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Applied and Environmental Microbiology, March 2000, p. 1026-1030, Vol. 66, No. 3
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
pH Regulation of Pectate Lyase Secretion Modulates
the Attack of Colletotrichum gloeosporioides on
Avocado Fruits
Nir
Yakoby,1,2
Ilana
Kobiler,1
Amos
Dinoor,2 and
Dov
Prusky1,*
Department of Postharvest Science of Fresh
Produce, Agricultural Research Organization, The Volcani Center,
Bet Dagan 50250,1 and Department of
Plant Pathology and Microbiology, Faculty of Agricultural Food and
Environmental Quality Sciences, Hebrew University of Jerusalem, Rehovot
76100,2 Israel
Received 30 June 1999/Accepted 24 December 1999
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ABSTRACT |
Growth of Colletotrichum gloeosporioides in pectolytic
enzyme-inducing medium (PEIM) increased the pH of the medium from 3.8 to 6.5. Pectate lyase (PL) secretion was detected when the pH reached
5.8, and the level of secretion increased up to pH 6.5. PL gene
(pel) transcript production began at pH 5.0 and increased up to pH 5.7. PL secretion was never detected when the pH of the inducing medium was lower than 5.8 or when C. gloeosporioides hyphae were transferred from PL-secreting
conditions at pH 6.5 to pH 3.8. This behavior differed from that of
polygalacturonase (PG), where pg transcripts and protein
secretion were detected at pH 5.0 and continued up to 5.7. Under in
vivo conditions, the pH of unripe pericarp of freshly harvested avocado
(Persea americana cv. Fuerte) fruits, resistant to C. gloeosporioides attack, was 5.2, whereas in ripe fruits, when
decay symptoms were expressed, the pericarp pH had increased to 6.3. Two avocado cultivars, Ardit and Ettinger, which are resistant to
C. gloeosporioides attack, had pericarp pHs of less than
5.5, which did not increase during ripening. The present results
suggest that host pH regulates the secretion of PL and may affect
C. gloeosporioides pathogenicity. The mechanism found in
avocado may have equivalents in other postharvest pathosystems and
suggests new approaches for breeding against and controlling
postharvest diseases.
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INTRODUCTION |
Colletotrichum
gloeosporioides (Penz.) Penz. & Sacc [teleomorph Glomerella
cingulata (Stonem). Spauld et Schrenk] infects avocado fruits,
via spore germination, appressorium formation, and penetration. The
pathogen attacks fruits early in their development and remains as a
germinated appressorium during fruit growth (quiescent infection)
(17). After harvest and fruit ripening, fast-developing brown-black spots on the pericarp and soft rot in the mesocarp, the
symptoms of anthracnose, appear (17). Prusky (17)
has suggested several hypotheses to explain the resistance mechanism of
unripe fruits: (i) nutrients available to the pathogen may be limited
in unripe hosts, (ii) preformed antifungal compounds present in unripe
fruits decline during ripening, (iii) inducible antifungal compounds in
unripe fruits decline during ripening, and (iv) fungal pathogenicity
factors may be activated mainly in ripening fruits. Antifungal
compounds are implicated in the resistance of avocado fruits to
C. gloeosporioides (19), but no reports have
described the role of enzyme secretion as a factor in fungal attack.
C. gloeosporioides produces an array of pectolytic enzymes,
including polygalacturonase (PG) (20), pectin lyase
(2), pectin methyl esterase (15), and pectate
lyase (PL) (28). No reports regarding the involvement of PG
or pectin methyl esterase in C. gloeosporioides attack have
been published. However, targeted disruption of pectin lyase from
C. gloeosporioides did not reduce virulence (2).
The importance of PL secretion during C. gloeosporioides attack in avocado fruits has been suggested by the inhibition of decay
development during coinoculation of C. gloeosporioides spores with PL antibodies and also by the reduced pathogenicity of a
Colletotrichum magna mutant with limited PL secretion
(26, 27). These results suggest that PL is a limiting factor
during pathogenesis (27).
Environmental conditions can affect protein production and secretion in
various organisms (1, 4, 12). Dean and Timberlake (5) found that Aspergillus nidulans secretes PG
at low pH values and PL when the medium pH increases and becomes
conducive to PL activity. St. Leger et al. (24) hypothesized
that ambient pH affects pathogenicity by altering the expression of
cuticle-degrading enzymes and hydrophobin in the insect pathogen
Metarhizium anisopliae.
Our working hypothesis is that host environmental conditions affect PL
secretion and prevent the activation of C. gloeosporioides colonization in unripe fruits. In the present study, we demonstrated the importance of pH as a regulator of pectolytic enzyme secretion in
C. gloeosporioides. Pathogenicity of C. gloeosporioides is dependent on its ability to secrete PL enzyme
and not on pel gene expression. These results indicate that
avocado susceptibility is regulated by more than one mechanism: a
decrease in the level of antifungal compounds and an increase in
pericarp pH which modulates PL secretion.
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MATERIALS AND METHODS |
Strains, media, and growth conditions.
C.
gloeosporioides isolate Cg-14 was obtained from decayed avocado
fruits and used in all experiments. Spores were stored in 10 mM Na
phosphate buffer (pH 7.2) in 40% glycerol at 
80°C. Cultures were
initiated with 0.5 × 106 to 1.5 × 106 spores/ml.
Spores from 10- to 20-day-old cultures were harvested from
M3S medium (25). Erlenmeyer flasks (500 ml)
containing 250 ml of medium with Na polypectate and pectin as the sole
carbon sources (pectolytic enzyme-inducing medium [PEIM])
(20) were inoculated with isolate Cg-14 (0.5 × 106 spores/flask) and grown at 24°C on an orbital shaker
(150 rpm). The PEIM consisted of the following (per liter):
polygalacturonic acid-Na, 5 g; pectin from citrus fruit (Sigma,
St. Louis, Mo.), 5 g; KNO3, 5 g;
KH2PO4, 4 g; MgSO4 · 7H2O, 2 g; CaCl2 · 7H2O, 0.3 g, and FeCl3, 10 mg. The initial
pH of the PEIM was 3.8. In some experiments, the medium was adjusted to
pH 6.0 with 5 N NaOH. In some experiments, mycelium was subsequently
transferred to fresh PEIM after 4 days of growth in liquid
M3S medium.
Fruits, inoculation conditions, and statistical analysis.
Avocado fruits, Persea americana Mill. var.
drymifolia (Schidl. and Cham.) S. F. Blake `Fuerte'
cultivars Fuerte, Ardit, and Ettinger, were harvested from an orchard
at Kibbutz Givat Brenner, Israel. Firmness, a parameter of fruit
ripening, was checked at two places on the longer transverse axis of
each fruit with a Penetrometer (Chatillon & Son Inc., Kew Gardens,
N.Y.) with a conical probe. Results are given in newtons.
Inoculation was carried out on 10 freshly harvested fruits by placing
10 µl of spore suspension (10
6 spores/ml) at six points,
three on each side of the longitudinal
axis of the fruit. The fruits
were then incubated at 22°C, in
90% humidity, for 10 to 12
days.
In vitro experiments were repeated five times. The results of one
representative experiment are presented. In vivo experiments
were
repeated at least three times during two consecutive harvesting
seasons. Standard deviations of the means were calculated, and
differences between means were analyzed by analysis of
variance.
pH measurements.
pH was measured with a flat electrode
(Sensorex, Stanton, Calif.) in 3-ml aliquots sampled at different times
after fungal inoculation. Pericarp pH was determined after an
approximately 0.2-mm-deep cut was made with a scalpel blade. Mesocarp
pH was determined after peeling the pericarp to a depth of 2 mm. pH
measurements were taken by placing the flat electrode directly against
the exposed tissue. The depth of the pericarp cut was determined based on the location of C. gloeosporioides infection
(17). All measurements were repeated on three fruits at
three different places (nine measurements) on the longer transverse
axis of each fruit. The standard error of the mean of pH measurements
was never higher than 2.5%. To test the hypothesis that direct pH
measurement was a reliable indicator of the environment within the
fruit, the direct measurement was compared to the pH determined by the
common homogenization method (10), in which 5 g of
pericarp tissue is ground in the presence of 10 ml of double-distilled
water. Direct and homogenatization method pH measurements were compared at three different stages of fruit growth in 10 P. americana
cv. Fuerte fruits (30 measurements). The regression coefficient
(r) between the measurements was 0.999. Similar results were
obtained with P. americana cultivars Ettinger and Ardit.
Detection of PL and PG in liquid media and fungal hyphae.
C. gloeosporioides hyphae were separated from the culture
medium by centrifugation (12,000 × g, 10 min) at
4°C. The hyphae were washed twice with sterile water (250 ml each
time), frozen under liquid nitrogen, lyophilized, and stored at
80°C until used. The culture medium supernatant was concentrated to
15 ml with a Rotavapor (Buchii, Flawil, Switzerland) at 42°C. The
concentrated culture filtrate was dialyzed overnight
(12,000-molecular-weight cutoff; Sigma) against 5 liters of Tris-HCl
(50 mM, pH 8.5) concentrated to 5 ml as above, and 5 µg per lane was
subjected to Western blot analysis.
Nonsecreted PL was extracted from fungal hyphae with TRI REAGENT
(Sigma). Lyophilized hyphae were ground three times with
a mortar and
pestle under liquid nitrogen. Proteins were extracted
by using 4 ml of
TRI REAGENT for every 0.2 g of lyophilized hyphae
in accordance
with the manufacturer's instructions (TRI REAGENT;
Sigma Technical
Bulletin MB-205). To determine the presence of
glycosylated PL, 10-µg
protein samples were subjected to
N-glycosidase
F in
accordance with the manufacturer's instructions (Boehringer
Mannheim
GmbH, Mannheim, Germany) and analyzed by Western blot
analysis.
Each protein sample was quantified by the Bio-Rad Laboratories
(Hercules, Calif.) protein assay, with bovine serum albumin
as the
standard. Samples were boiled for 4 min in loading buffer
as described
by Sambrook et al. (
22), with 10%
-mercaptoethanol
as a
reducing agent. Samples were loaded onto a sodium dodecyl
sulfate
(SDS)-12.5% polyacrylamide gel electrophoresis (PAGE)
gel
(Mini-Protean II; Bio-Rad Laboratories) and run for 1 h at
a
constant 150 V. Western blot analysis was performed with PL
(
27) and PG (D. Prusky and N. T. Keen, unpublished
data) antibodies
diluted 1:500. For PL, preimmune ascitic fluid was
used as a control,
and anti-mouse immunoglobulin (IgG)-alkaline
phosphatase (AP)
conjugate (Promega, Madison, Wis.) was used as a
secondary antibody.
PG antibodies were prepared by injecting the
glycosylated purified
PG into a rabbit and collecting the resultant
serum. Nonimmune
serum was used as a control. Anti-rabbit IgG-AP
conjugate (Promega)
was used as the secondary antibody. Both secondary
antibodies
were used at a dilution of 1:6,000.
RNA extraction and Northern blot analysis.
Lyophilized
hyphae were ground three times with a mortar and pestle under liquid
nitrogen, and total RNA was extracted by using 4 ml of TRI REAGENT
(Sigma) for every 0.2 g of lyophilized hyphae. Following
homogenization, samples were prepared in accordance with the
manufacturer's instructions (Sigma Technical Bulletin MB-205). RNA was
quantified by GeneQuant (Pharmacia Biotech, Cambridge, United Kingdom).
Northern blot analysis was conducted by running 10 µg of total RNA on
a 1.1% formaldehyde denaturing agarose gel (
22). The
RNA
was blotted to a Hybond+ nylon membrane (Amersham, Buckinghamshire,
United Kingdom) by the capillary method (
22), using 20× SSC
(1× SSC is 0.017 M NaCl plus 0.17 M sodium citrate). The RNA was
fixed
by baking for 2 h at 80°C and subjected to hybridization
using
the 1.1-kb
pel full-length clone (GenBank accession no.
U329242) as a probe. All hybridizations were carried out at 63°C,
and
the products were washed with 0.1× SSC. PG was probed with
the 487-bp
putative PG clone (
cgpg1; GenBank accession no.
AF116507)
obtained by PCR cloning. The
cgpg1 clone was obtained by PCR
from
3 µg of genomic DNA of
C. gloeosporioides, using two
oligonucleotides
(5'-CCTCAACGGCATCAAGGTACC-3' as the forward
primer and 5'-CAGGCATGCGTCCTGGTTGTA-3'
as the reverse
primer). The primers were constructed on the basis
of the alignment of
clpg1 and
clpg2 from
Colletotrichum
lindemuthianum (
3). The following cycles were used:
95°C for 5 min, 1 cycle;
95°C for 1 min, 60°C for 1.5 min, 72°C
for 1 min, 10 cycles; 94°C
for 1 min, 60°C for 1.5 min, 72°C for
1 min, 20 cycles; and 72°C
for 10 min, 1 cycle. The fragment was
subcloned into pGEM-T Easy
(Promega) and transferred to DH5-
by
means of a Gene Pulser II
(Bio-Rad Laboratories). Colonies were
selected on Luria-Bertani
(LB) medium supplemented with 100 µg of
ampicillin per ml, with
blue or white color selection. DNA was
sequenced at the Weizmann
Institute's Biological Services, Rehovot,
Israel. The results
were analyzed with the Wisconsin package, GCG
version, by comparison
with GenBank data. An ethidium bromide-stained
gel was used for
RNA
quantification.
Antifungal diene extraction.
A 10-g sample of avocado
pericarp (1- to 2-mm deep) was homogenized in 95% ethanol in an
Omni-Mixer (Sorvall; DuPont Company, Newtown, Conn.) at full speed for
3 min. The ethanol extract was dried in a rotary evaporator at 40°C
and redissolved in 10 ml of distilled water, and the organic phase was
extracted by fractionation with dichloromethane. Following two
extractions, the organic phases were pooled, dried with anhydrous
MgSO4 (Riedel-deHaen, Seelze, Germany), and evaporated to
dryness. Samples were redissolved in 1 ml of ethanol (analytical grade;
Bio Lab, Jerusalem, Israel) and analyzed by high-performance liquid
chromatography (21). The average values of three separate
extractions are presented.
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RESULTS |
pH changes and PL secretion in PEIM by C. gloeosporioides.
Three days after inoculation of PEIM cultures
with C. gloeosporioides spores, the pH of the medium was
3.8, increasing to 6.5 by day 6 (Fig.
1A). We detected PL in the culture
filtrate when the medium pH exceeded 6.0 (Fig. 1B), and the amount
secreted increased with further increases in pH. PL activity at pH 6.5 was 3.2 × 10
3 U/min. To determine whether the
absence of PL secretion was dependent on pH rather than on limited
fungal development, the following experiments were initiated from
fungal mass.
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FIG. 1.
Changes in pH values (A) and PL protein secretion (B) in
PEIM by C. gloeosporioides. Spores (0.5 × 106) were inoculated into 250 ml of PEIM, and the medium
was shaken at 150 rpm at 22°C. The culture medium was concentrated,
dialyzed, and further concentrated before being analyzed for the
presence of PL, by Western blot analysis (5 µg of protein/lane).
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Relationship between pH and PL secretion.
C.
gloeosporioides mycelium from a culture grown in PEIM for 6 days,
from pH 3.8 to PL secretion conditions at pH 7.0, was divided equally
and transferred to PEIM at either pH 3.8 or 6.0. The hyphae did not
secrete PL when transferred to pH 3.8 and grown to pH 5.2, 24 h
later. However, when the mycelium was transferred to pH 6.0 and grown
to pH 6.5, PL secretion was detected within 24 h (Fig.
2). The dry weights of the hyphae
developed under both pH conditions during the 24-h period were the
same. When mycelium grown under noninducing conditions (M3S
medium) was transferred to PEIM at pH 3.8 and grown for 4 days to a pH
not higher than 4.9 or 5.4, PL was not secreted. PL was detected in
PEIM only when the pH reached 5.8 (Fig.
3). At the end of the experiment, the dry
weight of the fungal hyphae at the low pH was 5 to 10% less than that
of the hyphae grown at the high pH. When mycelium grown under
noninducing conditions was transferred to PEIM adjusted to pH 6.0, PL
was secreted within 24 h (Fig. 3).
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FIG. 2.
Effect of PEIM pH on PL secretion by C. gloeosporioides following transfer from a 6-day-old culture grown
in PEIM. Spores (1.5 × 106) were inoculated into PEIM
and allowed to grow for 6 days until the pH reached 7.0. The mycelium
mass was then washed, split equally, and transferred to PEIM at pH 3.8, and the pH of PEIM was adjusted to 6.0. The mycelium transferred to
PEIM at pH 3.8 was grown for 24 h until the pH reached 5.2 and
then the medium was sampled. The mycelium transferred to PEIM at pH 6.0 was grown for 24 h until the pH reached 6.5 and then the medium
was sampled. The culture media were concentrated, dialyzed, and further
concentrated for SDS-PAGE (5 µg of protein/lane), and then they were
analyzed for the presence of PL by Western blot analysis.
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FIG. 3.
Secretion of PL by C. gloeosporioides when
grown on PEIM up to specific pH values. Spores (1.5 × 106) were inoculated into M3S medium and
allowed to grow for 4 days. The mycelium was then washed thoroughly and
transferred to PEIM that was adjusted with 32% HCl during growth to pH
values not exceeding 4.9, 5.4, and 5.8. At the same time, the pH of the
control changed from 6.0 to 7.0. The culture medium was concentrated,
dialyzed, further concentrated before being loaded onto an SDS-PAGE gel
(5 µg of protein/lane), and analyzed for the presence of PL by
Western blot analysis.
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pH levels in pericarp and mesocarp of avocado fruit during
development and ripening.
The pH of the pericarp, from 50 days
after fruit set (immature fruits) to 200 days later (mature fruits),
was approximately 5.2 (Fig. 4). No
symptoms of decay were observed during this period. When P. americana cv. Fuerte fruits were harvested at the regular harvesting period and stored at 20°C to ripen, the pH in the pericarp increased from 5.2 to 6.1, with decay initiation (decay diameter < 0.5 cm) at pH 5.8 and decay symptoms (decay diameter > 0.5 cm) at pH 6.1 (Fig. 5). Mesocarp pH values
averaged 6.4 during the ripening period (data not shown).
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FIG. 4.
Changes in the pericarp ( ) and mesocarp ( ) pH
values of cultivar Fuerte avocado fruits on different days after fruit
set. Bars represent the standard deviations of the mean of three
independent tests from one representative experiment. The differences
between the means of pericarp and mesocarp pH are significant
(P < 0.05).
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FIG. 5.
Changes in pH values of the pericarp () of cultivar
Fuerte avocado fruits during postharvest ripening. Firmness ( ) is
presented as a parameter of ripening. Arrow indicates the time of decay
initiation (DI) and of decay symptoms (DS) of C. gloeosporioides, following inoculation of freshly harvested
fruits. Bars represent the standard deviations of the mean from one
representative experiment. Cultivar Fuerte fruits were harvested at
midseason.
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The concentrations of the preformed antifungal diene in the pericarp of
resistant cultivars Ardit and Ettinger was subfungitoxic
(210 ± 10 and 330 ± 20 µg/g [fresh weight {FW}], respectively,
compared to 960 ± 110 µg/g [FW] in cultivar Fuerte fruits).
Interestingly,
cultivar Ardit and Ettinger fruits had average pericarp
pH levels
of 5.5 and 5.1, respectively, and the average pH of the
mesocarp
of both cultivars was 6.5 (Fig.
6).
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FIG. 6.
Changes in pH values of the pericarp () and mesocarp
() of cultivar Ardit (A) and Ettinger (B) avocado fruits during
postharvest ripening. Firmness () is presented as a parameter of
ripening. Bars represent standard deviations of the mean from one
representative experiment. Cultivars Ettinger and Ardit were harvested
at the same time, Ettinger late in its harvesting season and Ardit
early in its harvesting season. The differences between the means of
pericarp and mesocarp pH values in both cultivars are significant
(P < 0.01).
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Level of PL and PG regulation.
Northern blot analysis of
C. gloeosporioides hyphae harvested from PEIM at final pH
levels of 3.8 to 5.8 detected pel transcripts at pH 5.0. The
transcript amount increased steadily up to pH 5.7 (Fig.
7B). pel expression was not
accompanied by detectable PL in the culture medium. PL secretion was
detected only when the medium pH rose above 5.8 (Fig. 7A). However, the
expression (Fig. 8B) and secretion
(Fig. 8A) of PG were detected from pH 5.0 to 5.7.
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FIG. 7.
Effect of PEIM pH on mRNA expression of pel
and secretion of PL from C. gloeosporioides. C. gloeosporioides was grown in PEIM whose pH was initially 3.8. At
different pH levels, hyphae and culture media were harvested. Hyphae
were subjected to RNA extraction, and culture medium was concentrated
and dialyzed before PL analysis. Hybridizations were carried out at
63°C. The radioactive probe for pel was a full-length cDNA
pel clone. (A) Western blot analysis; (B) Northern blot
analysis; (C) ethidium bromide (EtBr) RNA loading.
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FIG. 8.
Effect of PEIM pH on pg expression and
secretion of PG from C. gloeosporioides. C. gloeosporioides
was grown in PEIM whose pH was initially 3.8. At different pH levels,
hyphae and culture media were harvested. Hyphae were subjected to RNA
extraction, and culture medium was concentrated and dialyzed before PG
analysis. Hybridization was carried out at 63°C. The radioactive
probe for pg was a 487-bp fragment obtained by PCR. (A)
Western blot analysis; (B) Northern blot analysis; (C) ethidium bromide
(EtBr) RNA loading.
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DISCUSSION |
Resistance of unripe avocado fruits to C. gloeosporioides depends on antifungal compounds (17-19,
23). However, two cultivars, Ettinger and Ardit, have unripe
fruits with concentrations of the antifungal diene that are
significantly lower than the 50% effective dose of 450 µg/ml (ca.
330 µg of diene/g [FW] of pericarp) but are still resistant to
C. gloeosporioides attack (17). This result
suggests that the absence of fungal colonization may depend on the
inhibition of secretion of pathogenicity factors, such as pectolytic
enzymes (17). In the Colletotrichum-avocado
system, high concentrations of the flavonoid epicatechin, an inhibitor of PL and PG, in the pericarp of unripe fruits was hypothesized to
affect fungal colonization (26). However, the specific
effect of host environmental conditions on the secretion of pectolytic enzymes has not been described.
C. gloeosporioides did not colonize avocado fruits when the
pericarp pH was lower than 5.8. These low pH values are detected in
cultivar Fuerte fruit pericarp during all periods of fruit growth
before ripening and in the ripe Ettinger and Ardit cultivars. However,
inoculation of C. gloeosporioides on either mesocarp or
pericarp tissue of ripe fruits with pH levels higher than 5.8 caused
decay symptoms within 2 days. C. gloeosporioides can cause disease in unripe mesocarp (8) that has a pH higher than
5.8, suggesting that the physiological stage of fruit ripening does not
determine susceptibility to fungal colonization. When mycelium grown in
PEIM at pH 6.5 was transferred to fresh PEIM at a pH of 
5.7, no PL
secretion was detected. PL also was not secreted if the medium was
adjusted to pH levels of 5.7 at any time during the 4-day incubation.
This behavior differed from that of PG, where pg transcripts
and protein secretion occur between pH 5.0 and 5.8.
How pH affects the secretion of PL in C. gloeosporioides is
still unknown. pH may affect a series of regulatory processes in fungi
and yeast (5-7, 9, 11, 13, 14, 16). In the present study,
the lack of PL secretion until transcript levels peaked suggests that
PL is translated but that the protein remains in the mycelia until a
secretion-permissive pH level is reached. The lack of PL secretion in
PEIM at low pH following the transfer of mycelium from conditions where
PL was being secreted further supports the hypothesis that PL secretion
is regulated by pH. The presence of PL in fungal hyphae grown in PEIM
at pHs lower than 5.7 suggests that PL is preformed and then released
from the fungal mycelium by a pH-dependent mechanism.
N-glycosidase F treatment of hyphal protein extracts
significantly reduced the molecular weight of the putative intermediate
PL-processed proteins, suggesting that the nonsecreted protein is
glycosylated (data not shown).
Since C. gloeosporioides easily macerates avocado tissue
(20), we hypothesize that the basal level of PG secretion
may locally digest avocado pericarp cell walls, disrupt cell
compartmentation, and expose the developing hyphae to the low-pH
environment of the vacuole. Present and previous results
(28) emphasize the importance of PL secretion for decay
development in ripening fruits. Our results suggest that modulation of
PL secretion by the host is an important mechanism that works together
with preformed antifungal compounds to inhibit fungal colonization
(17). The reduction in acidity and pH increase in ripening
and senescing fruits and the consequent decay development occurring
during this period also suggest that the effect of pH on pectolytic
enzyme secretion may have a broader significance than for avocado
fruits alone and could be used for breeding and control of decay
occurring in other postharvest pathosystems.
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ACKNOWLEDGMENTS |
This research was supported by the Binational Agricultural
Research and Development Fund (BARD), the German-Israel Agricultural Research Agreement (GIARA) for the Benefit of the Third World, and the
U.S.-Israel Cooperative Development Research (CDR) Program.
We thank N. T. Keen for critically reviewing the manuscript and
for suggesting significant changes.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Postharvest Science of Fresh Produce, Agricultural Research
Organization, The Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel.
Phone: 972-3-9683610. Fax: 972-3-9683622. E-mail:
prusky33{at}netvision.net.il.
Contribution 407-2000 from the Agricultural Research Organization,
The Volcani Center, Bet Dagan, Israel.
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Applied and Environmental Microbiology, March 2000, p. 1026-1030, Vol. 66, No. 3
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
