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Plant Pathology (2011) 60, 977985

Doi: 10.1111/j.1365-3059.2011.02450.x

Unstable green fluorescent protein for study of

Xanthomonas citri subsp. citri survival on citrus
J. Cuberoa, I. Gella, E. G. Johnsonb, A. Redondob and J. H. Grahamb*

Instituto Nacional de Investigacion y Tecnologa Agraria y Alimentaria (INIA), Ctra, De La Coruna, 28040 Madrid, Spain; and
University of Florida, Citrus Research and Education Center (CREC), 700 Experiment Station Road, Lake Alfred, FL 33850, USA

Xanthomonas citri subsp. citri (Xac) is the causal agent of citrus bacterial canker, an important disease for the citrus industry. Studies of Xac survival in environments outside of the lesion performed in the past may have underestimated the viable
population because the recovery was based on the ability of the bacterium to grow on culture media. This study monitored
survival of Xac that express green fluorescent protein (GFP) in two different forms: the native protein, and a protein that is
unstable due to a specific oligopeptide tail targeted by proteases within the bacterium. Transformed strains of Xac were verified to be stable in their expression of GFP and to show no differences in virulence and fitness compared to wild type strains.
Evaluation of protein stability confirmed that strains with unstable GFP only expressed and fluoresced in metabolically
active cells, and not in dead bacteria. Fluorescence of unstable GFP strains under confocal microscopy was used to track bacterial survival and biofilm formation on leaf and fruit surfaces. After spray inoculation, aggregates of fluorescing cells of
unstable GFP strains formed biofilms on leaves and fruit. Bacterial cells that aggregated on the surfaces only survived when
protected from desiccation. Aggregation of viable bacteria in biofilms confirms their role in pathogen survival outside of
lesions and protection from bactericide treatments in the field or in the fruit disinfection process.
Keywords: biofilm, citrus bacterial canker, epiphytic, GFP, persistence, phyllosphere

Citrus species are affected by several xanthomonad
pathogens, Xanthomonas citri subsp. citri (formerly
X. axonopodis pv. citri), X. fuscans subsp. aurantifolii
(formerly X. axonopodis pv. aurantifolii) and X. alfalfae
subsp. citrumelonis (formerly X. axonopodis pv.
citrumelo). The A type of citrus bacterial canker (CBC) is
caused by strains of X. citri subsp. citri (Xac), and the B
and C types are caused by strains of X. fuscans subsp. aurantifolii (Xfa). These diseases are characterized by erumpent, corky lesions on leaves, stems and fruits. CBC is a
serious disease of many commercial varieties, causing
unsightly blemishes on fruit destined for the fresh market
and premature fruit drop and yield losses on varieties for
juice processing. Most subtropical citrus areas of the
world have CBC, whereas areas with Mediterranean climates do not. Markets without CBC maintain quarantine
regulations preventing importation of fresh citrus (Gottwald et al., 2001; Graham et al., 2004).

*E-mail: jhg@crec.ifas.ufl.edu
Present address: Instituto Valenciano de Investigaciones
Agrarias, 46113 Moncada, Valencia, Spain.

Published online 24 March 2011

2011 The Authors

Plant Pathology 2011 BSPP

Studies evaluating Xac on plant tissues have demonstrated very limited survival outside of the lesions on leaf
and fruit surfaces (Graham et al., 1987, 2004; Gottwald
et al., 2009). Bacteria multiply in lesions, and when there
is free water on leaf surfaces, they egress from the stomata
to disperse by windblown rain droplets to other plants
where a new infection is initiated after stomatal penetration (Gottwald et al., 1989; Pruvost et al., 2002). Little is
known about the role of bacterial colonization of the
plant surface as a prelude to infection, but this needs clarification because of the potential role of this process in disease transmission. The combined effect of temperature
and leaf wetness duration plays a role in development of
CBC; however, it is unclear whether free moisture is necessary for bacterial infection through the stomata (Dalla
Pria et al., 2006; Christiano et al., 2009). In addition, evidence is limited or absent for survival of Xac on fruits and
the potential of surviving bacteria on harvested fruit surfaces to produce new infections (Gottwald et al., 2009).
Studies of citrus xanthomonad survival in different
environments have been conducted using bacterial isolation on semi-selective media which depends on the ability
of the bacterium to grow on culture media (Graham et al.,
1987, 1989, 1990; Gottwald et al., 1992; Pruvost et al.,
2002). However, for multiple bacteria, including
Xanthomonas, a starvation condition called viable but
non-culturable (VBNC) has been proposed (Byrd et al.,
1991; Manahan & Steck, 1997; Alexander et al., 1999;



J. Cubero et al.

Cho & Kim, 1999; Ghezzi & Steck, 1999; Grey & Steck,
2001; Pruvost et al., 2002; Del Campo et al., 2009). The
potential for the VBNC condition to occur in bacterial
populations means that studies based on bacterial growth
on culture media may underestimate or fail to detect viable bacteria. Recovery of the target bacterium by culture
plating is often compromised by the presence of competing microflora or physiological stress that prolongs or
inhibits the development of bacterial colonies on the culture plate. For these reasons, culture independent
approaches are required for assessing bacterial viability.
Green fluorescent protein (GFP) from the jellyfish Aequorea victoria (Chalfie et al., 1994) has been used extensively for studies of bacterial colonization of plants in situ
(Errampalli et al., 1999; Bloemberg et al., 2000; Stuurman et al., 2000). GFP is an excellent marker to locate
bacteria in the tissue, but is unsuitable for viability studies
because native GFP is stable in the cell for an extended
period. Therefore, dead or dying bacteria containing GFP
continue to fluoresce in the absence of current gfp expression (Andersen et al., 1998). Unstable GFP variants have
been used for gene expression studies because they have a
short half-life due to a C-terminal oligopeptide extension
that targets them for degradation by specific proteases
(Andersen et al., 1998). In the present study, strains with
native (stable) and unstable GFP were compared by evaluation of the location, colonization and survival of Xac
on citrus leaves and fruit. First, the stability of the unstable GFP protein in Xac strains grown in suspensions or on
solid media was assessed and the stability correlated with
gfp transcription. The fitness and pathogenicity of the
unstable and native GFP Xac strains were compared to
the non-transgenic wild type strains. Finally, the survival
of GFP Xac strains was tracked on citrus leaf and fruit
surfaces using confocal laser scanning microscopy

Materials and methods

Bacterial strains and transformation
Bacterial strains and the plasmids used for transformation are listed in Table 1. Strains 306 and MI of Xac were
used to construct the transgenic strains. Bacterial strains

Table 1 Bacterial strains used in the study








Wild type
Wild type
Native GFPa
Unstable GFP
Native GFP
Unstable GFP

Strains that express native (stable) or unstable green fluorescent
proteins (GFP) harbour pUFZ75 and pUFZJLVA plasmids,

were transformed by electroporation to incorporate plasmids that constitutively express GFP (Zhang et al., 2009).
Competent cells from the strains were transformed with
plasmids constructed as described below, using a Micropulser Electroporator (Bio-Rad). Selection of transformed strains was performed on LB agar (Difco)
medium with the addition of kanamycin (LB + Km) at
50 lg mL)1.

Plasmid construction
pUFZJLVA was constructed from pUFZ75 (Zhang et al.,
2009) using a protocol similar to that previously
described to obtain unstable GFP protein (Andersen
et al., 1998). PCR amplification with primers J-gfpup
(5-ATG AGT AAA GGA GAA GAA CT-3) and J-gfplva
GTA TAG TTC ATC CAT GCC A-3) was performed in
50 lL (total volume) mixtures containing 1 Taq buffer,
15 mM MgCl2, each primer at a concentration of 1 lM,
each deoxynucleoside triphosphate at 02 mM, 2 U of
Taq polymerase (Promega) and pUFZ75 as the template.
Amplified product included restriction sites for BamHI
and MscI at the 3 and 5 ends for cloning and a nucleotide
sequence at the 3 end that encodes an amino acid tail rendering GFP protein susceptible to degradation by tail-specific proteases produced endogenously by the bacterium
(Keiler et al., 1996; Andersen et al., 1998). This fragment
was subsequently ligated into the BamHI and MscI sites
of pUFZ75. Sequencing of the resultant plasmid verified
that no mutations had occurred. The modified plasmid
was designated pUFZJLVA and used for Xac transformation as described above.

Pathogenicity and plasmid stability tests

Citrus medica or Citrus paradisi leaves were inoculated
with bacterial suspensions (103104 CFU mL)1) in phosphate buffer obtained from an overnight culture in Nutrient Broth (NB; Difco). Inoculations were performed as
previously reported using a 1 mL syringe without a needle by pressure infiltration of the bacterial suspension in
the abaxial surface of the leaf (Viloria et al., 2004).
Lesions were counted at 30 days after inoculation and
means calculated for each strain. Lesion counts for the
strains were compared by analysis of variance and means
were separated by StudentNewmanKeuls (SNK) multiple range test (P < 005).
Plasmid stability was determined by evaluation of plasmid presence in a bacterial culture without antibiotics by
assessing resistance to kanamycin (Km). Bacteria were
grown in liquid LB medium with no antibiotics. A 100 lL
aliquot of a bacterial suspension was adjusted by spectrophotometer to an absorbance of 01 and the suspension
plated onto LB agar with or without Km (50 lg mL)1)
once a day for 3 weeks. The number of colonies appearing on LB + Km was used to calculate the amount of
bacterial cells that retained pUFZ75 or pUFZJLVA
Plant Pathology (2011) 60, 977985

Xanthomonas survival on citrus leaf and fruit

plasmids. Population data obtained from the plates with

or without antibiotics were subjected to analysis of variance and the population means separated by the SNK
multiple-range test. Two different assays and three replicates were performed for each evaluation date.
In addition, bacteria were isolated from leaves previously inoculated with the transformed strains for each
evaluation date. Bacteria were isolated by comminuting
10 mg of leaf tissue per mL of sterile water and plating
serial dilutions of the extract onto LB agar plates with or
without Km (50 lg mL)1) up to 3 weeks after inoculation. Population data obtained from the plates with or
without Km were compared and statistically analysed as

Monitoring fluorescence from single colonies

To evaluate the stability of fluorescence in transformed
bacteria, two different approaches were followed. First,
transformed strains of Xac were streaked on LB + Km
plates and colonies of the bacteria containing native and
unstable GFP were visualized in time course experiments
using CLSM for strain MI or epifluorescence microscopy
for strain 306. Secondly, fluorescence distribution in single colonies of each transformed strain was evaluated by
CLSM. For the first evaluation, strains of Xac carrying
pUFZ75 and pUFZJLVA were streaked onto plates of
LB + Km medium. From 2 to 21 days of incubation at
26C, the green fluorescent phenotypes of single colonies
on plates were recorded as maximum projection images
by CLSM using a Leica TCS-SL confocal microscope with
objective HC PL FLUOTA 100 030 PHI (Leica Microsystems) or with a Leica DC 500 camera mounted on a
Leica DMRE epifluorescence microscope equipped with
an ebq 100 dc epifluorescent condenser and a 5 lens.
Images were acquired using Leica IM500 v12 software
(Leica Microsystems).
In addition, single colonies of Xac strains carrying
pUFZ75 and pUFZJLVA plasmids were analysed by
CLSM as above to determine fluorescence as a measure of
cell activity in the colony. Distribution of fluorescence in
colonies was determined for 48-h-old plate cultures.

Fluorescence and relative concentration of mRNA

from gfp in dead cells
The relationship between gfp mRNA and fluorescence
was evaluated in Xac by quantitative reverse transcription real-time PCR (qRT-PCR). Primers J-RTgfp1up
TG-3) were used to amplify mRNA from gfp and
GTTTCT-3) for the 16S rRNA gene which was used as
an internal reference gene.
Total RNA extraction was performed using the
RNeasy Mini kit (QIAGEN) with the RNAprotect Bacteria Reagent (QIAGEN). Five hundred microlitres of
Plant Pathology (2011) 60, 977985


bacterial suspension were mixed with 1 mL of

QIAGEN RNAprotect Bacteria Reagent and treated
and centrifuged according to kit instructions. The
resulting pellet was resuspended in 200 lL TrisEDTA
buffer (10 mM TrisHCl pH 75, 1 mM EDTA) with
lysozyme (1 mg mL)1) and incubated for 5 min at
room temperature before performing the protocol
according to the manufacturers instructions (QIAGEN). After RNA extraction, the suspension was treated with 2 U lL)1 of DNase (TURBO DNA free;
Ambion) at 37C for 30 min. RNA concentration was
determined and adjusted to 10 ng lL)1 by a spectrophotometer and the preparation stored at )80C until
further use. Reverse transcriptase reactions were carried out using the Ambion RETROscript kit by adding
10 lL of the extracted RNA in a reaction mixture containing 2 lL of 10 RT Buffer (Ambion), 1 lL MMLV
Reverse Transcriptase (100 U lL)1; Ambion), 1 lL
RNase inhibitor (10 U lL)1; Ambion), 4 lL dNTP mix
(25 mM each; Ambion) and 2 lL of 50 lM Random
Decamer primer (Ambion). The reverse transciptase
reaction was incubated at 42C for 1 h followed by
reverse transcriptase deactivation at 95C for 10 min.
qPCR reactions were carried out by adding 1 lL
cDNA in a reaction mixture containing 125 lL PCR
universal SYBR Green master mix (Applied Biosystems), 025 lL of 10 lM forward and reverse primers,
in a total volume of 25 lL. Some qPCR samples were
run with RNA template instead of cDNA to rule out
DNA contamination. The amplification profile for all
primers consisted of an initial activation step of
10 min at 95C, followed by 40 cycles at 95C for 15 s
and 60C for 1 min. Amplification and data analysis
were performed in a 7000 Sequence Detection System
(Applied Biosystems).
After qRT-PCR reactions, melting point analyses were
performed for the PCR products generated to confirm the
positive samples and the absence of non-specific products.
A relative quantification method was used to relate the
PCR signal (Ct value) of the target RNA from a treatment
to that of a nontreated control. The 2)DDCt method was
used to analyse the relative quantity of RNA (Livak
et al.,1995). The 16S rRNA gene was used to normalize
the quantification of the RNA targets for differences in
the amount of total RNA.

Survival of Xac on leaf and fruit surfaces

Bacterial suspensions of strains carrying pUFZ75 and
pUFZJLVA made in water or phosphate buffer
(108 CFU mL)1) were spray-inoculated onto leaves or
fruit surfaces and analysed under the CLSM using a Leica
TCS-SL confocal microscope and the objective HCX
PLAPO630X132 OIL UV (Leica Microsystems). Xac
was evaluated on the leaf surface of Swingle citrumelo
and the fruit surface of Sunburst tangerine (C. reticulata
Blanco hybrid) at 28C and greenhouse conditions,


J. Cubero et al.



Characterization of Xac strains transformed with

Fluorescence by Xac strains transformed with pUFZ75
and pUFZJLVA was observed using epifluorescence
microscopy and CLSM. Strains transformed with pUFZJLVA containing the unstable GFP protein fluoresced
less strongly than strains transformed with pUFZ75. No
auto-fluorescence was observed from non-transformed
strains, and fluorescence from gfp-transformed bacteria
was easily differentiated from plant auto-fluorescence by
CLSM. Strains 306 and MI of Xac transformed with
pUFZ75 and pUFZJLVA were as virulent as the respective wild type strains on C. medica and C. paradisi,
respectively. No significant differences (P < 005) in
lesion number occurred between wild type strains of Xac
or the transformed strains with plasmids pUFZ75 or
pUFZJLVA (Fig. 1). Plasmids pUFZ75 and pUFZJLVA
were confirmed to be stable after repeated transfers
in vitro and in planta in Xac.
To verify the relationship between gfp gene expression
and fluorescence, the relative amount of gfp mRNA in
culture cells was evaluated by quantitative reverse transcription real-time PCR and compared with the incidence
of fluorescent cells. Elevated gfp mRNA from strains containing pUFZ75 and pUFZJLVA plasmids was accompanied by strong fluorescence (Fig. 2). After 3 days
incubation, a small amount of gfp mRNA was detected
concurrent with strong fluorescence in all cells of the MIpUFZ75 strain compared to weak fluorescence in a few
cells of the MIpUFZJLVA strain (Fig. 2).
To evaluate fluorescence distribution in colonies,
transformed strains of Xac were streaked on culture
plates and observed with epifluorescence or CLSM
microscopy. Single colonies of the transformed strains
306pUFZ75 and MIpUFZ75 of Xac fluoresced strongly

1 day

2 days

3 days





Figure 2 Determination of gfp mRNA concentration and

fluorescence of MI strain of Xanthomonas citri subsp. citri
transformed with plasmids pUFZ75 (MIpUFZ75; GFP) and
pUFZJLVA (MIpUFJLVA; LVA) of a bacterial suspension kept at
room temperature for 3 days. The graph shows normalized gfp
mRNA amounts from suspensions after 1, 2 and 3 days (a) and the
confocal laser scanning microscopy images show fluorescence of
the suspensions after 1 day (two pictures on the left) and 3 days
(two pictures on the right) (b).

after 9 days and were still fluorescing after 21 days

(Fig. 3). In contrast, strain 306pUFZJLVA and
MIpUFZJLVA, containing the unstable GFP, did not
fluoresce after 4 days. Single colonies of a 2-day-old culture of strains containing plasmid pUFZ75 produced
homogeneous fluorescence, whereas fluorescence was
confined to the borders of colonies of Xac transformed
with pUFZJLVA (Fig. 4). This contrast in distribution
and intensity of fluorescence between strains confirmed
that only the most active cells located in the periphery of
the colony of the pUFZJLVA strains accumulated sufficient GFP to be detected. Cells in colonies of the pUFZ75
strain were uniformly fluorescent, irrespective of their
metabolic activity (Fig. 4).

Survival of Xac on the leaf and fruit surface

Figure 1 Mean number of citrus canker lesions per inoculation site

after pressure infiltration of wild type (wt) and transformed strains
with plasmids pUFZ75 (GFP) and pUFZJLVA (LVA). Xanthomonas
citri subsp. citri strains 306 (white) and MI (grey) were tested in
Citrus medica and Citrus paradisi, respectively. No significant
differences were obtained among the means (P < 005) according
to StudentNewmanKeuls multiple range test.

Citrus leaves sprayed with bacterial suspensions were

examined after different time periods in order to locate
the bacterial strains on the surface and to determine the
number of living Xac surviving on the leaf surface compared with the total number of Xac cells on the leaf, using
strains with native and unstable GFP.
Xac was evaluated on citrus leaves of plants grown
at 28C. Leaves were spray-inoculated with bacteria
suspended in water and the leaf surface examined by
CLSM during the 9 days after inoculation (Fig. 5).
After 3 days, leaves inoculated with suspensions of
Xac initially showed scattered fluorescent cells on the
leaf surface. A few fluorescent cells inoculated with the
unstable GFP strain were observed, and those were
Plant Pathology (2011) 60, 977985

Xanthomonas survival on citrus leaf and fruit






2 days

4 days

7 days

9 days

21 days

Figure 3 Epifluorescence images of single colonies of Xanthomonas citri subsp. citri (Xac) strain 306 (top two rows) and maximum projection
confocal laser scanning microscopy images of Xac strain MI (bottom two rows). Xac colonies were expressing native GFP protein (GFP:
306pUFZ75, MIpUFZ75) or unstable GFP protein (LVA: 306pUFZJLVA, MIpUFZJLVA). Fluorescence was evaluated after 2, 4, 7, 9 and
21 days on culture plates.



Figure 4 Confocal laser scanning microscopy images of single colonies of Xanthomonas citri subsp. citri strain MIpUFZ75 expressing native
GFP protein (a) or MIpUFZJLVA expressing unstable GFP protein (b). The main image represents a single section inside the colony. Two
lateral views (xz and yz planes) corresponding to cross sections of image series are shown below and on the right of the main image.

always in lower numbers compared to the strain with

native GFP (Fig. 5a,b). The inoculated strain with
native GFP fluoresced more strongly than the strain
with unstable GFP. After 7 and 9 days, aggregates
were detected on the leaf surface inoculated with either
native (Fig. 5c,e) or unstable (Fig. 5d,f) GFP strains.
Fluorescent cells in the aggregates of the unstable GFP
strain indicated that metabolically active bacteria were
present. Fluorescent cells in aggregates were mainly
located in the depressions between epidermal cells and
surrounding the stomata. Fluorescent cells were also
observed inside the stomata.
Plant Pathology (2011) 60, 977985

After inoculation of the fruit surface with a water suspension of Xac strain MI with native and unstable GFP
under greenhouse conditions, only a few scattered cells
initially established on the surface (Fig. 6a), and a small
proportion of those cells with unstable GFP fluoresced
(Fig. 6b). After 05 days, activity of the bacteria appeared
to rebound for both native (Fig. 6e) and unstable (Fig. 6f)
GFP strains. After 1 (Fig. 6i,j) and 2 (Fig. 6m,n) days, little
change in number and activity was observed compared to
05 days. However, after 3 days, bacterial aggregates
developed which contained numerous fluorescent cells
(Fig. 6q,r). After 7 days, aggregates of the native (Fig. 6u)


J. Cubero et al.









3 days

7 days

9 days

Figure 5 Evaluation of Xanthomonas citri subsp. citri survival on the leaf surface. Water suspension of the strains MIpUFZ75 (GFP) or
MIpUFZJLVA (LVA) were sprayed on the surface of Swingle citrumelo leaves. The samples were observed by confocal laser scanning
microscopy at 3, 7 and 9 days after inoculation. The main image of each picture represents a single section of a part of the leaf surface.
Two lateral views (xz and yz planes) corresponding to cross sections of image series are shown below and on the right of the main image.
Parts (a) to (f) refer to comments in the text.

and unstable (Fig. 6v) GFP strains on the fruit surface

were still evident, although locating the aggregates was
more difficult because of their patchy distribution.
Inoculation of Xac strain MI in phosphate buffer
resulted in a higher incidence of fluorescent cells on the
fruit surface than when inoculated in a water suspension.
Metabolically active cells were prevalent at 0, 05 and
1 day after inoculation of the native (Fig. 6c,g,k) and
unstable (Fig. 6d,h,l) GFP strains. After 2 and 3 days,
scattered fluorescent cells were observed for the native
GFP strain (Fig. 6o,s), but cells for the unstable GFP strain
were located only in aggregates (Fig. 6p,t). After 7 days,
aggregates of both native (Fig. 6x) and unstable (Fig. 6y)
GFP strains contained pockets of fluorescent cells, but
were found in more restricted areas on the fruit surface.

Knowledge of the survival and potential for multiplication of Xac on citrus plant surfaces is fundamental to
understanding the CBC disease cycle. The role of epiphytic bacteria in the epidemiology of the disease has
important implications for the quarantine regulations
currently in place that exclude canker-infected fruit from
markets without CBC because the fruit may harbour
residual levels of Xac on their surfaces (Graham et al.,
2004; Gottwald et al., 2009). Previous studies of the ability of Xac to survive on the surfaces of citrus tissues (Graham et al., 1987, 1989; Pruvost et al., 2002) were based

on detection of bacteria on culture media; however, interpretation of survival based on bacterial isolation is constrained. Non-xanthomonad, yellow-pigmented bacteria
are ubiquitous on citrus plants and may be incorrectly
counted as epiphytic Xac unless their identity is verified
(Graham et al., 2004; Gottwald et al., 2009). Bacteria
stressed by environmental factors such as temperature,
humidity or exposure to chemicals may be compromised
in their ability to multiply, resulting in slow or nongrowth of bacteria on culture media after isolation. This
stressed condition results in a VBNC condition described
for several bacterial species including Xac (Oliver, 2005;
Del Campo et al., 2009). The VBNC condition means
that bacteria may not be detected by conventional isolation methods and that other methodologies should be
used to confirm bacterial viability (Oliver, 2005).
For these reasons, the survival of Xanthomonas pathogens in situ on citrus fruit and leaves was studied to
directly assay their establishment and viability. GFP protein has been used for bacterial localization (Bokman &
Ward, 1981; Ward & Bokman, 1982) but is highly stable
and, therefore, inappropriate for assessment of bacterial
cell survival. The stability of GFP protein in Xac was confirmed when suspensions of dead cells fluoresced like viable and active cells. This result supports studies of other
bacteria such as Pseudomonas fluorescens, in which a
high proportion of the dead cells retained GFP fluorescence until cell membrane integrity was completely lost
(Lowder et al., 2000).
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Xanthomonas survival on citrus leaf and fruit































0 day

05 day

1 day

2 days

3 days

7 days

Figure 6 Evaluation of bacterial survival on the fruit surface. Water (W) or phosphate buffer (PB) suspensions of Xanthomonas citri subsp. citri
strains MIpUFZ75 (GFP) or MIpUFZJLVA (LVA) were sprayed on the surface of Sunburst tangerine fruit. The samples were shown by confocal
laser scanning microscopy immediately after inoculation (0 day) and after 05, 1, 2, 3 and 7 days. The main image of each picture represents
a single section of a part of the fruit surface. Two lateral views (xz and yz planes) corresponding to cross sections of image series are
shown below and on the right of the main image. Parts (a) to (y) refer to comments in the text.

To overcome the stability of native GFP, strains of Xac

expressing unstable GFP were constructed based on a previously described method (Andersen et al., 1998). Native
and unstable GFP strains of Xac were as pathogenic as
the wild strains and were verified to maintain the gfp plasmid through several generations in vitro or in planta. Distribution of fluorescent cells within bacterial colonies of
unstable GFP strains was related to greater metabolic
activity at the perimeter of the colony, whereas fluorescence was uniform in colonies of native GFP strains. In
old cultures of unstable GFP strains, low concentrations
of gfp mRNA were detected concurrent with little or no
fluorescence. Expression of mRNA is potentially a good
viability marker because of its short half-life (Sheridan
et al., 1998; Rigano et al., 2007). Since gfp is expressed in
a constitutive manner, absence or reduced levels of
mRNA indicates little or no metabolic activity. Fluorescence from the unstable GFP protein is indicative of metabolic activity and, consequently, VBNC bacteria should
fluoresce. Therefore, fluorescence of the bacterial cells
under CLSM was validated as a viability assay for unstable GFP strains. A recent study of Xac survival on the
plant surface by Rigano et al. (2007) reported bacterial
aggregates; however, the viability of the aggregate cells
was not determined. To assess bacterial viability in aggregates, unstable GFP was used to locate bacteria on leaf or
Plant Pathology (2011) 60, 977985

fruit surfaces and to define the conditions for their survival as previously determined for the plant pathogen,
Pseudomonas syringae (Lindow & Brandl, 2003).
When suspensions of the unstable GFP strains were
sprayed on surfaces, bacterial aggregates formed. Persistence of fluorescence in these aggregates confirmed the
presence of metabolically active cells, but these cells had
limited survival without osmotic protection from desiccation by phosphate buffer. After inoculation of bacteria in
water, scattered cells of the unstable strains on the leaf or
fruit surfaces rapidly lost fluorescence, i.e. viability. Only
cells in aggregates continued to fluoresce and maintain
viability. As previously described by Lindow & Brandl
(2003) for aggregates on leaves, bacteria were not randomly distributed but located primarily in depressions
surrounding stomata or in plant cell junctions. Thus,
aggregates presumably were restricted to sites on the fruit
or the leaf surfaces that conserved moisture and nutrients.
Phosphate buffer which increased bacterial viability may
have acted to mitigate cell desiccation by reducing matrix
and osmotic potential on the surface and facilitating
aggregate formation. The results confirm the model
described for Pseudomonas syringae which maintained
high populations on the leaf surface under moist conditions irrespective of aggregation, but after desiccation,
stressed populations were significantly reduced and


J. Cubero et al.

viable cells located primarily in aggregates (Monier &

Lindow, 2003).
Free moisture is essential for Xac dissemination and
the initial steps of the infection process (Dalla Pria et al.,
2006; Christiano et al., 2009) and, as shown in the present study, for survival as an epiphyte. Leaf wetness duration on plant surfaces may influence epiphytic survival by
affecting aggregate formation, because Xac requires
some time for cell adaptation to unfavourable conditions
such as desiccation. During this period, processes may be
activated that are involved in bacterial survival and or
the first steps of infection, such as chemotaxis, adhesion
or biofilm formation. Studies to elucidate these processes
and their regulation are currently underway.
The aggregates observed on leaf and fruit surfaces
resemble what have previously been described as biofilms, i.e. structures produced by living bacteria for survival under a variety of conditions (Danhorn & Fuqua,
2007). Biofilms not only play a key role in maintaining
bacterial viability on leaf and fruit surfaces but also function in the pathogen infection process (Karatan & Watnick, 2009). As such, biofilms on leaf and fruit surfaces
may play a key role in the disease cycle and control strategies for CBC. Bacterial cells in biofilms may be protected
from bactericide compounds used for disease control
(Monier & Lindow, 2004). Persistence of viable cells in
biofilms explains the occasional isolation of Xac from
surfaces of symptomless CBC exposed fruits, even after
rigorous disinfection of postharvest fruit with chlorine
and sodium ortho-phenylphenate (Golmohammadi
et al., 2007; Gottwald et al., 2009). Recently, Gottwald
et al. (2009) demonstrated that prewashing with a detergent was the most effective treatment for disinfection of
fruit infested with Xac. Detergents may disrupt aggregates, exposing Xac to disinfectants such as chlorine. The
persistence of biofilms and viable Xac cells within them
after washes and bactericidal treatments of fruit surfaces
needs to be established since they constitute a possible
inoculum source that may be transmissible. Methods
developed in this study can be used to follow the fate of
viable bacteria in biofilms under different environmental
stresses and ultimately to define the risk, if any, for Xac in
biofilms to multiply, disseminate and transmit CBC.

The research was supported by the Florida Citrus Production Research Advisory Council, FDOC contract
00079694, and Instituto Nacional de Investigaciones
Agrarias projects RTA2006-00149 and RTA200800048. We thank J.B. Jones for kindly providing plasmid
pUFZ75. We also thank D. Achor for her support with
the CLSM and E. Ferragud and M. Saez for their technical

Alexander E, Pham D, Steck TR, 1999. The viable-butnonculturable condition is induced by copper in Agrobacterium

tumefaciens and Rhizobium leguminosarum. Applied and

Environmental Microbiology 65, 37546.
Andersen JB, Sternberg C, Poulsen LK, Bjorn SP, Givskov M,
Molin S, 1998. New unstable variants of green fluorescent
protein for studies of transient gene expression in bacteria.
Applied and Environmental Microbiology 64, 22406.
Bloemberg GV, Wijfjes AHM, Lamers GEM, Stuurman N,
Lugtenberg BJJ, 2000. Simultaneous imaging of Pseudomonas
fluorescens WCS365 populations expressing three different
autofluorescent proteins in the rhizosphere: new perspectives for
studying microbial communities. Molecular Plant-Microbe
Interactions 13, 11706.
Bokman SH, Ward WW, 1981. Renaturation of aequorea greenfluorescent protein. Biochemical and Biophysical Research
Communications 101, 137280.
Byrd JJ, Xu HS, Colwell RR, 1991. Viable but nonculturable
bacteria in drinking water. Applied and Environmental
Microbiology 57, 8758.
Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC, 1994.
Green fluorescent protein as a marker for gene expression.
Science 263, 8025.
Cho JC, Kim SJ, 1999. Green fluorescent protein-based direct
viable count to verify a viable but non-culturable state of
Salmonella typhi in environmental samples. Journal of
Microbiological Methods 36, 22735.
Christiano RSC, Dalla Pria M, Jesus Jr WC, Amorim L, Bergamin
Filho A, 2009. Modelling the progress of Asiatic citrus canker on
Tahiti lime in relation to temperature and leaf wetness.
European Journal of Plant Pathology 124, 17.
Dalla Pria M, Christiano RCS, Furtado EL, Amorim L, Bergamin
Filho A, 2006. Effect of temperature and leaf wetness duration
on infection of sweet oranges by Asiatic citrus canker. Plant
Pathology 55, 65763.
Danhorn T, Fuqua C, 2007. Biofilm formation by plant-associated
bacteria. Annual Review of Microbiology 61, 40122.
Del Campo R, Russi P, Mara P et al., 2009. Xanthomonas
axonopodis pv. citri enters the VBNC state after copper
treatment and retains its virulence. FEMS Microbiology Letters
298, 1438.
Errampalli D, Leung K, Cassidy MB et al., 1999. Applications of
the green fluorescent protein as a molecular marker in
environmental microorganisms. Journal of Microbiological
Methods 35, 18799.
Ghezzi JI, Steck TR, 1999. Induction of the viable but nonculturable condition in Xanthomonas campestris pv. campestris
in liquid microcosms and sterile soil. FEMS Microbiology
Ecology 30, 2038.
Golmohammadi M, Cubero J, Penalver J, Quesada JM, Lopez
MM, Llop P, 2007. Diagnosis of Xanthomonas axonopodis pv.
citri causal agent of citrus canker, in commercial fruits by
isolation and PCR-based methods. Journal of Applied
Microbiology 103, 230915.
Gottwald TR, Timmer LW, McGuire RG, 1989. Analysis of
disease progress of citrus canker in nurseries in Argentina.
Phytopathology 79, 127683.
Gottwald TR, Graham JH, Richie SM, 1992. Relationship of leaf
surface populations of strains of Xanthomonas campestris pv.
citrumelo to development of citrus bacterial spot and persistence
of disease symptoms. Phytopathology 82, 62532.
Gottwald TR, Hughes G, Graham JH, Sun X, Riley T, 2001. The
citrus canker epidemic in Florida: the scientific basis of

Plant Pathology (2011) 60, 977985

Xanthomonas survival on citrus leaf and fruit

regulatory eradication policy for an invasive species.

Phytopathology 91, 304.
Gottwald T, Graham JH, Bock C et al., 2009. The epidemiological
significance of post-packinghouse survival of Xanthomonas citri
subsp. citri for dissemination of Asiatic citrus canker via infected
fruit. Crop Protection 28, 50824.
Graham JH, McGuire RG, Miller JW, 1987. Survival of
Xanthomonas campestris pv. citri in citrus plant debris and soil
in Florida and Argentina. Plant Disease 71, 10948.
Graham JH, Gottwald TR, Civerolo EL, McGuire RG, 1989.
Population dynamics and survival of Xanthomonas campestris in
soil in citrus nurseries in Maryland and Argentina. Plant
Disease 73, 4237.
Graham JH, Gottwald TR, Fardelmann D, 1990. Cultivar specific
interactions for strains of Xanthomonas campestris from Florida
that cause citrus canker and citrus bacterial spot. Plant Disease
74, 7536.
Graham JH, Gottwald TR, Cubero J, Achor DS, 2004.
Xanthomonas axonopodis pv. citri: factors affecting successful
eradication of citrus canker. Molecular Plant Pathology 5,
Grey BE, Steck TR, 2001. The viable but nonculturable state of
Ralstonia solanacearum may be involved in long-term survival
and plant infection. Applied and Environmental Microbiology
67, 386672.
Karatan E, Watnick P, 2009. Signals, regulatory networks, and
materials that build and break bacterial biofilms. Microbiology
and Molecular Biology Reviews 73, 31047.
Keiler KC, Waller PRH, Sauer RT, 1996. Role of a peptide tagging
system in degradation of proteins synthesized from damaged
messenger RNA. Science 271, 9903.
Lindow SE, Brandl MT, 2003. Microbiology of the phyllosphere. Applied and Environmental Microbiology 69, 1875
Livak KJ, Flood SJA, Marmaro J, Giusti W, Deetz K, 1995.
Oligonucleotides with fluorescent dyes at opposite ends provide
a quenched probe system useful for detecting PCR product and
nucleic acid hybridization. PCR Methods and Applications 4,
Lowder M, Unge A, Maraha N, Jansson JK, Swiggett J, Oliver JD,
2000. Effect of starvation and the viable-but-nonculturable state
on green fluorescent protein (GFP) fluorescence in GFP-tagged

Plant Pathology (2011) 60, 977985


Pseudomonas fluorescens A506. Applied and Environmental

Microbiology 66, 31605.
Manahan SH, Steck TR, 1997. The viable but nonculturable state
in Agrobacterium tumefaciens and Rhizobium meliloti. FEMS
Microbiology Ecology 22, 2937.
Monier JM, Lindow SE, 2003. Differential survival of solitary and
aggregated bacterial cells promotes aggregate formation on leaf
surfaces. Proceedings of the National Academy of Sciences of
the USA 100, 1597782.
Monier JM, Lindow SE, 2004. Frequency, size, and localization of
bacterial aggregates on bean leaf surfaces. Applied and
Environmental Microbiology 70, 34655.
Oliver JD, 2005. The viable but nonculturable state in bacteria.
Journal of Microbiology 43, 93100.
Pruvost O, Boher B, Brocherieux C, Nicole M, Chiroleu F, 2002.
Survival of Xanthomonas axonopodis pv. citri in leaf lesions
under tropical environmental conditions and simulated splash
dispersal of inoculum. Phytopathology 92, 33646.
Rigano LA, Siciliano F, Enrique R et al., 2007. Biofilm formation,
epiphytic fitness, and canker development in Xanthomonas
axonopodis pv. citri. Molecular Plant-Microbe Interactions
20, 122230.
Sheridan GEC, Masters CI, Shallcross JA, Mackey BM, 1998.
Detection of mRNA by reverse transcription PCR as an indicator
of viability in Escherichia coli cells. Applied and
Environmental Microbiology 64, 13138.
Stuurman N, Bras CP, Schlaman HRM, Wijfjes AHM, Bloemberg
G, Spaink HP, 2000. Use of green fluorescent protein color
variants expressed on stable broad-host-range vectors to visualize
rhizobia interacting with plants. Molecular Plant-Microbe
Interactions 13, 11639.
Viloria Z, Drouillard DL, Graham JH, Grosser JW, 2004.
Screening triploid hybrids of Lakeland limequat for resistance to
citrus. Plant Disease 88, 105660.
Ward WW, Bokman SH, 1982. Reversible denaturation of
aequorea green-fluorescent protein physical separation and
characterization of the renatured protein. Biochemistry 21,
Zhang Y, Callaway M, Jones JB, Wilson M, 2009. Visualization
of hrp gene expression in Xanthomonas euvesicatoria in the
tomato phyllosphere. European Journal of Plant Pathology
124, 37990.