The General Secretory Pathway of Burkholderia gladioli pv. agaricicola BG164R Is Necessary for Cavity Disease in White Button Mushrooms

Abstract

Cavity disease in white button mushrooms is caused by Burkholderia gladioli pv. agaricicola. We describe the isolation and characterization of six mutants of the strain BG164R that no longer cause
this disease on mushrooms. The mutations were mapped to genes of the general secretory pathway (GSP). This is the first report
of the association of the type II secretion pathway with a disease in mushrooms. Phenotypes of the six avirulent mutants were
the following: an inability to degrade mushroom tissue, a highly reduced capacity to secrete chitinase and protease, and a
reduced number of flagella. Using these mutants, we also made the novel observation that the factors causing mushroom tissue
degradation, thereby leading to the expression of cavity disease, can be separated from mycelium inhibition because avirulent
mutants continued to inhibit the growth of actively growing mushroom mycelia. The GSP locus of B. gladioli was subsequently cloned and mapped and compared to the same locus in closely related species, establishing that the genetic
organization of the gsp operon of B. gladioli pv. agaricicola is consistent with that of other species of the genus. We also identify the most common indigenous bacterial
population present in the mushroom fruit bodies from a New Zealand farm, one of which, Ewingella americana, was found to be an apparent antagonist of B. gladioli pv. agaricicola. While other investigators have reported enhanced disease symptoms due to interactions between endogenous
and disease-causing bacteria in other mushroom diseases, to the best of our knowledge this is the first report of an antagonistic
effect.

Full text

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 2006, p. 3558–3565 Vol. 72, No. 5
0099-2240/06/$08.00⫹0 doi:10.1128/AEM.72.5.3558–3565.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
The General Secretory Pathway of Burkholderia gladioli pv. agaricicola
BG164R Is Necessary for Cavity Disease in White Button Mushrooms
Piklu Roy Chowdhury† and Jack A. Heinemann*
School of Biological Sciences, University of Canterbury, Christchurch, New Zealand
Received 26 September 2005/Accepted 26 February 2006
Cavity disease in white button mushrooms is caused by Burkholderia gladioli pv. agaricicola. We describe the
isolation and characterization of six mutants of the strain BG164R that no longer cause this disease on
mushrooms. The mutations were mapped to genes of the general secretory pathway (GSP). This is the first
report of the association of the type II secretion pathway with a disease in mushrooms. Phenotypes of the six
avirulent mutants were the following: an inability to degrade mushroom tissue, a highly reduced capacity to
secrete chitinase and protease, and a reduced number of flagella. Using these mutants, we also made the novel
observation that the factors causing mushroom tissue degradation, thereby leading to the expression of cavity
disease, can be separated from mycelium inhibition because avirulent mutants continued to inhibit the growth
of actively growing mushroom mycelia. The GSP locus of B. gladioli was subsequently cloned and mapped and
compared to the same locus in closely related species, establishing that the genetic organization of the gsp
operon of B. gladioli pv. agaricicola is consistent with that of other species of the genus. We also identify the
most common indigenous bacterial population present in the mushroom fruit bodies from a New Zealand farm,
one of which, Ewingella americana, was found to be an apparent antagonist of B. gladioli pv. agaricicola. While
other investigators have reported enhanced disease symptoms due to interactions between endogenous and
disease-causing bacteria in other mushroom diseases, to the best of our knowledge this is the first report of an
antagonistic effect.
Burkholderia gladioli,a␤-proteobacter, was initially identi-
fied as a pathogen of gladiolus (23). It was subsequently asso-
ciated with diseases in other plants, such as onions, iris, freesia,
dendrobium, cymbidium, tulip, green gram, and rice (22). Dis-
ease symptoms in the different plant hosts varied from the
spotting of foliar parts to scabbing and rotting of storage tis-
sues. In the last decade, different strains of B. gladioli have
been demonstrated to have the ability to infect animals, in-
cluding humans, causing food poisoning and severe pulmonary
infections in cystic fibrosis and other immunocompromised
human patients (11, 19).
B. gladioli pv. agaricicola is an important pathogen in the
mushroom industry. It causes soft rotting symptoms on a num-
ber of commercially important mushrooms, such as Lentinula
edodes,Pleurotus ostreatus,Flammulina velupies,Pholiota nameko,
Hypsizygus marmoreus, and Grifola frondos in Japan and on
different cultivated Agaricus species in New Zealand and Eu-
rope (14). While soft rot appears to be less prevalent than most
of the other mushroom diseases and is only sporadically re-
ported from farms in the United Kingdom and other European
countries (9), when it occurs, it can cause devastating effects
within very short periods of time. Thus, B. gladioli pv. aga-
ricicola is now considered to be a pathogen that has the
potential to cause significant crop losses in the mushroom
industry (9, 14).
Cavity disease was first reported in 1992 (13). The causal
microorganism was initially identified as Pseudomonas cepacia.
The strain, designated CANU-PMS164, was isolated from New
Zealand mushrooms with symptoms ranging from mild lesions
to deep pitting (13), and it was shown to inhibit the growth of
mushroom mycelia in vitro (14). Strain CANU-PMS164 rap-
idly degraded mushroom sporocarps, which resulted in marked
tissue damage within 72 h of infection. Although cavity disease
was originally thought to be a novel disease (18), it was later
reclassified (as reported by Gill and Tsuneda [14]) as “rapid
soft rot disease of edible mushroom.” The causative agent was
subsequently renamed Pseudomonas gladioli pv. agaricicola
(14) and is now known as B. gladioli pv. agaricicola following
the new genus nomenclature of members of the “pseudomallei
group” proposed by Yabucchi et al. (35) and accepting the
proposal of Lincoln et al. (21) that assigns the mushroom soft
rotting bacteria to a third pathovar, “agaricicola” (21, 35).
In this study, we identify four genes necessary for the viru-
lence of B. gladioli pv. agaricicola BG164R, the causative agent
of a mushroom soft rot disease (21), also described as cavity
disease of the white button mushroom Agaricus bitorquis (13).
Avirulent mutants of B. gladioli pv. agaricicola were generated
to answer three fundamental questions arising from previous
work. First, what are the genes essential for the expression of
cavity disease symptoms? Second, are the virulence factors
necessary for cavity disease also required to inhibit mycelial
growth? Last, why is there such a marked variation in disease
severity? In other words, are environmental factors responsible
for this variation, or is the differential intensity of disease an
attribute of multiple pathovars that exhibit different but over-
lapping symptoms? We anticipated that identification of the
virulence genes and investigating conditions related to disease
* Corresponding author. Mailing address: School of Biological Sci-
ences, University of Canterbury, Private Bag 4800, Christchurch, New
Zealand. Phone: 64 3 364 2500. Fax: 64 3 364 2590. E-mail: jack
.heinemann@canterbury.ac.nz.
† Present address: Bioprotection and Ecology Division, P.O. Box 84,
Lincoln University, Christchurch, New Zealand.
3558

expression would possibly provide an explanation for the vari-
ation in disease severity and explain why the disease is so
infrequently reported.
Our findings reconcile the confusing history associated with
the observed variation in the intensity of disease expression by
the causative agent and advance the understanding of viru-
lence at a molecular level. The ability of B. gladioli pv. agar-
icicola to inhibit mushroom mycelia has also always been as-
sociated with its ability to cause cavity disease (14). From these
observations, it has been thought that the pathogen produces
both hypha-degrading enzymes and toxins and that cavity dis-
ease symptoms are a combined effect of the toxin and the
enzymes. The avirulent mutants are affected in the ability to
secrete some virulence factors required for the symptoms of
cavity disease yet retain inhibitory activity toward fungal my-
celia.
(This research was conducted by P. Roy Chowdhury in ful-
fillment of the requirements for a Ph.D. from the University of
Canterbury, Christchurch, New Zealand, 2004.)
MATERIALS AND METHODS
Strains and plasmids. The main characteristics of the strains, plasmids, and
cosmids used are listed in Table 1. All bacteria were grown in Luria-Bertani (LB)
medium in either liquid broth or solid agar (24). B. gladioli,Pseudomonas
aureofaciens,Burkholderia cepacia,Pseudomonas putida,Pseudomonas fluorescens,
Serratia entomophila,and Ewingella americana were cultured at 30°C, while
Escherichia coli strains were grown at 37°C. When required, plates were supple-
mented with the following: ampicillin, 100 ␮gml
⫺1
; chloramphenicol, 25 ␮g
ml
⫺1
; gentamicin, 30 ␮gml
⫺1
; kanamycin, 50 ␮gml
⫺1
; nalidixic acid, 15 ␮g
ml
⫺1
; rifampin, 50 ␮gml
⫺1
; tetracycline 15 ␮gml
⫺1
; and X-Gal (5-bromo-4-
chloro-3-indolyl-␤-D-galactopyranoside), 25 ␮gml
⫺1
.
Mushroom bioassay. The bioassay system was modified from the basic tech-
nique of Gandy (12). In routine assays, fresh mushrooms, supplied by Meadow
Mushrooms (Christchurch, New Zealand), were surface sterilized, cut into slices
of approximately 1.5 to 2 mm thick under sterile conditions, immersed immedi-
ately in ice-cold distilled water (to prevent tissue browning), and transferred to
plastic lunch boxes lined with wet UV-sterilized paper towels. Either fixed vol-
umes of the test bacterial suspensions or single colonies transferred by toothpicks
were used as inocula and incubated for 16 h at 30°C. Within that time frame,
wild-type bacteria caused distinct holes of approximately 4 to 5 mm in diameter
on the mushroom pieces, while mutants failed to show any similar symptoms.
For screening of mutants, 10,000 transconjugants arising from 66 independent
experiments carried out in 16 attempts were screened for the transposition of the
mini-Tn5, resulting in “no-cavity” phenotypes.
Transposon mutagenesis. The transposon mini-Tn5KmlacZ2 (7), borne by
pUTZ2 (Table 1) and maintained in E. coli S17-1 ␭pir (32), was introduced into
the recipient, BG164R, by conjugation. The conjugation procedure was as fol-
lows. The donor, E. coli S17-1 ␭pir, and the recipient, BG164R, were grown in
liquid broth supplemented with antibiotics for 18 h at the respective tempera-
tures mentioned above. One milliliter of donor and recipient bacteria, harvested
by centrifugation (2,000 ⫻gfor 3 min in a Bio-Rad mini benchtop centrifuge),
was washed with fresh LB broth to remove antibiotics. The recipient was heat
shocked at 43°C for 15 min, mixed with the donor in a ratio of 4:1 (1 ml of
recipient to 250 ␮l of donor), and concentrated to 400 ␮l. Aliquots of 200 ␮l were
applied on a sterilized filter paper placed on top of LB plates as a single droplet
without any antibiotics and incubated at 30°C for 4 h. The mating mixture was
recovered from filter paper by washing the filter with 1.5 ml of fresh LB broth;
the mixture was concentrated to 200 ␮l, and 100-␮l aliquots were spread on
plates supplemented with rifampin, kanamycin, and chloramphenicol (15 ␮g
ml
⫺1
) and incubated at 30°C for 36 h to select for BG164R transconjugants.
Screening and isolation of avirulent mutants. Transconjugants arising from
independent matings were screened for mutations with an avirulent no-cavity
phenotype in the mushroom bioassay. The number of mutations resulting in at
least one auxotrophy was determined by transferring transconjugants to minimal
agar plates.
Phenotyping. All phenotypes were determined in comparison to wild-type
BG164R.
Morphological changes in mutants were assessed using transmission electron
microscopy. Bacterial cells from a 24-h incubation on LB plates were stained with
1% phosphotungstic acid and observed under bright-field conditions with either
a Hitachi H-600 electron microscope or a JEOL JEM-1200EX electron micro-
scope.
Motility, or the capacity of mutant bacteria to move away from the point of
inoculation on the motility agar plates (0.1% [wt/vol] Bacto tryptone, 0.05%
[wt/vol] yeast extract, and 0.5% [wt/vol] NaCl) supplemented with 0.3% agar, was
monitored to assess any change in this ability.
Any alteration in the capacity of mutants to interact with the host tissue was
monitored with scanning electron microscopy using a Leica s440 SEM, and tissue
samples were prepared according to Atkey et al. (2).
The ability of the mutants to produce chitinase was monitored on 1% chitin
extract plates (19) incubated at 30°C for 4 days and then scored in terms of the
clearing of colloidal chitin around the colonies spotted on the plates. Protease
secretion was studied by transferring the test colonies onto 0.1% skim milk agar
plates and looking for the capacity of the strains to clear casein around the test
colonies.
DNA manipulation and cloning. The genomic DNA was prepared by the
guanidium thiocyanate method (27), and plasmid DNA was prepared by the
standard alkaline lysis method (29). For sequencing, plasmid DNA isolated using
the alkaline lysis method was further purified by lithium chloride precipitation
(29). DNA samples were routinely quantified using a deuterium lamp LKB
Ultraspec Plus spectrophotometer. Restriction digestions were set up using the
manufacturer’s recommendations, analyzed by agarose gel electrophoresis (29),
and visualized on a Sigma T2210 UV transilluminator. Gels were photographed
using a Kodak Electrophoresis Documentation and Analysis System 120.
For regular cloning, vector DNA was dephosphorylated using calf intestinal
phosphatase (Bo¨ehringer Mannheim) prior to ligation with the inserts following
standard conditions described by Sambrook et al. (29).
Shotgun clones of the mutated genes in BG164R mutants were isolated by
transforming DH5␣electrocompetent cells (36) with ligation mixtures contain-
ing SalI-digested genomic DNA and the vector (pBluescript KS
⫹
). Desired
clones were isolated by selecting colonies resistant to ampicillin (on vector) and
kanamycin (in the transposon). The fact that each mutant had a single transpo-
son insertion was confirmed by a Southern hybridization, in which genomic DNA
of the mutants was hybridized to a probe (Table 1) of the kanamycin resistance
(nptII) gene originally isolated from Tn5(data not shown). SalI-generated DNA
fragments of variable sizes (Table 1) spanning the transposon insertion sites
in the genome of each of the avirulent mutants were “shotgun” cloned into
pBluescript KS⫹.
Construction of a genomic library. A genomic library of BG164R in cosmid
pLAFR
3
(33) was constructed and maintained in E. coli DH5␣lacking recA
activity (16) according to the method outlined by Fleischman et al. (10), except
for the extraction of genomic DNA, which was done according to the guanidium
thiocyanate method (27). Ligated DNA was packaged in vitro into phage heads
using Packagene Lambda DNA Packaging System packaging extract (Promega),
following the manufacturer’s protocol (Promega Technical Bulletin 005).
Cosmid isolation by colony hybridization. A total of 3,200 individual clones
representing the B.gladioli pv. agaricicola library were screened by colony hy-
bridization (24) with a 1.5-kb EcoRI/SalI probe constructed from pSPRC12. A
random primed DNA labeling kit from Bo¨ehringer Mannheim was used to label
50 ng of the probe DNA with
32
P, following the manufacturer’s protocol. Ap-
proximately 200,000 cpm of [␣-P
32
]dCTP-labeled probe was used, and mem-
branes were hybridized to the probes for 16 h following the standard techniques
of Sambrook et al. (29).
Cosmid mapping by Southern hybridization. The 1.5-kb SalI-EcoRI fragment
from the clone pSPRC12, which served as the gspF probe, had a single BamHI
site and hybridized to two bands, 2.3 kb and 7.2 kb (see Fig. 3B), in the BamHI-
digested cosmid DNA. Hence, these two bands were placed contiguously on the
physical map of the cosmid. The gspK probe, constructed from pSPRC88, hy-
bridized with a 6.5-kb BamHI fragment, as predicted from the restriction map.
Complementation of mutants. Avirulent mutants were transformed with the
cosmid by triparental matings with an E. coli DH5␣donor harboring the cosmid
in the presence of a helper plasmid pRK2013 and were maintained in E. coli
HB101 (3) following Carruthers et al. (4). The resultant transconjugants were
selected from plates supplemented with appropriate antibiotics, purified, and
checked for complementation using the assays described above.
DNA sequencing. The clones were sequenced with IRD41-labeled T3 and T7
primers with a SequiTherm Long-Read Sequencing Kit LC from Epicenter
Technologies in a Li-COR automated sequencer following the manufacturer’s in-
structions, exploiting the dideoxy chain termination method of Sangers et al. (30).
Sequences were analyzed using DNAMAN, version 4.02 (Lyonnon BioSoft). The
VOL. 72, 2006 GSP AND CAVITY DISEASE 3559

BLASTN and BLASTX (www.ncbi.nlm.nih.gov/BLAST) programs were used to
search for similar sequences in public databases (1).
Antifungal assay. Colonies of test bacteria were streaked with a sterile loop
approximately 1 cm away from 7-day-old actively growing mushroom mycelia on
compost malt agar plates (15) and further incubated for 5 days at 23°C.
Mutant rescue experiments. Overnight cultures of BG4-12Cos, BG12-88Cos,
and BG15-40Cos were diluted 100-fold and grown at 30°C in LB broth in the
absence of any antibiotic selection. Aliquots (100 ␮l each) were aseptically
removed from the cultures at 24-h intervals for 3 days, serially diluted 10
6
-fold,
spread on LB plates, and incubated for a further 24 h. Individual colonies arising
TABLE 1. List of strains, plasmids, cosmids, and vectors used
Name Genotype or description
a
Reference
or source
Strains
B.gladioli pv. agaricicola
BG164 Wild type; Cav
⫹
Af
⫹
Rif
s
Prot
⫹
18
BG164R Spontaneous Rif
r
mutant of BG164; Cav
⫹
Af
⫹
Rif
r
Prot
⫹
This study
BG4-12 BG164R gspF::mini-Tn5KmlacZ2 Rif
r
Km
r
Cav
⫺
Af
⫹
Prot
⫺
This study
BG12-88 BG164R gspK::mini-Tn5KmlacZ2 Rif
r
Km
r
Cav
⫺
Af
⫹
Prot
⫺
This study
BG12-147 BG164R gspK::mini-Tn5KmlacZ2 Rif
r
Km
r
Cav
⫺
Af
⫹
Prot
⫺
This study
BG15-40 BG164R gspE::mini-Tn5KmlacZ2 Rif
r
Km
r
Cav
⫺
Af
⫹
Prot
⫺
This study
BG15-87 BG164R gspD::mini-Tn5KmlacZ2 Rif
r
Km
r
Cav
⫺
Af
⫹
Prot
⫺
This study
BG16-787 BG164R gspE::mini-Tn5KmlacZ2 Rif
r
Km
r
Cav
⫺
Af
⫹
Prot
⫺
This study
BG4-12Cos BG4-12 complemented with cosmid pCosGSP; Rif
r
Kan
r
Tet
r
Cav
⫹
This study
BG12-88Cos BG12-88 complemented with cosmid pCosGSP; Rif
r
Kan
r
Tet
r
Cav
⫹
This study
BG15-40Cos BG15-40 complemented with cosmid pCosGSP; Rif
r
Kan
r
Tet
r
Cav
⫹
This study
BG-LAF
3
BG164R with pLAFR
3
; Rif
r
Tet
r
Cav
⫹
Prot
⫹
This study
BG4-12LAF
3
BG4-12 with pLAFR
3
; Rif
r
Kan
r
Tet
r
Cav
⫺
Prot
⫺
This study
E.coli
S17-1 ␭pir thi pro hsdR hsdM
⫹
⌬recA RP4-2::TcMu
⫺
Km::Tn732
DH5␣supE44 ⌬lacU169 (␾80lacZ⌬M15)hsdR17 thi-1 relA1 recA1 16
HB101 supE44 hsdS20 (r
B
⫺
m
B
⫺
)recA13 ara-14 rspL20 proA2lacY1 galK2 xyl-5 myl-13
P.aureofaciens
PA147-2 Wild type; Af
⫹
Bfm
⫹
Rif
r
Cm
r
4
PAE639 PA147-2; yeiJ::mini-Tn5KmlacZ2 Af
⫹
Bfm
⫺
Fla
⫺
Mot
⫺
Rif
r
Km
r
H. K. Mahanty
P.cepacia B111 Wild type; Chi
⫺
A. L. J. Cole
P. putida Wild type; Prot
⫺
H. K. Mahanty
S.entomophila A
1
MO
2
Derivative of A1 wild type; Amp
r
Chi
⫹
Path
⫹
36
Plasmids and cosmids
pUTZ2 pUT containing mini-Tn5KmlacZ2 7
pSPRC12 8.5-kb SalI fragment containing gspF::mini-Tn5KmlacZ2 from BG4-12 in
pBluescript KS⫹;Ap
r
Kan
r
This study
pSPRC40 5.4-kb SalI fragment containing gspE::mini-Tn5KmlacZ2 from BG15-40 in
pBluescript KS⫹;Ap
r
Kan
r
This study
pSPRC87 6.0-kb SalI fragment containing gspD::mini-Tn5KmlacZ2 from BG15-87 in
pBluescript KS⫹;Ap
r
Kan
r
This study
pSPRC88 5.2-kb SalI fragment containing gspK::mini-Tn5KmlacZ2 from BG15-88 in
pBluescript KS⫹;Ap
r
Kan
r
This study
pSPRC147 5.3-kb SalI fragment containing gspK::mini-Tn5KmlacZ2 from BG12-147 in
pBluescript KS⫹;Ap
r
Kan
r
This study
pSPRC787 5.4-kb SalI fragment containing gspE::mini-Tn5KmlacZ2 from BG12-787 in
pBluescript KS⫹;Ap
r
Kan
r
This study
pROBE 1.5-kb NotI fragment containing the kanamycin gene from mini-Tn5KmlacZ2
blunt-end cloned into the EcoRV site of pBluescript KS⫺;Ap
r
S. R. Giddens
pCosGSP 23.4-kb Sau3AI partial fragment containing the GSP gene cluster from BG164R
into pLAFR
3
This study
pGSP ME H-H16.7 16.7-kb HindIII subclone from pCosGSP in pME6001; Gen
r
This study
pGSP ME H-H2 2-kb HindIII subclone from pCosGSP in pME6001; Gen
r
This study
pGSP ME H-E1.7 1.7-kb HindIII-EcoRI subclone from pCosGSP in pME6001; Gen
r
This study
pGSP KS B-B2.2 2.2-kb BamHI subclone from pCosGSP in pBluescript KS⫹;Ap
r
This study
pGSP KS B-B7.2 7.2-kb BamHI subclone from pCosGSP in pBluescript KS⫹;Ap
r
This study
Vectors
pBluescript ColE1 ori lacZ␣/KS polylinker; T3/T7; Ap
r
(KS, M13⫹)Stratagene
pLAFR3 pRK290 derivative; RP4(IncP-1) ori ␭cos; pUC9 multicloning site and lacZ␣Tc
r
33
a
Cav, cavity disease; Prot, protease; Chi, chitinase; Bfm, biofilm; Af, antifungal.
3560 ROY CHOWDHURY AND HEINEMANN APPL.ENVIRON.MICROBIOL.

on the plates were screened for kanamycin (on transposon) and tetracycline (on
pCosGSP) susceptibilities, indicating loss of both the transposon and the cosmid.
All such kanamycin- and tetracycline-susceptible colonies were tested using the
mushroom bioassay and scored for strains exhibiting wild-type phenotypes, sug-
gesting the recreation of the wild-type strain by a double-crossover event or
allelic exchange between the mutated genes and the complementing fragment of
DNA present in pCosGSP. Recreation of wild-type strains was later confirmed
by Southern blot analysis.
RESULTS
Selection of avirulent mutants. Following mini-Tn5transpo-
son mutagenesis, 10,000 individual BG164R transconjugants
were screened in the modified mushroom bioassay for the
cavity-forming phenotype indicative of virulence. On average,
1.5% of transconjugants were auxotrophic mutants. Six pro-
totrophic but no-cavity-forming mutants were isolated (Fig. 1
and Table 1). Generation times of the mutants and parental
strain were estimated by measuring the change in optical den-
sity (at 600 nm) of the cultures growing in LB medium over a
12-h period. The doubling time of both the wild type and the
mutants was approximately 75 min (data not shown).
Phenotypes of the BG mutants are consistent with a defect
in protein secretion. The BG mutants did not degrade mush-
rooms, had different numbers of flagella, did not secrete pro-
tease, and had a highly reduced capacity to secrete chitinase
(Fig. 2). In contrast to the five polar flagella in the wild-type
strain, four mutants (BG4-12, BG12-147, BG15-87, and BG16-
787) out of the six had a single flagellum. Two flagella were
visible in transmission electron micrographs of the other two
mutants, BG12-88 and BG15-40 (data not shown). Likewise,
the mutants and the wild type (BG164R) looked the same on
plates with various proportions of agar, ranging from 0.2%
to 0.6%, which indicated that there were no differences in
motility.
The six BG164R mutants formed a continuous sheath on the
surface of the mushroom pieces, with occasional breaks under
which intact mushroom hyphae could be seen in scanning elec-
tron micrographs (data not shown). In comparison, mushroom
slices inoculated with the wild-type bacteria remained attached
to the skeletal remains of degraded hyphal filaments. Thus,
under the standardized assay conditions, mutants failed to
degrade the hyphae of mushrooms. Both the secreted casein-
degrading protease and chitinase activities were highly reduced
in the six BG164R mutants, which strongly suggested that the
mutations affected the protein secretion pathway.
Cloning and identification of transposon-tagged genes in
cavity disease mutants. To identify the mutated genes, DNA
flanking the transposon was sequenced in all the clones.
BLAST analysis of the sequences suggested that the transpo-
son insertions were located in homologs of different general
secretory pathway (gsp) genes. The mutations were mapped to
the gspF gene (BG4-12), gspK gene (BG12-88 and BG12-147),
gspE gene (BG15-40 and BG16-787), and gspD gene (BG15-
87) (Fig. 3). The DNA sequences of these four genes were 83
to 98% identical to corresponding gsp genes of two other
species belonging to the genus Burkholderia, namely, B. cepacia
strain KF1 (GenBank accession number AB050004.1) and
FIG. 1. The mushroom bioassay that demonstrates the effect of
mutations on cavity disease symptoms: control (A), BG164R (wild
type) (B), BG4-12 (C), BG12-88 (D), BG12-147 (E), BG15-40 (F),
BG15-87 (G), and BG16-787 (H).
FIG. 2. Complementation assays with the wild-type BG164R (A),
the representative GSP mutant BG4-12 (B), the complemented mu-
tant BG4-12Cos (C), and BG4-12LAF
3
(D)
.
Results shown are for the
following: mushroom assay (row 1), transmission electron microscopy
observations (row 2), protease assay (row 3), and chitinase assay (row 4).
The arrow (row 4) indicates the region of chitin degradation around
the complemented BG4-12Cos colony.
FIG. 3. (A) Clones aligned to the corresponding fragment of
genomic DNA on the cosmid map of pCosBG. (B) Cosmid map. Sites
on the map are as follows: B, BamHI; E, EcoRI; H, HindIII; S, SalI.
(C) Arrangement of gsp gene cluster in B. cepacia strain KFI, which
has 83 to 98% sequence similarity with BG164R gsp genes. The arrows
indicate the positions of insertions of the transposon into the GSP
operon.
VOL. 72, 2006 GSP AND CAVITY DISEASE 3561

Burkholderia pseudomallei strain 1026b (GenBank accession
number AF110185).
Each mutation could be complemented using a single
genomic clone carried by the cosmid pCosGSP. The genomic
fragment of 23.4 kb was isolated from a pLAFR
3
-based
genomic library of B. gladioli BG164R. pCosGSP was intro-
duced by triparental mating, and the presence of the cosmid in
the mutants was confirmed by subsequent reisolation and re-
striction analysis. The cosmid restored the virulent cavity-caus-
ing phenotype in each mutant. Since all the mutations mapped
to the gsp operon (Fig. 3), only three of four mutants—BG4-12
(gspF), BG12-88 (gspK), and BG15-40 (gspE)—were used for
further experiments. The extent of degradation of milk pro-
teins and colloidal chitin by the mutants and the numbers of
flagella (Fig. 2) were restored back to the levels of the wild type
when mutants were complemented in trans by the cosmid
pCosBG. All three representative gsp mutants could be res-
cued by recombination of the cosmid with the chromosome,
which was confirmed by Southern hybridization (data not
shown), and when they were compared in a bioassay to the
original mutants BG4-12, BG12-88, and BG15-40, no differ-
ences in the intensity of cavity formation were observed.
Mapping of the gsp genes in the cosmid pCosGSP. The
relative order of the genes coding for the different components
of the type II GSP machinery appears to be highly conserved,
as does the size of the gsp operons in the different species of
Burkholderia, which varies from 9 to 14 kb (8) (GenBank ac-
cession number AB050004). The gspF,gspK, and gspE genes
also define the ends and middle of the GSP cluster in closely
related members of Burkholderia (28). The cosmid pCosGSP
probably carried the full GSP locus of BG164R, because it
complemented these three genes in the different BG164R mu-
tants. The GSP locus in B. gladioli was also physically mapped
by Southern hybridization, and the relative order of the differ-
ent SalI fragments of DNA cloned from the mutants could be
arranged along the 23.4-kb cosmid pCosGSP.
The genomic insert in the cosmid was later subcloned (pGSP
ME H-H16.7, pGSP ME H-H2, pGSP ME1.7, pGSP KS B-B
2.2, and pGSP KS B-B 7.2) as smaller fragments, and the ends
of each fragment were sequenced. The above data, together
with the end-sequencing data of the cosmid subclones, were
used to map (Fig. 3B) the entire 23.4-kb cosmid, pCosGSP,
which revealed the presence of the entire gsp gene cluster of
BG164R on the cosmid.
Factors involved in the inhibition of mushroom mycelia are
different from those involved in the expression of cavity dis-
ease. The no-cavity-forming gsp mutants, BG4-12 and BG15-
40, still retained the capacity to inhibit mushroom mycelia (Fig.
4). The extent of inhibition was similar to that expressed by the
wild-type cavity-forming strain, BG164R, thereby indicating
that the two processes are independent of each other.
Qualitative observations on disease progression. As indi-
cated above, cavity disease symptoms can vary from infection
to infection. This could be due to any number of reasons. For
example, differences in the severity of symptoms could be due
to different pathogen concentrations. These concentrations
could be determined by environmental conditions and/or the
effect of other microbes. We manipulated these variables using
the mushroom bioassay.
It is normally expected that a small number of pathogenic
bacteria would initially colonize a mushroom and multiply to
reach the threshold concentration to cause disease. To test this
hypothesis, mushrooms were inoculated with various concen-
trations of BG164R, and disease progression was observed at
regular intervals over 5 days (Table 2). Surprisingly, no disease
symptoms were observed when as few as 20 cells of BG164R
were used. This was in contrast to the results presented by
Atkey et al. (2), who initiated soft rot disease within 72 h using
only 3 to 5 cells of the strain RR3.
Disease symptoms were consistently seen in bioassays using
larger numbers of bacteria, so we tested whether the indige-
nous flora was inhibiting establishment of the pathogen. The
indigenous flora was isolated from sporocarp samples. Three dis-
tinct types of bacteria were identified by different colony mor-
phologies, and Biolog tests and 16S rRNA sequence analysis
using the universal primers U16A and U16B followed. Two
(PRC121 and PRC122) were identified as different strains of P.
fluorescens (GenBank accession numbers DQ383803 and
DQ383804), while the third (PRC120) was E. americana (Gen-
FIG. 4. Inhibition of mushroom mycelia by BG164R and its no-
cavity-forming mutants: BG164R (A), BG4-12 (B), BG15-40 (C), and
P. putida (D).
TABLE 2. Cavity formation as a function of inoculum size
Dilution
factor
Estimated
inoculum
size (no.
of cells)
Symptoms observed after incubation (h) for
a
:
16 32 48 64 80 96 112 120
10
0
24 ⫻10
6
ⴱⴱⴱⴱⴱ⫹xxxxx
10
2
24 ⫻10
4
—ⴱⴱⴱ ⴱⴱ⫹xx x x
10
4
24 ⫻10
2
——— — H H H A
10
6
24 ——— — —— — A
10
8
——— — —— — A
a
ⴱ, cavity formation; ⴱⴱ, massive tissue degradation; ⴱⴱ⫹, complete tissue
degradation of mushrooms; —, no reaction observed; H, hypersensitive reaction
in 30% of the mushroom slices; A, assay terminated after mushroom slice started
degenerating; x, tissue disappearance due to excess degradation by the pathogen.
3562 ROY CHOWDHURY AND HEINEMANN APPL.ENVIRON.MICROBIOL.

Bank accession number DQ383802). Different combinations of
the pathogen, the three isolated mushroom bacteria, a mutant,
and a nonpathogenic Pseudomonas strain (PA147-2) were di-
luted to the same optical density from saturated cultures,
mixed in equal proportions, and spotted on mushroom slices,
and qualitative changes of the mushroom slices were observed
over a period of 3 days (Table 3). All combinations in which
strain PRC120 was present appeared to have a significantly
reduced cavity-forming capacity on mushroom slices, which
indicated a possible inhibition of BG164R by PRC120. Follow-
ing this qualitative test, a quantitative assay was done with only
strains BG164R and PRC120, which showed that disease es-
tablishment was inhibited for at least 72 h when mushrooms
were inoculated with 1:1 mixtures of BG164R and PRC120
totaling approximately 10
7
cells of each strain. The observation
was in contrast to observations recorded in Table 2, where
2.4 ⫻10
7
BG164R cells could cause cavity disease within 16 h.
This indicated that BG164R was being inhibited by strain
PRC120. Antagonism between BG164R and PRC120 was fur-
ther demonstrated in an in vitro plate assay. In this assay,
PRC120 was spotted onto a lawn of BG164R. The lawn of
BG164R around the PRC120 colony was inhibited, producing
a clear zone without growth. The observation confirmed the
ability of this isolate of E. americana to inhibit the cavity
disease pathogen, BG164R.
DISCUSSION
We have found that the type II protein secretion system, or
GSP, of B. gladioli pv. agaricicola BG164R is necessary for
mushroom cavity disease. The GSP is apparently responsible
for secreting the protease and chitinase activities that are nec-
essary for disease symptoms. It could not be known in advance
that secretion of these proteins was associated with cavity dis-
ease expression and that the GSP itself was necessary for the
disease.
The GSP of gram-negative bacteria is the main terminal
branch of a two-step secretion process. It generally consists of
12 invariant proteins spanning the cell envelope to form the
core secreton, plus some associated proteins present in specific
cases for the efficient functioning of the secretory machinery
(26). The GSP apparatus in the different bacteria studied
shows a high species specificity. The gsp operon of B.
pseudomallei (1026b) is a well-characterized and mapped
operon in the genus Burkholderia. The gspC gene in B.
pseudomallei 1026b is transcribed in a direction opposite to the
other genes in the gsp gene cluster (8). The two boundaries of
this gsp operon were identified by constructing mutants specif-
ically in the orfC and the orfD genes present in the left and
right ends of the gene cluster, respectively, which did not have
any effect on secretion. We present a physical map of the gsp
operon of B. gladioli pv. agaricicola which demonstrates that
the organization of the gsp genes in B. gladioli is similar to that
of B. pseudomallei 1026b.
In all members of the genus Burkholderia studied so far, the
virulence factors that are secreted through the GSP include
proteases, lipases, and phospholipase C. The most common
virulence factors in all of these cases have been the different
types of proteases (5, 8, 20, 31). Although the above-men-
tioned secreted factors have been reported to be dependent
TABLE 3. Interactions between bacteria on mushrooms influence severity of cavity disease
Strain or combination Observations noted after interaction for:
24 h 48 h 72 h
a
BG164R Distinct cavity Slice degraded —
PA147-2 No cavity No cavity No cavity
BG15-40 No cavity No cavity No cavity
BG164R⫹PRC120 Hypersensitive reaction Indent Prominent indent
BG164R⫹PRC121 Distinct cavity Slice degraded —
BG164R⫹PRC122 Distinct cavity Slice degraded —
PA147-2⫹PRC120 No cavity No cavity No cavity
PA147-2⫹PRC121 No cavity No cavity No cavity
PA147-2⫹PRC122 No cavity No cavity No cavity
BG15-40⫹PRC120 No cavity No cavity No cavity
BG15-40⫹PRC121 No cavity No cavity No cavity
BG15-40⫹PRC122 No cavity No cavity No cavity
BG164R⫹PRC120⫹PRC121 No cavity Hypersensitive reaction Indent
BG164R⫹PRC120⫹PRC122 No cavity Hypersensitive reaction Indent
BG164R⫹PRC121⫹PRC122 Distinct cavity Slice degraded —
PA147-2⫹PRC120⫹PRC121 No cavity No cavity No cavity
PA147-2⫹PRC120⫹PRC122 No cavity No cavity No cavity
PA147-2⫹PRC121⫹PRC122 No cavity No cavity No cavity
BG15-40⫹PRC120⫹PRC121 No cavity No cavity No cavity
BG15-40⫹PRC120⫹PRC122 No cavity No cavity No cavity
BG15-40⫹PRC121⫹PRC122 No cavity No cavity No cavity
BG164R⫹PRC120⫹PRC121⫹PRC122 Indent Prominent indent Cavity
PRC120 No cavity No cavity No cavity
PRC121 No cavity No cavity No cavity
PRC122 No cavity No cavity No cavity
Control
b
No cavity No cavity No cavity
a
—, mushroom slice fully degraded.
b
Mushroom slice without bacteria.
VOL. 72, 2006 GSP AND CAVITY DISEASE 3563

on the GSP for secretion, there are two different schools of
thought about their roles in pathogenesis. In some cases, the
secreted products could be directly linked to pathogenesis (31,
8), while in others they were not (12). Reports on the secretion
of virulence factors by the GSPs in the different Burkholderia
strains have always been correlated to the pathogenicity in
animal models. This is the first report linking the type II se-
cretion system to a disease in mushrooms. We have shown
through the characterization of avirulent mutants that the GSP
in BG164R is necessary for secretion of the cavity disease
virulence factors, which may or may not be the protease and
chitinase activities monitored in this study.
The complementation experiments confirm that each tested
mutant carried a mutation in only the 23.4-kb region corre-
sponding to the gsp locus. Therefore, we expect that the
virulence factors themselves are probably still produced by
BG164R avirulent mutants. We also agree with Gill and
Tsuneda (14), who hypothesized that cavity disease is a man-
ifestation of the combined action of more than one factor. The
secreted proteins are capable of causing disease symptoms only
when present together on the mushroom. Their hypothesis is
strengthened by the finding that gsp mutants were avirulent
because the inability to secrete certain proteins would affect
several virulence factors simultaneously.
Gill and Tsuneda (14) also proposed that the expression of
cavity disease is a combined effect of mushroom tissue-degrad-
ing enzymes and toxic compounds that inhibit the growth of
the mycelia. We provide evidence that the mechanism of inhi-
bition of mushroom mycelia is different from that of degrada-
tion of sporocarps, since the avirulent mutants still inhibited
mushroom mycelia.
The avirulent mutants isolated in the course of this study
had mutations in gspD,gspE,gspF, and gspK. The role and
localization of these proteins are known by comparison to their
description in other species. The GspD protein (BG15-40) is
the only outer membrane-associated protein forming the se-
creton. The GspF and GspK proteins (BG4-12, BG12-88, and
BG12-147) are associated with the inner membrane (26). The
GspE protein (BG15-40 and BG15-87) is cytoplasmic. It pos-
sesses a conserved ATP-binding motif providing an autokinase
activity; evidently it energizes the secretion process or assem-
bly of the secretory apparatus (34).
This is the first observation associating a reduction in flagella
number to mutations in the gsp genes. The only gene that is
known to be linked to flagella number is the fleN gene in
Pseudomonas aeruginosa. The FleN protein regulates flagella
number by acting as a negative regulator of fleQ, the transcrip-
tional activator of the flagellar synthesizing genes (6). The fleN
mutants of P. aeruginosa strains PAK and PAO1 had an in-
crease in flagella number. Interestingly, Dasgupta et al. (6) also
suggested that motility and flagella number were not necessar-
ily linked. All our mutants were motile (data not presented)
despite a reduction in the number of flagella.
The occurrence and severity of cavity disease are highly
variable, and the reason for the variability has gone unex-
plained until now. We provide evidence that antagonistic in-
teractions between B. gladioli and endogenous flora account
for this variability. Healthy New Zealand mushrooms appear
to be colonized consistently by E. americana. This bacterium
significantly impaired B. gladioli from creating the conditions
necessary for cavity disease symptoms. Possibly E. americana
prevents B. gladioli from reaching the necessary population
density to cause symptoms, either passively through resource
competition or actively by expression of a toxin. Other mush-
room diseases, such as brown blotch caused by Pseudomonas
tolaasii (25) and internal stipe necrosis caused by E. americana
(18), require a very high number of cells (⬃10
8
) for their
initiation under test conditions. Thus, either quantitative or
qualitative differences in the native flora from mushroom to
mushroom may account for all or some of the variability in the
disease.
E. americana PRC120 is the first report of a New Zealand
isolate of E. americana from mushrooms, and it has been
deposited in the New Zealand Culture Collection (accession
number of NZRM 4225). Interestingly, PRC120 isolated from
button mushrooms in New Zealand did not cause any necrotic
symptoms (17, 18) in our assay.
While we present evidence that E. americana inhibits cavity
disease on New Zealand mushrooms, it remains formally pos-
sible that other bacteria, not as consistently distributed, aug-
ment the ability of B. gladioli to establish the conditions nec-
essary for the disease. Moquet et al. (25) in their study of
blotch disease observed a species-specific difference in symp-
tom intensity to disease caused by P. tolaasii. Inglis et al. (18)
mentioned a variation in the intensity of disease symptoms by
E. americana and reported an interaction between P. fluore-
scens and E. americana in the formation of internal stipe ne-
crosis of mushrooms. They also suggested a contributory role
played by P. fluorescens in the expression of disease symptoms.
To the best of our knowledge, this is the first report of an
interaction on mushrooms that is antagonistic.
ACKNOWLEDGMENTS
P.R.C. was supported by NZAID and University of Canterbury
doctoral scholarships.
We are indebted to H. K. Mahanty for his mentorship in this project.
We thank A. L. J. Cole for providing the strain BG164, Mark Silby for
critical review of the manuscript, and Scott Godfrey and Mark Braith-
waite (MAF-New Zealand) for the identification of the mushroom
isolates PRC120, PRC121, and PRC122.
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