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ABSTRACT
Duan, Y. P., Sun, X., Zhou, L. J., Gabriel, D. W., Benyon, L. S., and Gottwald, T. 2009. Bacterial
brown leaf spot of citrus, a new disease caused by Burkholderia andropogonis. Plant Dis.
93:607-614.
A new bacterial disease of citrus was recently identified in Florida and is here named bacterial
brown leaf spot (BBLS) of citrus. BBLS-infected citrus leaves from the field displayed circular,
brownish, flat lesions with slightly raised and water-soaked margins surrounded by a chlorotic
halo. Based on Biolog carbon source metabolic fingerprinting, fatty acid analysis, and sequence analysis of partial 16S rDNA, gyrB, and rpoD genes, the causal agent of the disease was
identified as Burkholderia andropogonis. Pathogenicity of these B. andropogonis isolates taken
from multiple citrus leaves with BBLS was tested by various inoculation methods on three species of citrus as well as on carnation, corn, and sorghum. All isolates infected carnation, corn,
and sorghum with varying degrees of pathogenicity. Variation among citrus isolates in pathogenicity was also observed in high titer (108 CFU/ml) inoculations of citrus leaves, ranging from
a hypersensitive-like response to canker-like lesions. When the inoculum concentration was low
(106 CFU/ml), only necrotic spots or small lesions slowly developed with all strains. Growth of
B. andropogonis in citrus was relatively slow, tissue wounding appeared necessary for symptom
appearance with many isolates, and field samples were recovered only after severe storms, indicating that this wide-host-range bacterium is a weak, opportunistic pathogen of citrus.
Burkholderia andropogonis was first described as the causal agent of stripe disease
of sorghum and leaf spot of velvet bean in
1911 (20); at that time, it was classified as
Pseudomonas andropogonis. Pseudomonas woodsii was also described, in the
same publication, as an important pathogen of carnation. These two pathogens
were reclassified to the genus Burkholderia based on DNA-rRNA hybridizations (13) and were shown to be synonymous based on phenotypic, genotypic, and
chemotaxonomic evidence (9,13). Symptoms of B. andropogonisinfected leaves
include brown, water-soaked, circular lesions with chlorotic halos. These lesions
can coalesce, resulting in severe blight or
leaf drop. When infected leaves are dissected in water, a stream of bacterial ooze
is observed. B. andropogonis is now
known to cause leaf spot, streaks, and
doi:10.1094 / PDIS-93-6-0607
This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. The American Phytopathological Society, 2009.
stripe on a wide variety of host plants covering 52 species from 15 families of unrelated monocotyledons and dicotyledons,
including ornamentals such as white clover, carnation (26), and bougainvillea (29),
and economically important crops such as
corn, coffee, and chick pea (15). Recent
reports show B. andropogonis to be a
causal agent of diseases of jojoba (10),
orchids (23), and golden cane palms (32).
It can also survive on weeds such as Johnsongrass and sudangrass (8).
Since 2002, on both residential and
commercial citrus plants, citrus leaves with
flat, circular, brownish lesions, with
slightly raised and water-soaked margins,
surrounded by a chlorotic halo have been
observed by citrus canker inspectors with
the Division of Plant Industry, Florida
Department of Agriculture and Consumer
Services (DPI-FDAC) (21). The disease
was observed only after storms that caused
a high degree of damage and wounding to
citrus trees. The flat lesions with slightly
raised margins appeared somewhat similar
to citrus canker (Xanthomonas citri pv.
citri) and were therefore sent to the DPIFDAC laboratory for further analysis and
identification. However, the lesions were
different from those of citrus canker disease, and were confirmed negative for X.
citri by polymerase chain reaction (PCR)
and enzyme-linked immunosorbent assay
(ELISA). Under the microscope, a bacterial stream was consistently observed from
607
Fig. 1. Field sample and phenotypic variations of citrus leaves inoculated with different isolates of Burkholderia andropogonis. A, Original field sample of
bacterial brown leaf spot (BBLS) on grapefruit. B, Grapefruit leaf inoculated with isolate 6269 at concentration 107 CFU/ml using infiltration. B was photographed 2 weeks after inoculation with 16 close-up. C, Key lime leaf inoculated with isolate 6269 at concentration 107 CFU/ml using needle puncture,
photographed 3 weeks after inoculation. D, Grapefruit leaf inoculated with isolate 6269 at concentration 107 CFU/ml using needle puncture, photographed 3
weeks after inoculation. E, Hypersensitive reaction (HR)-like and chlorosis response on grapefruit leaves inoculated with isolate 6177 at 108 CFU/ml (1 and
2) and 106 CFU/ml (3), respectively, photographed 4 days after inoculation. F, Canker-like lesion on grapefruit inoculated with isolate 6367 at concentration
108 CFU/ml (1) and 105 CFU/ml (2), respectively, and healthy control (3), photographed 2 weeks after inoculation.
608
Syn.
Species
Origin
Host
1894
6173
6177
6268
6269
6367
6368
6369
6370
6758
1068
7665
1976
D294
D502
DNA
GMI 1000
3213
1660
C340
1377
DH 5
XS2002-0001
XC2004-00267
XI2004-00506
XN2005-1111
XI2004-856
XI2005-0014
XC2005-0161
XC2005-0162
X2005-1008-2
XC2005-283
P1998-2547
P2006-15
B. andropogonis
B. andropogonis
B. andropogonis
B. andropogonis
B. andropogonis
B. andropogonis
B. andropogonis
B. andropogonis
B. andropogonis
B. andropogonis
B. andropogonis
B. andropogonis
B. andropogonis
B. glumaeb
B. glumaeb
B. gladiolib
Ralstonia solanacearum
Xanthomonas citri pv. citri
X. citri pv. citri
X. citri pv. aurantifolia
X. axonopodis pv. citrumelo
Escherichia coli
Guyana
Florida
Florida
ATCC 51302
Florida
New England Biolabs
Citrus sp.
C. sinensis
Citrus sp.
C. sinensis
Citrus sp.
Citrus sp.
Citrus paradisi
C. sinensis
C. reticulata
C. sinensis
Dianthus caryophyllus
Bougainvillea sp.
Sorghum vulgare
Rice
Rice
Tomato
Citrus
Citrus
Citrus
Citrus
a
b
Fig. 2. Lesions induced by Burkholderia andropogonis isolate 6367 and thin-section microscopy of the infected tissue. A, Lesion on sweet orange. B, Thin
section of a 2-week-old lesion on sweet orange. Note hypertrophy and absence of hyperplasia. C, Thin section of a healthy control leaf of sweet orange. D,
Lesion on grapefruit. E, Thin section of a 2-week-old lesion on grapefruit. Note hypertrophy and absence of hyperplasia. F, Thin section of a healthy control
leaf of grapefruit.
Plant Disease / June 2009
609
DNA analyzer (Applied Biosystems, Foster City, CA). Sequences were compared
with previously identified sequences in the
NCBI database using BLAST (2). The
sequences of 16S rDNA obtained in this
study were aligned with closely matched
sequences from the NCBI database using
the pileup function of the GCG Package
(Accelrys Inc., San Diego, CA) and adjusted manually with ClustalX version 1.8
(24). Phylogenetic trees were generated
with Treecon (27,28) using a neighborjoining method. Bootstrap analysis was
performed with 1,000 resamplings of the
DNA sequences to estimate the confidence
of the tree topology.
PCR-based genotyping and sequence
analysis of the PCR products. The genetic diversity of citrus isolates was analyzed using a single primer, DG04, which
targets an inverted terminal repeat sequence of X. citri pv. citri (11). The amplified DNA fragments generated from the
PCR reaction were separated on a 1.0%
agarose gel in TAE buffer, and visualized
with ethidium bromide staining. PCR
products from selected isolates were
cloned into the pCRII-TOPO cloning vector and identified by direct PCR amplification using M13 forward and reverse primers. The identified clones were grown in
LB broth overnight, and DNA was isolated
using a plasmid mini kit (Qiagen). DNA
sequencing of the clones was performed by
the DNA Sequencing Core Laboratory,
Interdisciplinary Center for Biotechnology
Research at the University of Florida. Putative functional homologues of the obtained
Table 2. Polymerase chain reaction (PCR) and sequencing primers used in this study
Primer
Target gene
Sequence (5-3)
Reference
fD1
rD1
LJ23f
LJ24r
LJ25f
LJ26r
DG04
16S rDNA
AGAGTTTGATCCTGGCTCAG
AAGGAGGTGATCCAGCC
GGGGGTGTCTTGCGTAAAC
ACACCAGCTTGTCCTTCGTCT
CGCCAAACGGATCGAGGAAGG
TCACCCAGCGGCAACACGAC
AAT CCC TGC CGA CCC TC
30
30
This study
This study
This study
This study
11
gyrB
rpoD
Invert repeats
Table 3. Pathogenicity tests on sweet orange, key lime, and grapefruit and selected known hosts of Burkholderia andropogonis following high level leaf
tissue infiltration inoculation with bacterial brown leaf spot (BBLS) isolates and selected strains of B. andropogonis
Grapefruit
Key lime
Isolate/strain
6173
6177
6268
6269
6367
6368
6369
6370
6758
1894
1068
1976
7665
a
b
Sweet orange
Ch+b
HR+++
Ch++
Ch++
C+++
HR+++
HR++
Ch++
HR+++
HR+++
HR+++
Ch++
HR+++
Infiltration
Ch++
HR+++
HR+++
C++
C++
HR+++
HR++
HR++
HR+++
HR+++
HR++
Ch+
HR+++
Needle-puncture
+
++
++
++
+
+
++
+
+
+
Infiltration
Ch++
HR+++
C++
C++
C++
Ch++
C++
C++
HR+++
HR+++
Ch++
HR+++
Ch+
Needle-puncture
Carnationa
Corna
Sorghuma
++
++
++
+
+
++
+
+
+
+++
+
++
+
+++
+++
++
++
+++
++
+++
+++
+++
+++
+
++
++
+++
+++
++
++
++
+++
+++
+++
+++
+
+
+++
+
+++
+
++
+
+++
+++
+
+++
++
610
Fig. 3. Population dynamics of Burkholderia andropogonis isolates 6269 and 6369 in grapefruit (Citrus paradisi). Data are means of three replications repeated twice (n = 6) standard error of the
mean.
Fig. 4. Dendrogram showing the relationships of Burkholderia andropogonis strains isolated from
citrus and other Burkholderia species based on the fatty acid analysis.
Plant Disease / June 2009
611
Fig. 5. Neighbor-joining phylogenetic placement of Burkholderia andropogonis and related plant pathogens. Analysis based on approximately 1,500-nt
sequences from positions 271543 (based on Escherichia coli numbering) of 16S rDNA with Agrobacterium tumefaciens as the outgroup taxon. Scale bar
represents 10% change. Numbers at nodes represent percentage of bootstrap resampling based on 1,000 replicates.
Table 4. Comparison of fatty acid analysis and gyrB, rpoD gene sequence analyses results for identification of Burkholderia andropogonis isolates
Isolate/strain
Biolog I.D.
6173
6177
6268
6269
6367
6368
6369
6370
6758
1894
1068
1976
7665
N/A
N/A
N/A
B. andropogonis
N/A
N/A
B. andropogonis
N/A
N/A
N/A
N/A
N/A
N/A
a
b
gyrB BLASTb
rpoD BLASTb
Fatty acid
sim indexa
Score
E-value
Acc. num.
Score
E-value
Acc. num.
N/A
N/A
0.739
0.696
0.692
0.908
0.934
0.734
N/A
N/A
N/A
N/A
N/A
1279
1279
1279
1279
1279
1279
1284
1279
1279
1279
1279
1279
1279
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
EU265649
EU265650
EU265651
EU265652
EU265653
EU265654
EU265655
EU265656
EU265657
EU265658
EU265659
EU265660
EU265661
1369
1369
1369
1369
1369
1369
1369
1369
1363
1369
1369
1369
1369
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
EU306162
EU306163
EU306164
EU265662
EU306165
EU306166
EU306167
EU306168
EU306169
EU306170
EU306171
EU306172
EU306173
A similarity index of fatty acid greater than 0.8 is considered an exact match.
All BLAST score values represent the highest match to B. andropogonis gyrB (GenBank accession no. AB190572) and rpoD (GenBank accession no.
AB190669).
612
Fig. 6. Polymerase chain reaction (PCR) test for the specificity of primer LJ23f/LJ24r designed from Burkholderia andropogonis gyrB gene. Lane M, 1-kb
DNA ladder; lanes 1-13, B. andropogonis 6173, 6177, 6268, 6269, 6367, 6368, 6369, 6370, 6758, 1068, 1894, ATCC23061, 7665; lane 14, Xanthomonas
axonopodis pv. citri 3213; lane 15, X. axonopodis pv. citri 1660; lane 16, X. axonopodis pv. citrumelo 1377; lanes 17-18, Burkholderia glumae D294, D502;
lanes 19-20, Burkholderia sp.; lane 21, Escherichia coli DH5; lane 22, Ralstonia solanacearum GMI 1000; lane 23, water control.
Fig. 7. Genetic diversity analysis of Burkholderia andropogonis isolates using polymerase chain reaction (PCR) amplification with DG04 primer. M, 1-kb
DNA marker (Promega, Madison, WI). Lanes 1-11, B. andropogonis isolates 1068, 1976, 6173, 6177, 6268, 6269, 6367, 6368, 6369, 6370, and 7665, respectively.
Plant Disease / June 2009
613
BBLS was discovered because these isolates were submitted by state inspectors as
citrus canker suspects. Thus, the lesions of
BBLS obviously can be confused with
those of citrus canker. Typical canker
symptoms include raised, erumpent lesions
with a water-soaked margin, and are histologically characterized by both hypertrophy and hyperplasia (5). BBLS elicits
small lesions that are flat with slightly
raised margins. Microscopically, B. andropogonis causes hypertrophy but does not
induce hyperplasia. The causal agent of
citrus bacterial spot, X. axonopodis pv.
citrumello, also causes flat, water-soaked
leaf spots on juvenile citrus (12,22), but
this nursery disease does not affect mature
trees. Although there are some symptom
similarities among these bacterial diseases,
BBLS lesions can be distinguished by a
reddish appearance on grapefruit (Fig. 1A)
and sweet orange. Since BBLS lesions on
citrus can be confused with atypical lesions of citrus canker, it is important for
inspectors and growers to be informed
regarding this new bacterial disease of
citrus in order to limit confusion. Also due
to the difficulty in differentiating BBLS
from atypical citrus canker and bacterial
spot by symptomology, serological and
molecular tools are necessary to aid in
diagnosis. The primers DG04 and those
that target the gyrB and rpoD genes of B.
andropogonis developed from this study
may be used for future PCR detection and
diagnosis.
ACKNOWLEDGMENTS
We are thankful to Debbie Jones and Lisa Jones,
Florida Department of Agriculture and Consumer
Services, Division of Plant Industry, and Kimberly
Poole, USDA-ARS-USHRL, for their excellent
technical support in this research.
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repeats that determines pathogenicity on citrus,
but none determine host-range variation. Mol.
Plant-Microbe Interact. 20:934-943.
2. Altschul, S. F., Gish, W., Miller, W., Myers, E.
W., and Lipman, D. J. 1990. Basic local
alignment search tool. J. Mol. Biol. 215:403410.
3. Bouzar, H., Jones, J. B., and Hodge, N. C.
1993. Differential characterization of Agrobacterium species using carbon-source utilization
patterns and fatty acid profiles. Phytopathology 83:733-739.
4. Bradbury, J. 1986. Guide to Plant Pathogenic
Bacteria. CABI Publishing, Wallingford, UK.
5. Brunings, A. M., and Gabriel, D. W. 2003.
Xanthomonas citri: Breaking the surface. Mol.
Plant Pathol. 4:141-157.
6. Burkholder, W. H., and Guterman, C. E. F.
1935. Bacterial leaf spot of carnations. Phytopathology 25:114-120.
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20.
21.
22.
23.
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