FEMS Microbiology Letters 61 (1989) 57-60
Published by Elsevier
57
FEMSLE 03667
Sclerotinia sclerotiorum growth and oxalic acid production
on selected culture media
P. Marciano
1, p.
Magro 2 and F. Favaron 1
1 Istituto di Patologia Vegetale, Padova, and 2 Istituto di Difesa delle Piante, Universitd della Tuscia, Viterbo, Italy
Received 15 March 1989
Accepted 25 April 1989
Key words: Sclerotinia sclerotiorum; Oxalic acid production
1. SUMMARY
Two isolates of Sclerotinia sclerotiorum, the
highly aggressive (B24) and the weakly aggressive
(SS41), were grown on liquid media containing
one of the following carbon sources: purified cell
walls obtained from onion or sunflower, pectin,
polygalacturonic acid, carboxymethylcellulose,
xylan or arabinogalactan. Isolates were equally
able to utilize these substrates for mycelial growth
but differed in their ability to utilize them for
oxalate production. B24 produces oxalic acid always to a substantial extent, SS41 only in traces.
The poor ability to produce oxalic acid by SS41
seems to be due to a lower efficiency in the
synthetic pathway.
2. I N T R O D U C T I O N
The ability to produce cell wall degrading enzymes (CWDE) and oxalic acid was found to be
Correspondence to: P. Marciano, Istituto di Patologia Vegetale,
Via Gradenigo 6, 35131 Padova, Italy.
an important factor in the pathogenesis of Selerotinia sclerotiorum Lib. (de Bary), pathogen of many
monocot and dicot plant species [1,2]. A positive
correlation seems to exist between aggressiveness
and 'in vivo' oxalic acid production of some isolates of the pathogen [3,4]. Two isolates, the highly
aggressive B24 and the weakly aggressive SS41,
have been particularly studied. They differ greatly
in the ability to produce oxalate in infected tissues, but they are equally able to synthethise and
release C W D E [3,4]. Moreover, they possess the
same ability in decarboxylating oxalic acid [5].
During disease development, mycelial growth
and various metabolic functions, including oxalic
acid synthesis, are probably supported by the
nutrients released following the activity of C W D E
on cell wall constituents. Therefore, the different
level of oxalic acid accumulated in host tissues by
isolates with different aggressiveness might depend on their differential ability to utilize host cell
wall components as nutrients.
To test this hypothesis, we studied the 'in vitro'
influence of cell walls and of several commercial
cell wall-like polysaccharides on oxalate accumulating ability and mycelial growth of the above
mentioned S. sclerotiorum isolates.
0378-1097/89/$03.50 © 1989 Federation of European Microbiological Societies
58
3. M A T E R I A L S A N D M E T H O D S
3.1. Growth conditions
Isolates were cultured on liquid media. The
mineral base was a modification of Czapek basal
salt medium, 3 g N a N O 3, 0.5 g MgSO 4 • 7 H 2 0 , 0.5
g KC1 and 0.01 g FeSO 4 • 7 H 2 0 dissolved in 1 litre
K phosphate buffer 0.1 M, p H 6.0. Buffer was
used instead of water since it has been demonstrated [6,7] that buffered media enhance oxalate
accumulation by S. sclerotiorum.
Carbon sources (0.5%, w / v ) were: pectin (Pe),
polygalacturonic acid (PGA), carboxymethylcellulose (CMC), xylan (XY), arabinogalactan (AG)
(all from Sigma) and plant cell walls. Cell walls
were obtained as previously described [8] from
sunflower hypocotyls (S-cw) and onion bulbs (Ocw). Onion and sunflower are used because of the
differences in cell wall composition between
monocot and dicot plants [9,10].
250-ml Erlenmeyer flasks containing 30 ml of
salt medium supplemented with one of each of the
carbon sources were autoclaved for 20 minutes
with saturated steam, adjusted to pH 6.0 and
inoculated with one agar disk (0.7 cm diameter)
cut from the margin of a 5-day-old culture of SS41
or B24 on potato dextrose agar. Cultures were
incubated at 20 ° C in the dark.
3.2. MyceHal dry weight, p H and oxalic acid content
in culture filtrates
At 3, 5, 7, and 9 days of growth, mycelial mats
from 4 flasks for each isolate and carbon source
were gently removed, floated in distilled water,
Table 1
pH values of B24 culture filtrates during the 9-day culture
period
Media
Days of culture
3
O-cw
S-cw
CMC
Pe
PGA
5
5.90+0.05 5.85+0.01
5.78_+0.10 4.81+0.09
5.82-+0.13 5.45-+0.07
5.71_+0.20 4.78_+0.24
5.10_+0.10 4.33_+0.08
7
9
4.92+0.20
4.25_+0.15
5.41_+0.10
4.53_+0.18
4.46+0.06
4.43_+0.13
4.00-+0.17
5.30_+0.08
4.41_+0.10
4.41_+0.09
Data represent the average of three experiments with four
determinations per experiment and their standard deviations.
filtered through a tared crucible and used for dry
weight determinations. Mycelial mats were dried
to constant weight at 70 ° C. Data are expressed as
mg dry weight, flask -1. The respective culture
media were filtered through Watman n° 4 paper to
remove debris, and their p H was determined using
a PHM64 radiometer. Filtrates were stored at
- 3 0 ° C until used for the determination of oxalic
acid content with a Boehringer Mannheim enzymatic kit according to Drawert [11]. Data are
expressed as mg oxalic acid - m l - 1
4. R E S U L T S A N D D I S C U S S I O N
Over the 9-day incubation period, there was no
noticeable difference between initial (pH 6.0) and
final p H values of SS41 culture media. In this
period of time, pH values of B24 filtrates changed
gradually (Table 1) in all but two media (containing A G and XY) where no changes in pH were
detected (data not shown). Furthermore, in all
culture media, the pH was above the value reported as inhibitory for S. sclerotiorum oxalic acid
production [6].
Table 2 reports the effect of the different carbon
sources on mycelial growth of B24 and SS41.
Onion cell walls and XY were the best carbon
sources for mycelial growth of both isolates. With
the exception of AG, which supported better
growth of SS41 than B24, all the tested substrates
seemed to affect the two isolates in the same way.
The effect of the different carbon sources on
oxalate production is reported in Table 2. It shows
that whichever was the carbon source, SS41 produced only small amounts whereas B24 always
produced substantial amounts of oxalic acid. Both
cell wall preparations and, among the polysaccharides, the pectic substances, were the most
favourable carbon sources for oxalate production
by B24. Similar results were obtained with another
pathogenic S. sclerotiorum isolate [12]. Data confirm that S. sclerotiorum produces oxalic acid
adaptively depending on the type of carbon sources
and to varying extent depending on the isolates
[4,6,7,12]. Results add further evidence to the lack
of correlation between oxalate production and
mycelial growth, as found for Botrytis cinerea and
59
Table 2
Mycelial growth and oxMic acid production by S. sclerotiorurn isolates in buffered medium containing different carbon sources
Carbon
B24
soHrces
5 days a
O-cw
S-cw
CMC
Pe
PGA
XY
AG
SS41
9 days a
5 days a
9 days a
Growth b
Oxalic
acid c
Growth b
Oxalic
acid ~
Growth b
Oxalic
acid c
Growth b
Oxalic
acid ~
25.1±2.0
12.4±1.6
15.2±1.6
14.5±1.9
11.5±0.4
18.7±0.7
11.5±1.5
0.38±0.08
0.76±0.05
0.27±0.05
0.52±0.08
0.72±0.08
0.10±0.05
0.10±0.03
26.2±1.5
24.4±1.8
12.8±1.9
12.7±1.2
20.5±0.6
30.1±0.9
5.2±1.0
1.24±0.05
1.16±0.04
0.36±0.04
0.58±0.06
0.64±0.06
0.30±0.03
0.25±0.03
17.2±1.9
13.7±1.5
15.7±0.8
11.6±1.3
12.2±0.7
18.2±0.8
19.5±1.3
0.06±0.01
0.04±0.02
0.04±0.06
0.04±0.06
0.04±0.05
0.05±0.04
0.02±0.03
24.8±1.7
22.7±2.0
15.0±1.2
12.2±0.7
19.8±0.8
30.3±1.2
15.6±1.9
0.06±0.02
0.04±0.02
0.02±0.04
0.04±0.04
0.04±0.05
0.04±0.03
0.02±0.04
Data represent averages of three experiments with four determinations per experiment and their standard deviations.
a Days of cultures.
b Expressed as mg dry weight-flask-1.
c Expressed as mg.ml- 1.
S c l e r o t i u m rolfsii [13-16]. The data indicate that
the possible differences i n p r i m a r y cell wall comp o s i t i o n seem to i n d u c e some differences i n oxalate
p r o d u c t i o n a n d mycelial growth o n l y in B24, 5
days after i n o c u l a t i o n . E q u i v a l e n t v a r i a t i o n in
mycelial growth, b u t n o c o m p a r a b l e differences in
oxalate p r o d u c t i o n b y SS41 were observed o n the
same range of c a r b o n sources.
It was previously f o u n d [5] that SS41 a n d B24
mycelia c o n t a i n e d equivalent a m o u n t s of oxalate
decarboxylase, the enzyme that destroys oxalic
acid. The enzyme is i n d u c e d b y the presence of
oxalate in the culture m e d i u m a n d is e n h a n c e d b y
acid p H [5,17]. I n the culture m e d i a tested i n this
study, the p H never d r o p p e d towards more acidic
values d u r i n g SS41 growth, m a k i n g it unlikely that
the low a m o u n t of oxalic acid f o u n d in SS41
culture filtrates could be explained i n terms of
high decarboxylating activity. O n the other h a n d ,
as previously d e m o n s t r a t e d [5], SS41 a c c u m u l a t e s
oxalic acid if grown in a m e d i u m c o n t a i n i n g succinic acid, a n e a r precursor of oxalic acid.
I n conclusion, o n the basis of the results rep o r t e d here, it seems likely that the p o o r ability of
SS41, in relation to B24, to p r o d u c e oxalic acid is
due to a lower efficiency in the synthetic p a t h w a y
rather than to a different ability to utilize host cell
wall c o m p o n e n t s as nutrients.
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