M.
Rodrigues
et al.
Environmental Microbiology (2006) 8(4), 747–754
Brief report
In situ probing of Xylella fastidiosa in honeydew of a
xylem sap-feeding insect using 16S rRNA-targeted
fluorescent oligonucleotides
Jorge L. M. Rodrigues,1* Maria E. Silva-Stenico,1
Adriane N. de Souza,1 João R. S. Lopes2 and
Siu M. Tsai1
1
Centro de Energia Nuclear na Agricultura, and
2
Departamento de Entomologia, Fitopatologia e Zoologia
Agrícola, ESALQ, Universidade de São Paulo, Piracicaba,
SP, 13400-970, Brazil.
Summary
Xylella fastidiosa is a plant pathogen that threatens a
US$ 4.6 billion worldwide wine and citrus industry.
Monitoring its presence and distribution in plants and
vectors is crucial for designing control strategies, as
well as for understanding its ecological role and fate.
We developed two fluorescent oligonucleotide probes
complementary to different regions of the 16S rRNA
gene of X. fastidiosa. The specificity of the newly
designed probes S-S-X.fas-0067-a-A-18 and S-S-
X.fas-1439-a-A-18 was demonstrated using fluores-
cence in situ hybridization (FISH) for 12 Xylella iso-
lates, 15 closely related microorganisms and three
plant endophytes. These probes were used to detect
and quantify X. fastidiosa in plant sap (average value
of 2.9 ± 0.3 ¥ 106 cells ml-1) from three different citrus
orchards. In a second experiment, cells were quanti-
fied in honeydew (2.2 ± 0.2 ¥ 104 cells ml-1) collected
from the insect vector Bucephalogonia xanthophis
during the acquisition access period on an infected
plant. The number of pathogen cells retained or
digested by the insect is 10 000 times greater than the
estimated minimum value to ensure an efficient trans-
mission. Polymerase chain reaction (PCR) amplifica-
tion using specific primers with plant sap and
honeydew samples, followed by sequencing, con-
firmed the presence of the plant pathogen. This is the
first demonstration of FISH being used for environ-
Received 31 December, 2004; accepted 30 August, 2005. *For cor-
respondence. E-mail rodrig76@msu.edu; Tel. (+1) 517 355 6463 ext.
1580; Fax (+1) 517 355 8957.
© 2005 Society for Applied Microbiology and Blackwell Publishing Ltd
doi:10.1111/j.1462-2920.2005.00958.x
mental samples, such as plant sap and insect honey-
dew, to estimate the abundance of a plant pathogen
during infection.
Introduction
Xylella fastidiosa is a Gram-negative plant pathogen 0.3–
0.5 µm in diameter by 1–5 µm in length (Wells et al.,
1987). Many agriculturally important plants have this
pathogen as the causal agent of diseases, making this
species sufficiently important to have its genome partially
or entirely sequenced for five different pathotypes (Simp-
son et al., 2000; Bhattacharyya et al., 2002; Van Sluys
et al., 2003).
Some groups of insects of the order Hemiptera, known
as sharpshooter leafhoopers (Cicadellidae: Cicadellinae)
or spittlebugs (Cercopidae), transmit this bacterium (Pur-
cell and Hopkins, 1996). These insects usually feed on
xylem sap of numerous plant species, many of which are
known to harbour X. fastidiosa in their xylem vessels.
Throughout the insect feeding period, X. fastidiosa
attaches to the food canal (precibarium) and pump cham-
ber (cibarium) in the foregut of the insect (Brlansky et al.,
1982a; 1983), forming a dense aggregation over time.
Because xylem sap is an unbalanced solution of amino
acids and organic acids (Douglas, 2003), these sap-suck-
ing insects ingest copious amounts of fluid, which is
excreted through the intestinal tract (Auclair, 1963). This
liquid droplet excretion is known as honeydew.
Identification of X. fastidiosa is based on cultivation fol-
lowed by biochemical and serological analyses of isolates
(Fry et al., 1990; Chang and Donaldson, 1993; Hartung
et al., 1994), being time-consuming and labour-demand-
ing. Conventional methods of whole-cell detection of
X. fastidiosa include phase contrast (Lima et al., 1997),
electron microscopy (Brlansky et al., 1983; Hartung et al.,
1994) and fluorescence microscopy (Brlansky et al.,
1982b), which either lack specificity or are expensive
owing to the requirement of monoclonal antibodies (Pur-
cell and Hopkins, 1996). Recently, a green fluorescent
recombinant strain of X. fastidiosa was successfully used
748 J. L. M. Rodrigues et al.
for analysis of bacterial colonization of grapes and insect
vector by confocal laser scanning microscopy (Newman
et al., 2003). However, the requirement of genetic trans-
formation of the bacterium for expression of the green
fluorescent protein makes this technique not applicable for
the detection of X. fastidiosa in field samples. Fluores-
cence in situ hybridization (FISH) combines both specific-
ity, adjustable to different phylogenetic levels with
appropriate oligonucleotide probes, and detection and
enumeration without prior isolation and cultivation of the
targeted bacterium (Amann et al., 1990a).
In this study, we report the development of two probes
targeting X. fastidiosa, on basis of the 16S rRNA
sequences of different isolates and compared these with
non-target, but phylogenetically related microorganisms,
and endophytes (Fig. 1). Specific target sequences were
identified using the small subunit rRNA database of the
ARB software package (Ludwig et al., 2004). Specificity for
each designed probe was tested against the public
databases using the CHECK PROBE function of the
Ribosomal Database Project II (RDP; http://
www.rdp.cme.msu.edu) (Cole et al., 2003) and the Basic
Local Alignment Search Tool (BLAST) program from the
National Center for Biotechnology Information (NCBI)
(Altschul et al., 1990). The target sequence for probe S-
S-X.fas-0067-a-A-18 (positions 67–84, according to
Escherichia coli numbering; Gutell, 1993) had two nucle-
otide mismatches with Xanthomonas albilineans,
X. hyacinthi, X. melonis and X. translucens, the closest
relatives to X. fastidiosa, and a Bacteroides species.
These mismatches were located at the central position 77
(U:G) and 3′ end position 84 (U:C) regions of the probe.
Probe S-S-X.fas-1439-a-A-18 targeted the region com-
prising nucleotides 1439 and 1456, in which one mis-
match was found at position 1448 (G:A) with 16S rRNA
sequences from Burkholderia andropogonis, Duganella

FISH signal
A B C D

B14 (AF536765) + + + –
0.1
SL1 (AF536766) + + + –
65
9a5c (AE003861) + + + –
CM1 (AF536767) + + + –
ALS-BC (AF536770) + + + –
PCE-RR (AF53461) + + + –
Xylella fastidiosa
MUL-1 (AF224749) + + + –
100
Temecula (AF635760) + + + –
PWT-22 (AF159578) + + + –

RGW-R (AF536762) + + + –
ELM-1 (AF536764) + + + –
PLM G83 (AF536761) + + + –

Xanthomonas vasicola (Y10755) – – + –

100 54
Xanthomonas axonopodis pv. citri (AE012039) – – + –
62
Xanthomonas cordiae (Y10765) – – + –
Xanthomonas bromi (Y10764) – – + –
Xanthomonas curcubitae (Y10760) – – + –
100 Xanthomonas campestris pv. campestris (AE012505) – – + –
Xanthomonas melonis (Y10756) – – + –
97
Xanthomonas translucens (Y10761) – – + –
99
Xanthomonas hyacinthi (Y10754) – – + –
Xanthomonas albilineans (X95918) – – + –
Pseudomonas fluorescens (AJ308308) – – + –
86 Methylobacterium mesophilicum (AY828569) – – + –
Curtobacterium flaccunfaciens (AY828578) – – + –
Escherichia coli (U00096) – – + –

Fig. 1. Phylogenetic tree generated by parsimony analysis based on shared nucleotides of the 16S rRNA gene of Xylella fastidiosa strains grown
in PW liquid medium (Davis et al., 1981), whereas closely related bacterial species were grown in 523 medium (Kado and Heskett, 1970) at
28°C. GenBank accession numbers follow species or strain designation. The scale is the expected number of substitutions per position. Numbers
at the nodes are presented only for percentage bootstrap values above 50% of 1000 re-samplings. Symbols on the right represent hybridization
signals experimentally observed for each isolate for oligonucleotide probes (A) S-S-X.fas-0067-a-A-18, (B) S-S-X.fas-1439-a-A-18, (C) Domain
Bacteria S-D-Bact-0338-a-A-18 and (D) Antisense Domain Bacteria S-D-Bact-0338-a-S-18. Symbols: +, strong hybridization signal; –, no
hybridization signal.

© 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 8, 747–754
 Fluorescence in situ hybridization of Xylella fastidiosa
zoogloeoides, Janthinobacterium lividum, Pseudomonas
lemoignei, Photorhabdus luminescens, Rhodoferax fer-
mentans, and Variovorax paradoxus and position 1447
(C:A) for Telluria mixta. None of these microorganisms
has been found in plant vascular systems.
Probe validations
Bacterial cells (Table 1) were harvested from liquid cul-
tures by centrifugation for 10 min at 3000 g and then
washed once with, and resuspended in, phosphate-buff-
ered saline (PBS) solution [10 mM sodium phosphate
buffer (pH 8.0) and 130 mM NaCl]. Cells were fixed for
12 h at 4°C with freshly prepared formaldehyde solution
[4% (w:v) paraformaldehyde]. After fixation, cells were
washed twice with PBS, resuspended in 1:1 (v:v)
PBS:ethanol and stored at −20°C until use (de los Reyes
et al., 1997).
Samples (3 µl) were spotted in duplicate on 10-well
Teflon-coated slides (Cel-Line Brand, Portsmouth, NH)
749
containing 1% gelatin and dried at room temperature over-
night (Santo Domingo et al., 1998). Slides were dehy-
drated for 3 min in 50%, 80% and 96% ethanol and air-
dried. Hybridization solution (9 µl) containing 100 mM
Tris-HCl (pH 8.0), 0.9 M NaCl, 20% formamide, 0.1%
sodium dodecyl sulfate (SDS), 50 ng of the oligonucle-
otide probe S-S-X.fas-0067-a-A-18 (5′-TACTACCAATGT
GCTGCC-3′) labelled with fluorescein or S-S-X.fas-1439-
a-A-18 (5′-CTCCTCGCGGTTAAGCTA-3′) labelled with
rhodamine (Invitrogen, São Paulo, SP, Brazil) were added
to each well. In addition, probes Domain Bacteria S-D-
Bact-0338-a-A-18 (5′-GCTGCCTCCCGTAGGAGT-3′)
(Amann et al., 1990b) and Antisense Domain Bacteria S-
D-Bact-0338-a-S-18 (5′-ACTCCTACGGGAGGCAGC-3′)
(Amann et al., 1995), labelled with the fluorophore
indocarbocyanin Cy3, were used in all experiments as
positive and negative controls respectively. Probes were
named according to nomenclature defined by Alm and
colleagues (1996). Hybridizations were conducted in
moisture chambers for 12 h at 37°C, in the dark. Slides

Table 1. List of 31 strains used for in situ probe specificity.

Speciesa Strain Biological origin Geographical origin Collectionb

Xylella fastidiosa 9a5c Citrus sinensis (citrus) São Paulo, Brazil INRA
CM1 Coffea arabica (coffee) São Paulo, Brazil CCT 6737
MUL-1 Morus nigra (mulberry) Massachusetts, USA ATCC 35868
ALS-BC Prunus amygdalus (almond) California, USA ATCC 35870
PLM G83 Prunus salicina (plum) Georgia, USA ATCC 35871
ELM-1 Ulmus americana (elm) Washington, USA ATCC 35873
RGW-R Ambrosia artimeisiifolia (ragweed) Florida, USA ATCC 35876
PWT-22 Vinca minor (periwrinkle) Florida, USA ATCC 35878
PCE-RR Vitis vinifera (grape) Florida, USA ATCC 35879T
Temecula Vitis vinifera California, USA ATCC 700964
SL1 Citrus sinensis Minas Gerais, Brazil CCSM
B14 Citrus sinensis São Paulo, Brazil CCSM
Xanthomonas albilineans Xa13 Saccharum officinarum São Paulo, Brazil E. Gigliote
Xanthomonas axonopodis pv. citri 306 Citrus sinensis São Paulo, Brazil CCSM
Xanthomonas bromi Bromus carinatus France LMG 947T
Xanthomonas campestris pv. campestris Brassica oleracea UK ATCC 33913T
Xanthomonas campestris pv. citri Citrus sinensis Brazil IBSBF 338
Xanthomonas campestris pv. malvacearum Malva brasiliensis Brazil IBSBF 555
Xanthomonas campestris pv. citrumelo Poncirus trifoliata Brazil IBSBF 1011
Xanthomonas campestris pv. pruni Prunus salicina Brazil IBSBF 1097
Xanthomonas campestris pv. phaseoli Phaseolus vulgaris Brazil IBSBF 1394
Xanthomonas cordiae Codiaeum variegatum USA LMG 8678T
Xanthomonas curcubitae Curcubita maxima New Zealand LMG 690T
Xanthomonas hyacinthi Hyacinthus orientalis The Netherlands LMG 739T
Xanthomonas melonis Cucumis melo Brazil LMG 8670T
Xanthomonas translucens pv. translucens Hordeum vulgare USA LMG 876T
Xanthomonas vasicola pv. holcicola Sorghum bicolor New Zealand LMG 736T
Curtobacterium flaccunfaciens Citrus sinensis Brazil W. Araújo
Methylobacterium mesophilicum Citrus sinensis Brazil W. Araújo
Pseudomonas fluorescens Rhizosphere USA ATCC 17467
Escherichia coli DH10B USA Invitrogen

a. Xylella fastidiosa strains were grown in PW liquid medium (Davis et al., 1981), whereas the other species were grown in 523 medium (Kado
and Heskett, 1970) at 28°C.
b. INRA, Institut National de la Recherche Agronomique et Université Victor Ségale, Bordeaux, France; CCT, Coleção de Culturas Tropical,
Fundação André Tosello, Campinas, Brazil; ATCC, American Type Culture Collection, Manassas, VA; CCSM, Centro de Citricultura Silvio Moreira,
Cordeirópolis, Brazil; LMG, Laboratorium voor Microbiologie, Universiteit Gent, Belgium; IBSBF, Instituto Biológico, Seção de Bacteriologia
Fitopatológica, Campinas, Brazil; Dr E. Gigliote, Universidade Federal de São Carlos, Araras, SP, Brazil; Dr W. Araújo, Universidade de São
Paulo, ESALQ, Departamento de Genética, Piracicaba, SP, Brazil; T, type strain.

© 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 8, 747–754
750 J. L. M. Rodrigues et al.
were washed with 100 mM Tris-HCl (pH 7.2), 10 mM
NaCl, 0.1% SDS and 5 mM ethylenediamine-tetraacetic
acid (EDTA) for 15 min at 39°C. The slides were dipped
in a cold DAPI solution (0.1 M Tris-HCl, 0.9 M NaCl and
10 µg ml−1 4′,6-diamidino-2-phenylindole) for 10 min,
rinsed with cold water, and air-dried at 37°C and mounted
with Vectashield (Vector Laboratories, Burlingame, CA).
Fluorescence signal was examined using a Zeiss
Axiophot2 epifluorescence microscope equipped with fil-
ter sets 15 (for rhodamine and Cy3), 9 (fluorescein) or 2
(DAPI) (Carl Zeiss, Jena, Germany). Images were cap-
tured with a charge-coupled device video camera VC44
(PCO AG, Kelheim, Germany) and processed with ISIS
software (Meta Systems Documentation, Gaithersburg,
MD). Digital images were imported into Adobe Photoshop
v.5 for final processing.
All 12 X. fastidiosa isolates were successfully visualized
with both probes (Fig. 1), suggesting that any pathotype
(Martinati et al., 2005) of this microorganism can be
detected using our protocol. Strains SL1 and B14 isolated
from citrus plants formed aggregates, clustering together
in situ and making it difficult to count individual cells. The
same was observed when cells from these strains were
grown in liquid medium. Aggregates were disrupted by
gentle vortexing (15 s) of samples.
The two probes did not hybridize with the 15 Xanthomo-
nas strains, or with the three citrus endophytes tested,
attesting the specificity of these probes (Fig. 1). Nonethe-
less, it can not be excluded that these probes might cross-
hybridize with other non-target microorganisms. All iso-
lates fluoresced with probe S-D-Bact-0338-a-A-18, which
is complementary to a highly conserved region of the 16S
rRNA molecule, indicating that all tested microorganisms
were suitable for FISH with the applied fixation protocol
and had sufficient ribosome content for their detection
with monolabelled oligonucleotide probes. No positive
signals were observed when the non-complementary
Domain Bacteria probe S-D-Bac-0338-a-S-18 was used
with any of the isolates (Fig. 1). In addition, all strains were
visualized when the appropriate filter (excitation and emis-
sion wavelengths of 365 and 420 nm) was set for DAPI
images (data not shown).
When dealing with fastidious or uncultured microorgan-
isms, Moter and Göbel (2000) suggest that accuracy and
reliability of a newly designed FISH probes should be first
tested with other hybridization assays. We believe that
taking a step further and assessing whole-cell hybridiza-
tions for 31 different isolates with both probes S-S-X.fas-
0067-a-A-18 and S-S-X.fas-1439-a-A-18 was appropriate
to address and overcome such problems as lack of spec-
ificity, higher-order structure of the ribosomal RNA among
X. fastidiosa isolates and other close relatives, or insuffi-
cient probe penetration among isolates of the target
microorganism. In doing so, we prevented designing
© 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 8, 747–754
probes that performed well in hybridization arrays with
denatured RNA but were not useful for FISH (Frischer
et al., 1996).
In situ enumeration in field-collected xylem sap and
insect honeydew
Samples were randomly collected from 10 plants with and
without symptoms from three different citrus orchards (cit-
ies of Gavião Peixoto, Neves Paulista and Paraíso) in the
State of São Paulo, Brazil. Samples of cut branches were
kept in humid dark plastic bags on ice and transported to
the laboratory within 2–4 h of collection. Four sap samples
of each citrus plant were collected from small branch
pieces (3 cm) by centrifugation at 16 000 g for 20 min.
Images of sap samples obtained from diseased citrus
plants were taken after FISH with probes for X. fastidiosa
(Fig. 2C and D). A minimum of five and a maximum of 20
microscopical fields were counted and combined for aver-
age values. We were able to use successfully both probes
to detect this microorganism in citrus sap samples taken
from three different orchards. Presence or absence of
fluorescent cells when either one of the probes was used
accords with the selected diseased or healthy plant sap
sample respectively. Pathogen cell counts in sap from
different orchards for diseased plants were similar and
had an average value of 2.9 ± 0.3 × 106 cells ml−1 (Fig. 3).
Although X. fastidiosa numbers in situ vary with host spe-
cies (Hopkins and Adlerz, 1988; Almeida and Purcell,
2003a; Lopes et al., 2003), sampling time after inoculation
(Oliveira et al., 2002; Almeida and Purcell, 2003b) and
plant sampling point (Fry et al., 1990), the abundance of
this pathogen in the plant vascular system agrees with
previous data for citrus, which were obtained by dilution
plating (Almeida et al., 2001). To date, population esti-
mates of X. fastidiosa have been based on dilution plating
followed by colony counts after 2 weeks. Fluorescence in
situ hybridization with our specific probes reduces the time
of analysis to 2 days, and still avoids errors that might
occur in the dilution plating method due to the presence
of contaminant bacteria in the plant sap. To our knowl-
edge, this is the first instance in which FISH has been
used for the detection of a plant pathogen in sap samples.
To date, only two plant pathogens have been detected by
the use of FISH, Ralstonia solanacearum (Wullings et al.,
1998) and Clavibacter michiganensis ssp. sepedonicus
(Li et al., 1997). However, these two microorganisms
required simultaneous use of FISH and immunofluores-
cence for their accurate identification, while in this study,
specific detection of X. fastidiosa was achieved by FISH
alone, preventing any cross-reactivity of antibodies.
We also frequently observed fluorescent aggregates of
X. fastidiosa cells in sap samples, similar to ones previ-
ously observed in pure cultures of strains SL1 and B14.
 Fluorescence in situ hybridization of Xylella fastidiosa 751
Fig. 2. Epifluorescence micrographs of Xylella
A B fastidiosa cells.
A and B. Pure culture cells of X. fastidiosa
strain 9a5c hybridized with fluorescein-labelled
S-S-X.fas-0067-a-A-18 and rhodamine-labelled
S-S-X.fas-1439-a-A-18 probes respectively.
C. Citrus sap sample hybridized with probe S-
S-X.fas-0067-a-A-18.
D. Identical micrograph shown in (C) but DAPI-
stained.
E. Insect (Bucephalogonia xanthophis) honey-
dew sample hybridized with probe S-S-X.fas-
0067-a-A-18.
F. Identical micrograph shown in (E) but DAPI-
stained.
C D

E F

107 Interestingly, these strains were previously isolated with-

out excessive passages in culture medium (H.D. Colleta-
Filho, pers. comm.). de Souza and colleagues (2003)
Xylella fastidiosa cells ml-1

have shown that X. fastidiosa genes involved in pathoge-

106 nicity, virulence and adaptation were expressed only in
freshly isolated cultures and loss of aggregation pheno-
type and virulent state were related to many consecutive
transfers on plates. Our results strengthen the hypothesis
105
that aggregation is an important characteristic for coloni-
zation, and hence, for the pathogenicity of X. fastidiosa
(Marques et al. 2002). In addition, our natural samples are
directly comparable to the aggregates observed with a
104
A B C D green fluorescent protein-labelled X. fastidiosa strain arti-
Sample (sap or honeydew) ficially inoculated into grapes (Newman et al., 2003;
2004).
Fig. 3. Quantification of Xylella fastiodiosa cells of citrus sap from In a further experiment, four healthy individuals of the
three different orchards and insect honeydew by FISH with species- sharpshooter vector Bucephalogonia xanthophis (Berg.)
specific probes: (A) Gavião Peixoto, (B) Neves Paulista, (C) Paraíso
and (D) Bucephalogonia xanthophis. Error bar represents standard were caged for a 24 h acquisition access period (AAP) on
error. an X. fastidiosa-infected citrus plant kept in greenhouse.
Healthy insects were reared in the laboratory as described
elsewhere (Marucci et al., 2003). Four insects from the
same rearing batch were caged for 24 h on control healthy

© 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 8, 747–754
752 J. L. M. Rodrigues et al.
citrus plants. During the AAP, insects were confined on
citrus branches by using a cage, which allowed collection
of liquid excretion (honeydew) as described by Andersen
and colleagues (1992). The total volume of the excreted
honeydew was measured and immediately stored on ice
for further FISH analysis. When the probe S-S-X.fas-
0067-a-A-18 was applied to insect honeydew,
X. fastidiosa was detected in all samples from insects fed
in diseased plants (Fig. 2E and F), yielding an average
number of 2.2 ± 0.2 × 104 cells per ml of excreted honey-
dew (Fig. 3). This number accounted for 98.3% of the total
DAPI-stained cells. The high proportion of plant pathogen
cells in comparison with the whole community, such as
endophytes, is explained by the high number of the same
X. fastidiosa cells in the sap of infected plants and hence
ingested by the insect. Based on the difference in cell
counts of X. fastidiosa between citrus xylem sap and
excreted honeydew, we calculate that approximately
2.7 × 106 cells would be retained and/or digested by the
sharpshooter B. xantophis in a 24 h AAP, explaining, with
no surprise, why only 1 h of access time on a diseased
grape plant is sufficient for the glassy-winged sharp-
shooter Homalodisca coagulate (Say.) to become infective
(Almeida and Purcell, 2003a). This population number is
10 000 times greater than the estimated minimum value
to ensure an efficient transmission (Hill and Purcell,
1995). As sap-feeding insects are generally thought not
to exhibit polymer digestion, but only hydrolysis of sucrose
and absorption of soluble nutrients and amino acids in the
luminal tract (Terra, 1990), X. fastidiosa can pass intact
through the insect gut, as quantified here.
Primers previously developed for the 16S rRNA gene
(Rodrigues et al., 2003) were used for the detection of
X. fastidiosa in sap of infected citrus plants and in honey-
dew of insects that had been fed for 1 day in the same
diseased plants. Positive results for honeydew samples
were obtained only after two rounds of polymerase chain
reaction (PCR) amplification. Products of approximately
1348 bp were amplified as expected (Fig. 4). Partial
sequencing of the PCR products resulted in 99% identity
to the X. fastidiosa 16S rRNA gene (data not shown),
confirming the presence of this pathogen in diseased
plants as well as in insect honeydew. No PCR amplifica-
tion occurred when honeydew samples were obtained
from insects reared on X. fastidiosa-free laboratory-grown
plants. The need for two rounds of PCR to detect
X. fastidiosa in honeydew samples agrees with our previ-
ous observations (Rodrigues et al., 2003) and might be
due to the variety of contaminants found in the insect
honeydew (Auclair, 1963). Nevertheless, positive amplifi-
cation of the X. fastidiosa 16S rRNA gene with specific
primers revealed that cells of this plant pathogen are
being excreted in the honeydew during the feeding time
of the insect vector.
© 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 8, 747–754
1 2 3 4 5 6 7 8 9
1375 bp 1348 bp
831 bp
564 bp
Fig. 4. Ethidium bromide-stained agarose gel of PCR products
obtained from insect honeydew and xylem sap samples using Xylella
fastidiosa 16S rRNA gene-specific primers. Honeydew samples were
concentrated through centrifugation, followed by addition of 100 µl of
0.05N NaOH, and then boiled for 10 min. Total DNA isolation from
sap was performed with CTAB buffer according to Doyle and Doyle
(1990). Polymerase chain reaction followed protocol described in
Rodrigues and colleagues (2003). The expected fragment size is
1348 bp. Numbers above lanes refer to: 1 – molecular marker λ
restricted with HindIII and EcoRI; 2 – X. fastidiosa DNA-positive con-
trol; 3, 4 – Bucephalogonia xanthophis fed on a pathogen free plant;
5, 6 – B. xanthophis fed on a diseased citrus plant; 7, 8 – diseased
citrus plant; and 9 – negative control (H2O).
Although only transmission tests can verify whether an
insect is a vector to a pathogen, to date, X. fastidiosa has
been detected in 39 species of sharpshooters (Cicadelli-
nae) collected from several crop plants (Redak et al.,
2004). Inasmuch as there is a large diversity of sharp-
shooters described in the world (approximately 1950 spe-
cies) (Mejdalani, 1998), we expect the number of known
species to carrying this pathogen to grow rapidly in the
future, once other methods are devised, such as the one
presented here, for the detection and quantification of
X. fastidiosa.
In summary, we designed and tested specific fluores-
cent oligonucleotide probes for X. fastidiosa. We demon-
strated their usefulness for in situ enumeration of cells in
environmental samples such as plant sap and insect hon-
eydew. The probes used in this study provided a tool to
examine the ecology of X. fastidiosa, especially their
acquisition and retention by an insect vector, as well as
providing a reliable technique to identify additional plant
and insect reservoirs for this plant pathogen.
Acknowledgements
This article is dedicated in memory of Olga V. Maltseva, an
outstanding scientist and dear friend. The authors thank Dr
Margarida L.R.A. Perecin for generously allowing microscope
utilization; Dr Helvécio D. Coletta Filho for providing strains
SL1 and B14; Dr Janay A. dos S. Serejo, Juliana D. de
Almeida, Miriam G. de Chaves and Sandra Miyasawa for
technical assistance with FISH; Wagner Piccinini for plant
sampling; Rodrigo N. Marques for insect rearing; and Dr J.A.
Breznak, B.P. Keough and S.A. Eichorst for critically reading
this manuscript. J.L.M.R. (00/13160-6 and 02/01719-0) and
M.E.S.-S. (00/08045-3) recognize financial support from the
 Fluorescence in situ hybridization of Xylella fastidiosa
Fundação de Amparo à Pesquisa do Estado de São Paulo
(FAPESP). This project was supported by FAPESP (Grants
98/16265-1 and 00/10168-6).
References
Alm, E.W., Oerther, D.B., Larsen, N., Stahl, D.A., and Raskin,
L. (1996) The oligonucleotide probe database. Appl Envi-
ron Microbiol 62: 3557–3559.
Almeida, R.P.P., and Purcell, A.H. (2003a) Biological traits
of Xylella fastidiosa strains from grapes and almonds. Appl
Environ Microbiol 69: 7447–7452.
Almeida, R.P.P., and Purcell, A.H. (2003b) Transmission of
Xylella fastidiosa strains to grapevines by Homalodisca
coagulata (Hemiptera: Cicadellidae). J Econ Entomol 96:
264–271.
Almeida, R.P.P., Pereira, E.F., Purcell, A.H., and Lopes,
J.R.S. (2001) Multiplication and movement of a citrus strain
of Xylella fastidiosa within sweet orange. Plant Dis 85:
382–386.
Altschul, S.F., Gish, W., Miller, W., Myers, E.W., and Lipman,
D.J. (1990) Basic local alignment search tool. J Mol Biol
215: 403–410.
Amann, R.I., Krumholz, L., and Stahl, D. (1990a) Fluores-
cent-oligonucleotide probing of whole cells for determina-
tive, phylogenetic, and environmental studies in
microbiology. J Bacteriol 172: 762–770.
Amann, R.I., Binder, B.J., Olson, R.J., Chisholm, S.W.,
Devereux, R., and Stahl, D.A. (1990b) Combination of 16S
rRNA-targerted oligonucleotide probes with flow cytometry
for analyzing mixed microbial populations. Appl Environ
Microbiol 56: 1919–1925.
Amann, R.I., Ludwig, W., and Schleifer, K.H. (1995) Phylo-
genetic identification and in situ detection of individual
microbial cells without cultivation. Microbiol Rev 59: 143–
169.
Andersen, P.C., Brodbeck, B.V., and Mizell, R.F., III (1992)
Feeding by the leafhopper, Homalodisca coagulata, in rela-
tion to xylem fluid chemistry and tension. J Insect Physiol
38: 611–622.
Auclair, J.L. (1963) Aphid feeding and nutrition. Ann Rev
Entomol 8: 439–490.
Bhattacharyya, A., Stilwagen, S., Ivanova, N., D’Souza,
M., Bernal, A., Lykidis, A., et al. (2002) Whole genome
comparative analysis of three phytopathogenic Xylella
fastidiosa strains. Proc Natl Acad Sci USA 99: 12403–
12408.
Brlansky, R.H., Timmer, L.W., and Lee, R.F. (1982a) Detec-
tion and transmission of a Gram-negative, xylem-limited
bacterium in sharpshooters from a citrus grove in Florida.
Phytopathology 66: 590–592.
Brlansky, R.H., Lee, R.F., Timmer, L.W., Purcifull, D.E., and
Raju, B.C. (1982b) Immunofluorescence detection of
xylem-limited bacteria in situ. Phytopathology 72: 1444–
1448.
Brlansky, R.H., Timmer, L.W., French, W.J., and McCoy, R.E.
(1983) Colonization of the sharpshooter vectors, Oncome-
topia nigricans and Homalodisca coagulate, by xylem-lim-
ited bacteria. Phytopathology 73: 530–535.
Chang, C.J., and Donaldson, R.C. (1993) Xylella fastidiosa:
© 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 8, 747–754
753
cultivation in chemically defined medium. Phytopathology
83: 192–194.
Cole, J.R., Chai, B., Marsh, T.L., Farris, R.J., Wang, Q.,
Chandra, S., et al. (2003) The Ribosomal Database Project
(RDP-II): previewing a new autoaligner that shows regular
updates and the new prokaryotic taxonomy. Nucleic Acids
Res 31: 442–443.
Davis, M.J., French, W.J., and Schaad, N.W. (1981) Axenic
culture of the bacteria associated with phony disease of
peach and plum leaf scald. Curr Microbiol 6: 309–314.
Douglas, A. (2003) The nutritional physiology of aphids. Adv
Ins Physiol 31: 73–140.
Doyle, J.J.T., and Doyle, J.L. (1990) Isolation of plant DNA
from fresh tissue. Focus 12: 13–18.
Frischer, M.E., Floriani, P.J., and Nierzwicki-Bauer, S.A.
(1996) Differential sensitivity of 16S rRNA targeted oligo-
nucleotides probes used for fluorescence in situ hybridiza-
tion is a result of higher order structure. Can J Microbiol
42: 1061–1071.
Fry, S.M., Mihlolland, R.D., and Huang, P.-Y. (1990) Isola-
tion and growth of strains of Xylella fastidiosa from
infected grapevines on nutrient agar media. Plant Dis 74:
522–524.
Gutell, R.R. (1993) Collection of small subunit (16S- and 16S-
like) ribosomal RNA structures. Nucleic Acids Res 21:
3051–3055.
Hartung, J.S., Beretta, J., Brlansky, R.H., Spisso, J., and
Lee, R.F. (1994) Citrus variegated chlorosis bacterium:
axenic culture, pathogenicity, and serological relationships
with other strains of Xylella fastidiosa. Phytopathology 84:
591–597.
Hill, B.L., and Purcell, A.H. (1995) Acquisition and retention
of Xylella fastidiosa by an efficient vector. Phytopathology
85: 209–212.
Hopkins, D.L., and Adlerz, W.C. (1988) Natural hosts of
Xylella fastidiosa in Florida. Plant Dis 72: 429–431.
Kado, C.I., and Heskett, M.G. (1970) Selective media for
isolation of Agrobacterium, Corynebacterium, Erwinia,
Pseudomonas and Xanthomonas. Phytopathology 60:
969–976.
Li, X., De Boer, S.H., and Ward, L.J. (1997) Improved
microscopic identification of Clavibacter michiganensis
subsp. cepedonicus cells by combining in situ hybridiza-
tion with immunoflorescence. Lett Appl Microbiol 24:
431–434.
Lima, J.E.O., de Miranda, V.S., Roberto, S.R., Coutinho, A.,
Palma, R., and Pizzolitto, A.C. (1997) Diagnose da clorose
variegada dos citros por microscopia ótica. Fitopatol Bras
22: 370–374.
Lopes, S.A., Marcussi, S., Torres, S.C.Z., Souza, V., Fagan,
C., França, S.C., et al. (2003) Weeds as alternative hosts
of the citrus, coffee, and plum strains of Xylella fastidiosa
in Brazil. Plant Dis 87: 544–549.
Ludwig, W., Strunk, O., Westram, R., Richter, L., Meier, H.,
Yadhukumar, et al. (2004) ARB: a software environment for
sequence data. Nucleic Acids Res 32: 1363–1371.
Marques, L.L.R., Cerri, H., Manfio, G.P., Reid, D.M., and
Olson, M.E. (2002) Characterization of biofilm formation by
Xylella fastidiosa in vitro. Plant Dis 86: 633–638.
Martinati, J.C., Pacheco, F.T.H., Miranda, V.F.O., and Tsai,
S.M. (2005) Phylogenetic relationships of Xylella fastidiosa
754 J. L. M. Rodrigues et al.
strains based on 16S−23S rDNA sequences. Curr Micro-
biol 50: 190–195.
Marucci, R.C., Giustolin, T.A., Miranda, M.P., Miquelote, H.,
Almeida, R.P.P., and Lopes, J.R.S. (2003) Identification of
a non-host plant of Xylella fastidiosa to rear healthy sharp-
shooter vectors. Scientia Agric 60: 669–675.
Mejdalani, G. (1998) Morfologia externa dos Cicadellinae
(Homoptera, Cicadellidae): Comparação entre Versigona-
lia ruficauda (Walker) (Cicadellini) e Tretogonia cribrata
Melichar (Proconiini), com notas sobre espécies e análise
da terminologia. Rev Brasil Zool 15: 451–544.
Moter, A., and Göbel, U.B. (2000) Fluorescence in situ
hybridization (FISH) for direct visualization of microorgan-
isms. J Microbiol Methods 41: 85–112.
Newman, K., Almeida, R.P.P., Purcell, A.H., and Lindow,
S.E. (2003) Use of a green fluorescent strain for analysis
of Xylella fastidiosa colonization of Vitis vinifera. Appl Envi-
ron Microbiol 69: 7319–7327.
Newman, K., Almeida, R.P.P., Purcell, A.H., and Lindow,
S.E. (2004) Cell–cell signaling controls Xylella fastidiosa
interactions with both insect and plants. Proc Natl Acad Sci
USA 97: 1737–1742.
Oliveira, A.C., Vallim, M.A., Semighini, C.P., Araújo, W.L.,
Goldman, G.H., and Machado, M.A. (2002) Quantification
of Xylella fastidiosa from citrus trees by real time poly-
merase chain reaction assay. Phytopathology 92: 1048–
1054.
Purcell, A.H., and Hopkins, D.L. (1996) Fastidious xylem-
limited bacterial plant pathogens. Annu Rev Phytopathol
34: 131–151.
Redak, R.A., Purcell, A.H., Lopes, J.R.S., Blua, M.J., Mizell,
R.F., and Andersen, P.C. (2004) The biology of xylem
fluid-feeding insect vectors of Xylella fastidiosa and their
relation to disease epidemiology. Annu Rev Entomol 49:
243–270.
de los Reyes, F.L., Ritter, W., and Raskin, L. (1997) Group
specific small-subunit RNA hybridization probes to charac-
© 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 8, 747–754
terize filamentous foaming in activated sludge systems.
Appl Environ Microbiol 63: 1107–1117.
Rodrigues, J.L.M., Silva-Stenico, M.E., Gomes, J.E., Lopes,
J.R.S., and Tsai, S.M. (2003) Diversity and detection
assessment of Xylella fastidiosa through 16S rRNA and
gyrB sequences in field-collected plant and insect sam-
ples. Appl Environ Microbiol 69: 4249–4255.
Santo Domingo, J.W., Kaufman, M.G., Klug, M.J., and
Tiedje, J.M. (1998) Characterization of the cricket hindgut
microbiota with fluorescently labeled rRNA-target oligonu-
cleotide probes. Appl Environ Microbiol 64: 752–755.
Simpson, A.J.G., Reinach, F.C., Arruda, P., Abreu, F.A.,
Acencio, M., Alvarenga, R., et al. (2000) The genome
sequence of the plant pathogen Xylella fastidiosa. Nature
406: 151–157.
de Souza, A.A., Takita, M.A., Coletta-Filho, H.D., Caldana,
C., Goldman, G.H., Yanai, G.M., et al. (2003) Analysis of
gene expression in two growth states of Xylella fastidiosa
and its relationship with pathogenicity. Mol Plan Microbe
Interact 16: 867–875.
Terra, W.R. (1990) Evolution of digestive systems of insects.
Ann Rev Entomol 35: 181–200.
Van Sluys, M.A., Oliveira, M.C., Monteiro-Vitorello, C.B., Miy-
aki, C.Y., Furlan, L.R., Camargo, L.E., et al. (2003) Com-
parative analysis of the complete genome sequences of
Pierce’s disease and citrus variegated chlorosis strains of
Xylella fastidiosa. J Bacteriol 185: 1018–1026.
Wells, J.M., Raju, B.C., Hung, H.Y., Weisburg, W.G., Man-
delco, P.L., and Brenner, D.J. (1987) Xylella fastidiosa gen.
nov., sp. nov. Gram-negative, xylem-limited, fastidious
plant bacteria related to Xanthomonas spp. Int J Syst Bac-
teriol 37: 136–143.
Wullings, B.A., Van Beuningen, A.R., Janse, J.D., and Akker-
mans, A.D.L. (1998) Detection of Ralstonia solanacearum,
which causes brown rot of potato, by fluorescent in situ
hybridization with 23S rRNA-targeted probes. Appl Environ
Microbiol 64: 4546–4554.