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Doi: 10.1111/j.1365-3059.2011.02450.x
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
Introduction
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.
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;
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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
(CLSM).
Species
Origin
Characteristic
306
MI
306pUFZ75
306pUFZJLVA
MIpUFZ75
MIpUFZJLVA
Xac
Xac
Xac
Xac
Xac
Xac
Brazil
USA
306
306
MI
MI
Wild type
Wild type
Native GFPa
Unstable GFP
Native GFP
Unstable GFP
a
Strains that express native (stable) or unstable green fluorescent
proteins (GFP) harbour pUFZ75 and pUFZJLVA plasmids,
respectively.
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
(5-CTC TCG GAT CCA TTA AGC TAC TAA AGC
GTA GTT TTC GTC GTT TGC TGC AGG CCT TTT
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.
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Results
(a)
1 day
2 days
3 days
(b)
GFP
LVA
GFP
LVA
981
306
GFP
306
LVA
MI
GFP
MI
LVA
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.
(a)
(b)
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.
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)
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(a)
(c)
(e)
GFP
(b)
(d)
(f)
LVA
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.
Discussion
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).
Plant Pathology (2011) 60, 977985
(u)
(q)
(m)
(i)
(e)
(a)
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GFP
(v)
(r)
(n)
(j)
(f)
(b)
LVA
(x)
(s)
(o)
(k)
(g)
(c)
GFP
PB
(y)
(t)
(p)
(l)
(h)
(d)
LVA
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.
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
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Acknowledgements
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
assistance.
References
Alexander E, Pham D, Steck TR, 1999. The viable-butnonculturable condition is induced by copper in Agrobacterium
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