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Interaction between endophytic bacteria from citrus plants
and the phytopathogenic bacteria Xylella fastidiosa,
causal agent of citrus-variegated chlorosis
P.T. Lacava
1
, W.L. Arau
´jo
1
, J. Marcon
1
, W. Maccheroni Jr
1
and J.L. Azevedo
1,2
1
Department of Genetics, Escola Superior de Agricultura ‘Luiz de Queiroz’, University of Sa
˜o Paulo, Piracicaba, SP, Brazil,
and
2
Nu
´cleo Integrado de Biotecnologia, University of Mogi das Cruzes, Mogi das Cruzes, SP, Brazil
2003/1097: received 1 December 2003, revised 30 March 2004 and accepted 5 April 2004
ABSTRACT
P.T. LACAVA, W.L. ARAU
´J O , J . M A R C O N , W . M A C C H E R O N I J R A N D J . L . A Z E V E D O . 2004.
Aims: To isolate endophytic bacteria and Xylella fastidiosa and also to evaluate whether the bacterial endophyte
community contributes to citrus-variegated chlorosis (CVC) status in sweet orange (Citrus sinensis [L.] Osbeck
cv. Pera).
Methods and Results: The presence of Xylella fastidiosa and the population diversity of culturable endophytic
bacteria in the leaves and branches of healthy, CVC-asymptomatic and CVC-symptomatic sweet orange plants and
in tangerine (Citrus reticulata cv. Blanco) plants were assessed, and the in vitro interaction between endophytic
bacteria and X. fastidiosa was investigated. There were significant differences in endophyte incidence between leaves
and branches, and among healthy, CVC-asymptomatic and CVC-symptomatic plants. Bacteria identified as
belonging to the genus Methylobacterium were isolated only from branches, mainly from those sampled from healthy
and diseased plants, from which were also isolated X. fastidiosa.
Conclusions: The in vitro interaction experiments indicated that the growth of X. fastidiosa was stimulated by
endophytic Methylobacterium extorquens and inhibited by endophytic Curtobacterium flaccumfaciens.
Significance and Impact of the Study: This work provides the first evidence of an interaction between citrus
endophytic bacteria and X. fastidiosa and suggests a promising approach that can be used to better understand CVC
disease.
Keywords: citrus plants, Curtobacterium flaccumfaciens, CVC, endophytes, Methylobacterium extorquens,
Methylobacterium mesophilicum.
INTRODUCTION
Citrus-variegated chlorosis (CVC) is a disease of sweet
orange (Citrus sinensis L.) trees caused by the xylem-limited
bacterium Xylella fastidiosa (Hartung et al. 1994), which is
transmitted by xylem-feeding suctorial insects (sharpshoot-
ers) or by seeds (Li et al. 2003). In spite of the fact that
X. fastidiosa was the first plant pathogen to have its genome
completely sequenced (Simpson et al. 2000) there is still no
effective control for CVC, although the presence of a few
asymptomatic plants in some infected orchards may lead to
new approaches to the investigation of the control of CVC.
These asymptomatic plants have the same genotype as
diseased plants and are located in the same grove under
similar climatic and edaphic conditions, suggesting that
some other factor is responsible for resistance to CVC. One
factor that may influence resistance to CVC is the nature of
the endophytic microbial community colonizing individual
C. sinensis plants (Arau´jo et al. 2002b).
Endophytic bacteria are those that live in the inner plants
parts (Hallmann et al. 1997) without causing apparent
damage to their hosts. The role of endophytic bacteria in
Correspondence to: Welington L. Arau
´jo, Departamento de Gene
´tica, ESALQ/USP,
Cx. P. 83, Piracicaba, SP, Brazil, 13400-970 (e-mail: wlaraujo@esalq.usp.br).
Present address: W. Maccheroni Jr, Allelyx Applied Genomics/Techno Park,
Km 104, Rod. Anhangu
¨era -13067-850- Campinas, SP, Brazil.
ª2004 The Society for Applied Microbiology
Letters in Applied Microbiology 2004, 39, 55–59 doi:10.1111/j.1472-765X.2004.01543.x
endophyte–plant associations has been extensively discussed
(Hallmann et al. 1997; Arau´jo et al. 2001, 2002b; Lode-
wyckx et al. 2002), but as endophytes colonize ecological
niches similar to those colonized by phytopathogens (Hall-
mann et al. 1997), interactions between these two groups are
possible. Research has shown that endophytic micro-organ-
isms isolated from surface disinfected plant tissues exhibit
potential as biocontrol agents against phytopathogens (Sturz
et al. 1998) and insects (Azevedo et al. 2000) and also
increase plant growth and hasten plant development
(Lodewyckx et al. 2002), although synergistic interactions
between endophytes and phytopathogens have not yet been
studied.
Citrus endophytic bacteria (Burkholderia cepacia,Citrob-
acter freundii,Achromobacter, Acinetobacter,Alcaligenes–Mor-
axella,Arthrobacter,Bacillus,Corynebacterium,Enterobacter
and Pseudomonas) were already isolated from the xylem of
lemon (Citrus jambhiri) roots (Gardner et al. 1982, 1985). In
more recent work, Arau´jo et al. (2001) isolated endophytic
bacteria from citrus rootstocks and showed a possible
interaction between Pantoea agglomerans,Bacillus pumilus
and the fungus Guignardia citricarpa (the causal agent of
citrus black spot), while Arau´jo et al. (2002b) suggested that
there may be an interaction between Curtobacterium flac-
cumfaciens,Methylobacterium species and X. fastidiosa.
The overall aim of this work was to determine the
culturable population of endophytic bacteria in the stems
and leaves of diseased and healthy citrus plants and to
evaluate, in vitro, the interaction between members of the
endophytic bacterial community and X. fastidiosa.
MATERIAL AND METHODS
Plant samples
Samples were taken from sweet orange (Citrus sinensis [L.]
Osbeck cv. pera) and tangerine (Citrus reticulata cv. Blanco)
trees growing in healthy orchards with no history of
infection with CVC and diseased orchards containing both
CVC-symptomatic and CVC-asymptomatic trees. Healthy
and diseased orchards were sampled in Catanduva and
Novais, two citrus-growing areas in the Brazilian State of
Sa
˜o Paulo. For each cultivar, orchard and location, samples
of branches and leaves were removed from four trees during
the winter and summer of 1999.
Isolation of culturable endophytic bacteria
Branches and leaves from sweet orange and tangerine plants
were surface sterilized (Arau´jo et al. 2002a,b), leaves and
peeled branches were cut into 4–6 mm pieces and placed
onto tryptic soy agar (TSA) medium (Merck, Darmstadt,
Germany) plates supplemented with the fungicide benomyl
(50 lgml
)1
), 10 replicates were analysed per plant. Incu-
bation was carried out at 28C for 1–12 days to allow growth
of endophytic bacteria and to determine the number of
infected fragments, endophyte incidence (EI) were calcula-
ted as the percentage of plant pieces exhibiting bacterial
growth.
Isolated colonies of recovered endophytic bacteria were
tested for colony characteristics (colour, growth rate,
opacity, shape, texture). Gram and motility tests were
performed as described by Arau´jo et al. (2002a). Some
isolates from each bacterial group of interest were further
identified using the MIDI system (Microbial Identification
System Inc., Newark, DE, USA) whole cell cellular fatty
acid methyl ester (FAME) gas chromatography analysis.
Isolation and PCR detection of X. fastidiosa
Leaf petioles from sweet orange and tangerine plants were
washed in running tap water and surface disinfected (Arau´jo
et al. 2002a,b), the disinfection process is checked by
pressing the disinfected plant material onto Periwinkle
medium (PW) agar (Chang et al. 1993). For isolation of X.
fastidiosa, the surface disinfected petioles were cut into small
pieces, placed into 0Æ2 ml of PW broth and centrifuged at
3000 gfor 15 min, aliquots of the supernatant being plated
onto PW agar and incubated at 28C for up to 20 days
(Lacava et al. 2001). The same method was used to quantify
the number of X. fastidiosa, except that the supernatant was
serially diluted in PW broth and titres calculated based on
which dilutions showed growth (i.e. visually apparent
turbidity) after incubation for up to 20 days.
DNA for PCR was extracted from disinfected sweet
orange and tangerine branches as described by Arau´jo et al.
(2002a). PCR was carried out in a 50-ll reaction volume
containing 2 ll of citrus branch DNA, 0Æ4lmol l
)1
each of
CVCP1 and 272-2 int primers (Pooler and Hartung 1995),
200 lmol l
)1
of each dNTPs, 3Æ75 mmol l
)1
of MgCl
2
,5U
Taq DNA polymerase (Invitrogen, Sao Paulo, Brazil) in
10 mmol l
)1
Tris–HCl (pH 8Æ3) and 10 mmol l
)1
KCl. The
amplification protocol consisted of an initial step at 94C for
4 min, followed by 30 amplification cycles of 94C for
1 min, 62C for 1 min and 72C for 1 min with a final
extension at 72C for 10 min. Amplification products were
separated by electrophoresis by spotting 5 ll of the PCR
reaction mixture onto 1Æ2% agarose gel and visualized under
ultra-violet light after staining with ethidium bromide
(0Æ5ugml
)1
).
Interaction between endophytic bacteria
and X. fastidiosa
We used two experiments to evaluate possible interactions
between endophytic isolates and the X. fastidiosa isolated in
56 P.T. LACAVA ET AL.
ª2004 The Society for Applied Microbiology, Letters in Applied Microbiology,39, 55–59, doi:10.1111/j.1472-765X.2004.01543.x
the present work. In one experiment, each endophyte was
streaked onto three PW agar plates which were incubated at
28C for 24 h to allow the endophyte to form a line of bacterial
growth on each plate, the X. fastidiosa being inoculated close
to this line and the plates incubated at 28C for up to 15 days.
Interaction between each endophyte and X. fastidiosa being
assessed visually based on the degree of growth of X.
fastidiosa. In another experiment, three of the endophytic
bacteria (Methylobacterium extorquens,M. mesophilicum and
Curtobacterium flaccumfaciens) were individually grown in PW
broth to the mid-log phase and the cells pelleted by
centrifugation at 3000 gfor 5 min, the supernatant being
collected, filtered through a 0Æ22-lm membrane filter (Mil-
lipore, Sao Paulo, Brazil) and added, individually, to PW
medium at a final concentration of 0Æ2, 2 or 20% (v/v) to
produce supplemented PW medium containing either M.
extorquens supernatant (PWSMe), M. mesophilicum superna-
tant (PWSMm) or C. flaccumfaciens supernatant (PWSCf).
Supplemented and unsupplemented (control) PW media
were inoculated with X. fastidiosa by placing 9 ml of PWSMe,
PWSMm or PWSCf in a 30-ml tube and inoculating it with
1 ml of PW broth containing 10
4
viable X. fastidiosa cells
(previously grown at 28C for 24 h without agitation). After
inoculation, the tubes were incubated at 28C for 20 days
without agitation, growth of X. fastidiosa being evaluated at
k¼600 gm using an Ultrospec 3000 spectrophotometer
(Amershan-Pharmacia Biotech, Cambridge, UK).
Statistical analysis
All statistical analyses were performed with the SAS
software package (Proc GLM in SAS, release 6Æ12; SAS
Institute, Cary, NC, USA) using completely randomized
analysis of variances (
ANOVAANOVA
) for unequally replicated
treatments at P<0Æ05 (Steel and Torrie 1980). Endophyte
incidence (EI) values were subjected to a square root + 0Æ5
transformation before
ANOVAANOVA
. Duncan’s tests for unequally
replicated means was used for further comparisons between
means (P<0Æ05).
RESULTS
Isolation of culturable endophytic bacteria
Endophytic bacteria were isolated from both the leaves and
branches of the sweet orange and tangerine plants sampled,
branches having a significantly (P<0Æ05) lower incidence of
endophytic bacteria than leaves (Fig. 1). Tangerine plants
generally showed lower levels of colonization by endophytes
than sweet orange plants, while for both citrus species
asymptomatic plants as well as healthy plants had a lower
incidence of colonization by endophytic bacteria than CVC-
symptomatic plants (Figs 1 and 2).
The total endophytic community isolated from the two
species of citrus plants included Bacillus pumilus,Curtobac-
terium flaccumfaciens,Enterobacter cloacae,Methylobacterium
0
10
20
30
40
50
60
70
80
90
100
CVC-symptomatic
plants
Asymptomatic
plants
Healthy
plants
Tangerine
Endophyte incidence (%)
Branches Leaves
Fig. 1 Total endophyte incidence of the leaves and branches of sweet
orange and tangerine plants. Bars represent the standard error
0
10
20
30
40
50
60
CVC-symptomatic
plants
Asymptomatic
plants
Healthy plants Tangerine CVC-symptomatic
plants
Asymptomatic
plants
Healthy plants Tangerine
Endophytes incidence in leaves (%)
Xanthomonas campestris
Actinomycetes
Enterobacter cloacae Curtobacterium flaccumfaciens
Others
0
10
20
30
40
50
60
Endophytes incidence in branches (%)
Xanthomonas campestris Curtobacterium
flaccumfaciens
Enterobacter cloacae
Actinomycetes
Methylobacterium
spp.
Nocardia
sp.
Others
Fig. 2 Incidence of the main endophytes isolated from leaves and branches of sweet orange and tangerine plants. Bars represent the standard error
ENDOPHYTIC BACTERIA AND X. FASTIDIOSA 57
ª2004 The Society for Applied Microbiology, Letters in Applied Microbiology,39, 55–59, doi:10.1111/j.1472-765X.2004.01543.x
extorquens,M. mesophilicum,Nocardia sp., Pantoea agglom-
erans,Streptomyces sp. and Xanthomonas campestris.
The frequency of isolation of specific genera and species
differed depending on the type of material sampled. For
leaves, C. flaccumfaciens was isolated more frequently from
CVC-asymptomatic than CVC-symptomatic orange and
tangerine plants, while pink-pigmented facultative methyl-
trophs (PPFMs) of the genus Methylobacterium were never
isolated (Fig. 2). However, for branches Methylobacterium
was the most frequently recovered bacterium from both
healthy and CVC-symptomatic orange and tangerine plants,
while C. flaccumfaciens was not isolated at all from the
branches of healthy tangerine plants (Fig. 2). The frequency
of these bacteria was stable at both the Catanduva and
Novais sampling sites.
Isolation and detection of X. fastidiosa
In the present study, X. fastidiosa was not isolated from
asymptomatic or healthy sweet orange branches or from
tangerine branches but was consistently isolated from the
petioles of sweet orange plants symptomatic for CVC, the
X. fastidiosa population of the different CVC-symptomatic
sweet orange samples ranging from 10
7
to 10
8
colony-
forming units (CFU) g
)1
of fresh tissue weight (FTW). At
serial dilutions of up to 10
)5
growth of X. fastidiosa was very
frequent, but growth also occurred in the higher dilutions
after 15 days incubation. The PCR method was successful in
detecting X. fastidiosa in both CVC-symptomatic and CVC-
asymptomatic plants.
Interaction between endophytic bacteria
and X. fastidiosa
Growth of some X. fastidiosa isolates on PW agar appeared to
be stimulated by Methylobacterium isolates, as indicated by the
presence of 2–3 mm more growth of X. fastidiosa in the
vicinity of M. extorquens (data not shown). The growth of
X. fastidiosa in liquid culture was not stimulated by the
presence of M. extorquens supernatant (Fig. 3), although the
presence of M. mesophilicum reduced the growth of
X. fastidiosa. Inhibition of X. fastidiosa was also observed in
the presence of C. flaccumfaciens supernatant, higher concen-
trations of which resulted in decreased growth of X. fastidiosa
(Fig. 3).
DISCUSSION
The serial dilution method using PW media showed high
titres of X. fastidiosa in CVC-symptomatic sweet orange
plants but not in CVC-asymptomatic or healthy sweet
orange plants or tangerine. However, as previously shown by
Arau´jo et al. (2002a,b), PCR detected X. fastidiosa in
CVC-asymptomatic sweet orange plants, indicating that
X. fastidiosa might colonize these plants without inducing
symptoms of CVC. The results also showed that the
X. fastidiosa population is lower in CVC-asymptomatic than
CVC-symptomatic plants, possibly because conditions for
growth inside the host plant are unfavourable in asympto-
matic plants.
The total isolation frequency of endophytic bacteria was
higher in leaves than in branches (Fig. 1), suggesting that
leaves are the preferential niche for endophytic bacteria
in sweet orange and tangerine. As mentioned above,
X. fastidiosa may colonize CVC-asymptomatic sweet orange
plants without inducing symptoms of CVC and it appears
(Fig. 2) that the diversity in these resistant plants is lower
than in CVC-symptomatic or healthy sweet orange plants or
tangerine plants. These results suggest that CVC-asympto-
matic sweet orange plants are more selective than CVC-
symptomatic or healthy sweet orange plants or tangerine
plants with the result that fewer endophytic groups are able
to colonize them. For example, Methylobacterium spp. were
only isolated from branches and mainly from CVC-symp-
tomatic and healthy plants, results which agree with those of
Arau´jo et al. (2002b) and show a possible interaction
between Methylobacterium spp. and CVC symptoms. The
in vitro interaction between X. fastidiosa and Methylobacte-
rium spp. seen by us suggests that in some instances some
Methylobacterium spp. could stimulate the growth of
X. fastidiosa, resulting, if occur in the field, a more intense
symptoms of CVC. However, we also found that
C. flaccumfaciens inhibited in vitro X. fastidiosa. If these
endophytes are able to produce the inhibitory compound
0
0·3
0·6
0·9
SR 1.6 AR 1.6 ER 1.6
Growth of
Xylella fastidiosa
(OD600nm)
00·2% 2% 20%
Fig. 3 Effect of cell-free supernatants of the endophytic bacteria
Methylobacterium mesophilicum (isolate SR1.6/6), Methylobacterium
extorquens (isolate AR1.6/2) and Curtobacterium flaccumfaciens (isolate
ER1/6) on the growth of Xylella fastidiosa. The supernatants were
individually tested by adding them to PM broth at final concentrations
of 0 (control), 0Æ2, 2Æ0 and 20% (v/v), inoculating the supplemented
PM broth with X. fastidiosa and evaluating growth spectrophotomet-
rically at k¼600 nm after 20 days
58 P.T. LACAVA ET AL.
ª2004 The Society for Applied Microbiology, Letters in Applied Microbiology,39, 55–59, doi:10.1111/j.1472-765X.2004.01543.x
inside the host plant, the CVC symptoms could be reduced
by a high titre of this endophytic bacterium.
In mature citrus plants the niche represented by the
endophytic environment becomes more stable and uniform,
resulting in selective pressure, which may preferably
promote some genotypes within each local microbial pop-
ulation. Consequently, bacteria living in an endophytic
environment may show a tendency to adapt themselves to a
more stable environment, resulting in more intense inter-
action between them. The present study highlighted the
relationship among bacterial population and suggests that
the CVC symptoms in citrus plants could be a result of
the population balance between Methylobacterium spp.,
C. flaccumfaciens and X. fastidiosa.
ACKNOWLEDGEMENTS
This work was supported by a grant from the Brazilian
agency FAPESP (Proc. 98/16262-2 and 02/08786-9) which
also provided fellowships to P.T.L. (no. 00/14987-1) and
W.L.A. (no. 00/10699-1).
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