The results from the other
samples were similarly negative (data not shown).
3.2. PCR detection of Ca. L. americanus and Ca. L.
asiaticus in symptomatic leaves
By July 2004, specific primers for PCR amplification of
Ca. L. americanus 16S rDNA (Fig. 1, GB1/GB3) became
available [3]. These primers, as well as the primers specific
for the PCR detection of Ca. L. africanus and Ca. L.
asiaticus (Fig. 1, OI1C OA1/OI2c) were used for the
detection of the three liberibacters in each leaf sample
unicipaloties, where citrus trees infected with HLB liberibacters have been
and a white dot, respectively.
Fig. 3. Agarose gel electrophoresis of symptomatic leaf DNA amplified with 16S rDNA primers (OI1C OA1)/OI2c, specific for Ca. L. asiaticus and Ca. L.
africanus. 1–31: DNA from symptomatic leaf samples 1–31, collected in April and June, 2004. AS and AF: DNA from symptomatic Hamlin sweet orange
leaves, respectively, infected with Ca. L. asiaticus and Ca. L. africanus. H: DNA from healthy sweet orange leaves. Leaves for AS, AF, and H were from the
Bordeaux greenhouse. O: amplification in the absence of DNA. M; DNA size markers.
D. do Carmo Teixeira et al. / Molecular and Cellular Probes 19 (2005) 173–179176
collected. One aliquot of the DNA from a leaf sample was
used for the detection of Ca. L. americanus, and a second
aliquot served for the detection of Ca. L. africanus and/or
Ca. L. asiaticus in the same leaf sample.
3.2.1. Leaf samples collected in April and June, 2004
The DNAs of these samples had been saved, and were
used again in August 2004, when the Ca. L. americanus
primers GB1/GB3 had become available. As before, all 43
samples tested negative for Ca. L. africanus and Ca. L.
asiaticus, but they were all positive when assayed for Ca. L.
americanus. Fig. 4 shows the results obtained, respectively
with samples 1–5, and 11–18. The other April–June samples
(samples 6–10, and 19–43) gave identical results (data not
shown). As illustrated on Fig. 4, the PCR reactions were
negative with healthy leaves (H), and in the absence of DNA
(O). Primers GB1/GB3 (Am on Fig. 4) were specific for
Fig. 4. Agarose gel electrophoresis of symptomatic leaf DNA amplified with 16S
africanus, and 16S rDNA primers GB1/GB3, specific for Ca. L. americanus. Sympt
As: primers (OI1C OA1)/OI2c. Am: primers GB1/GB3. (a) DNA aliquots amplifie
Fig. 3. AM: DNA from symptomatic sweet orange leaves infected with Ca. L. am
Ca. L. americanus, as no amplification was obtained with
DNA from control leaves infected with Ca. L. africanus (AF
on Fig. 4) or Ca. L. asiaticus (AS on Fig. 4). Inversely,
primers (OA1C OI1)/OI2c (As on Fig. 4), specific for Ca.
L. africanus and Ca. L. asiaticus, gave no amplification with
Ca. L. americanus-infected citrus leaves (Fig. 4, lanes ‘b’).
Finally, as seen on the gels of Fig. 4–6 and 8, the 16S rDNA
amplicon from Ca. L. americanus, with a size of 1027 bp,
was easily distinguishable from the 1160 bp amplicon
characteristic of the other two liberibacters.
3.2.2. Leaf samples collected in August, 2004
Additional symptomatic citrus leaf samples were col-
lected in August 2004. Among these samples, 171 gave
the same results than the previous 43 samples: the PCR
reactions with the primers for Ca. L. americanus were
positive, but negative with the primers for Ca. L. africanus
rDNA primers (OI1C OA1)/OI2c, specific for Ca. L. asiaticus and Ca. L.
omatic citrus leaf samples 1–5 and 11–18 were collected in April/June 2004.
d with Am. (b) DNA aliquots amplified with As. AS, AF, H, M, and O: as in
ericanus.
Fig. 5. Agarose gel electrophoresis of symptomatic leaf DNA amplified with 16S rDNA primers (OI1C OA1)/OI2c, specific for Ca. L. asiaticus and Ca. L.
africanus, and 16S rDNA primers GB1/GB3, specific for for Ca. L. americanus. Symptomatic citrus leaf samples 50, 64–67, and 69–71 were collected in July,
2004. DNA aliquots a and b, AM, Am and As: as in Fig. 4. AS, M, and O: as in Fig. 3.
D. do Carmo Teixeira et al. / Molecular and Cellular Probes 19 (2005) 173–179 177
and Ca. L. asiaticus. Fig. 5 shows the results for some of these
samples. Eventually however, four samples gave a positive
PCR reaction with the primers for Ca. L. africanus and Ca. L.
asiaticus, and the Xba1 test identified the liberibacter as Ca.
L. asiaticus. Two of these samples, sample 51, from a
Murcott tangor orchard, and sample 121, from a ‘Hamlin’
sweet orange orchard, were infected with Ca. L. asiaticus
only, but sample 34, from a backyard ‘Lima’ sweet orange
tree, and sample 322, from a Pera sweet orange tree, were
positive for both Ca. L. asiaticus and Ca. L. americanus. The
results from samples 34 and 51 are illustrated on Fig. 6. The
results from samples 34, 51 and 121, were confirmed a first
time with additional leaves of these samples, which had been
left over and kept at 4 8C, and a second time with a new batch
of leaves collected on the same trees.
In total, Ca. L. americanus was detected in 216 citrus leaf
samples, of which five were from Ponkan mandarin trees, one
from a Murcott tangor tree, two from Cravo mandarin trees,
and 208 from sweet orange trees. All sweet orange varieties,
including ‘Chamout’, ‘Hamlin’, ‘Lima’, ‘Natal’, ‘Pera’,
‘Valencia’ and ‘Westin’, were found to be infected. The 216
samples were from 47 farms in 35 municipalities (Fig. 2).
Only four samples were found to be infected with
Ca. L. asiaticus, of which two were also infected with
Ca. L. americanus. The four farms in which Ca. L. asiaticus
Fig. 6. Agarose gel electrophoresis of symptomatic leaf DNA amplified with 16S
africanus, and 16S rDNA primers GB1/GB3, specific for for Ca. L. americanus. S
from samples 34 and 51, amplified with Am. 34b and 51b: DNA aliquots from sam
and O: as in Fig. 3.
was detected, had also trees infected with Ca. L. americanus.
No evidence was obtained for the presence of Ca. L.
africanus.
3.3. Search for Ca. L. americanus, Ca. L. africanus and Ca.
L. asiaticus in symptomless leaves by PCR
Samples of symptomless leaves were collected on three
types of trees: (i) on symptomatic trees, opposite the
affected sector, (ii) on symptomless trees adjacent to
symptomatic trees, and (iii) on symptomless trees from a
region not affected by HLB. As expected from previous
experience, all symptomless leaves gave negative PCR
reactions (data not shown).
3.4. Search for Ca. L. americanus, Ca. L. africanus and
Ca. L. asiaticus in D. citri psyllids by PCR
D. citri psyllids were collected on three Pera sweet
orange trees with severe symptoms of HLB and shown by
PCR to be infected with Ca. L. americanus only.
In the experiment of Fig. 7, 22 batches of psyllids (10
insects per batch) were tested by PCR for the presence of
liberibacters. The 22 batches gave negative PCR reactions
with the primers specific for Ca. L. africanus and
rDNA primers (OI1C OA1)/OI2c, specific for Ca. L. asiaticus and Ca. L.
amples 34 and 51 were collected in July 2004. 34a and 51a: DNA aliquots
ples 34 and 51, amplified with As. AM, Am and As: as in Fig. 4. AS, H, M,
Fig. 7. Agarose gel electrophoresis of Diaphorina citri psyllid DNA amplified with 16S rDNA primers (OI1C OA1)/OI2c, specific for Ca. L. asiaticus and Ca.
L. africanus (A), and 16S rDNA primers GB1/GB3, specific for for Ca. L. americanus (B). 1–22: amplified DNA from 22 psyllid batches (10 psyllids per
batch). Arrows indicate lanes with a DNA band amplified.from psyllid DNA. AF, AS, H, M, and O: as in Fig. 3. AM: as in Fig. 4.
D. do Carmo Teixeira et al. / Molecular and Cellular Probes 19 (2005) 173–179178
Ca. L. asiaticus (Fig. 7A), as expected, since the insects were
collected on trees that were not infected with these
liberibacters. However, with primers specific for Ca. L.
americanus, several batches of insects gave positve PCR
signals (Fig. 7B, batches 3, 5, 7, 10, 17 and 18). Fig. 8 shows
that the amplicons from batches 3, 5, 7, 10 and 18 (lanes 3–7)
have the same size and electrophoretic mobility than the
1027 bp amplicon characteristic of Ca. L. americanus (Fig. 8,
lanes 2 and 8), and confirms that at least one psyllid in each
positive batch was infected with Ca. L. americanus.
Fig. 8. Electrophoretic characterization of the DNA amplified from psyllid
batches 3, 5, 7, 10, and 18 (see Fig. 7) with 16S rDNA primers GB1/GB3,
specific for Ca. L. americanus. M1 and M2: DNA size markers. Lanes 1 and
9: amplified 16S rDNA of Ca. L. asiaticus. Lanes 2 and 8: amplified 16S
rDNA of Ca. L. americanus. Lanes 3–7; DNA amplified with primers
GB1/GB3 from psyllid DNA of batches 3, 5, 7, 10, and 18, respectively.
4. Discussion and conclusion
With primers GB1 and GB3, it has been possible to
specifically amplify the 16S rDNA of Ca. L. americanus
from symptomatic citrus leaves, and the amplicon obtained
had the expected size of 1027 bp. With the same primers, no
amplification was obtained with DNA from Ca. L. africanus
or Ca. L. asiaticus. As shown on Fig. 1, the sequences of
GB1 and GB3 were chosen in such a way that annealing
with 16S rDNA of Ca. L. africanus and Ca. L. asiaticus
cannot occur. In particular, GB1 has three mismatches,
indicated by ‘!’ on Fig. 1, and a one-base insertion (G),
while GB3 has a deletion of 17 bases. Inversely, the primers
specific of Ca. L. africanus and Ca. L. asiaticus, (OA1COI1)/OI2c, do not permit amplification of Ca. L. americanus
16S rDNA, and this fact explains why so many symptomatic
leaf samples, known today to be infected with Ca. L.
americanus, gave negative PCR reactions when tested with
primers (OA1C OI1)/OI2c. When these ‘negative’ samples
were later analysed with primers GB1/GB3, they all turned
positive.
Before Ca. L. americanus was discovered, the first
liberibacter detected in Sao Paulo State was Ca. L. asiaticus
(M. A. Machado, personal communication).
Since primers GB1/GB3 and (OA1C OI1)/OI2c are
specific of their respective liberibacters, it has been possible
to analyse the same symptomatic leaf sample for the
presence of Ca. L. americanus by a PCR reaction with
GB1/GB3, as well as of Ca. L. africanus and/or Ca. L.
asiaticus by a PCR reaction with (OA1C OI1)/OI2c. In this
way, 218 symptomatic leaf samples could be analysed. Ca.
L. americanus was detected in 214, Ca. L. asiaticus in 2, Ca.
L. africanus in none, and two samples were infected with
both Ca. L. americanus and Ca. L. asiaticus. The proportion
D. do Carmo Teixeira et al. / Molecular and Cellular Probes 19 (2005) 173–179 179
of Ca. L. asiaticus to Ca. L. americanus is thus 4 to 216, or
1.9%. Another way to get a rough estimate of this
percentage is as follows. Ca. L. americanus has been
detected in as many as 35 municipalities, and Ca. L.
asiaticus in only 3. In these three municipalities, the number
of samples in which, respectively, Ca. L. asiaticus and Ca.
L. americanus were detected, amounts to 4 and 99, thus
giving a proportion of 4 to 99, or 4%. These percentages
indicate that Ca. L. americanus is probably today the most
widely distributed HLB pathogen in Sao Paulo State.
Even though, HLB was reported only in March of 2004,
the disease is probably present since many years in the
Araraquara area, where some citrus orchards with a high
proportion of affected trees have been identified recently. In
one of these orchards, according to the owner, the symptoms
of HLB, as seen now, were already present some 9 years
ago. This would explain the relatively wide spatial
extension of the disease, as observed today, only 5 months
after the first report. However, except for some orchards in
the Araraquara area, the incidence of the disease in most
farms is still low, with approximately 0.2% of affected trees.
However, surveys have to be carried out in order to estimate
the incidence of the disease more precisely.
In the frame of this work, all symptomatic leaf samples
showing blotchy mottle gave positive PCR reactions, and
Ca. L. americanus was the liberibacter detected most
frequently. However, even though blotchy mottle is
characteristic, it is not specific of HLB, and certain leaf
patterns might resemble HLB symptoms without any
liberibacters being involved. In such cases, the PCR tests
described here are most valuable for HLB identification.
Also, control of HLB will require removal of infected trees
in order to reduce inoculum pressure. In orchards affected
by a number of diseases, such as blight, leprosis, citrus
variegated chlorosis, and sudden death, the PCR detection
method will undoubtedly help identify the trees to be
removed.
D. citri, the Asian psyllid vector of Ca. L. asiaticus, is
present in Sao Paulo State since 60 years. It is very likely
that this insect is also responsible for the spread of Ca. L.
americanus. In support of this hypothesis, the presence of
the liberibacter in psyllids could be demonstrated.
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