Download Extracellular Pectinolytic Enzymes of Fungi Elicit

Journal of General Microbiology ( I986), 132, 77 1-777.
Printed in Great Britain
77 1
Extracellular Pectinolytic Enzymes of Fungi Elicit Phytoalexin
Accumulation in Carrot Suspension Culture
By M A G D Y A M I N , F U M I Y A K U R O S A K I * A N D A R A S U K E N I S H I
Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Sugitani,
Toyama 930-01, Japan
(Received 17 June 1985; revised 7 October 1985)
A heat-labile elicitor of phytoalexin accumulation in carrot (Daucus curota L.) was detected in
culture filtrate of Chaetomium globosum grown in Czapek-Dox medium. The elicitor activity
correlated with the activity of pectinolytic enzymes in the culture filtrates. The culture filtrates
induced the release of heat-stable elicitors from carrot cell homogenates, pectic fraction of carrot
cell walls and citrus pectin. The coincidence of heat sensitivity and sodium periodate
inactivation profiles of elicitor and pectinolytic activity suggested that the activity of
pectinolytic enzymes is essential for the release of the heat-stable elicitor. The culture filtrates of
Botrytis cinerea, Fusarium rnoniliforrne and Helrninthosporium oryzue showed similar elicitation
patterns.
INTRODUCTION
Phytoalexins are low-M, antimicrobial compounds which have been shown to accumulate in
many species of higher plants after infections and appear to be a widespread mechanism by
which plants attempt to protect themselves against microbes (Kui., 1972; Grisebach & Ebel,
1978). Molecules of microbial origin which trigger phytoalexin accumulation in plants have
been called elicitors (Keen et al., 1972). Many elicitors have been reported, including fungal wall
polysaccharides, glycoproteins and lipids (West, 1981). Lee & West ( I98 1 a, b) and Davis et al.
(1984) showed that pectinolytic enzymes released into the culture filtrates of Rhizopus stolonifer
and Erwinia carotovora elicited phytoalexin accumulation in castor bean and soybean,
respectively.
Kurosaki & Nishi (1983, 1984) showed that carrot produces 6-methoxymellein as a common
phytoalexin after being challenged by various fungi, and that wall fragments released from
carrot cells by a commercial pectinase or protease elicit the production of the phytoalexin. These
findings suggested that fungal pectinolytic enzymes play a role in the elicitation of phytoalexin
production in carrot-fungi interactions. In this work, we present evidence that the extracellular
pectinolytic enzymes of fungi serve as elicitors of phytoalexin accumulation in carrot by
releasing fragments from pectic polysaccharides of the plant cell wall.
METHODS
Micro-organisms and cultures. Chaetomium globosum and Botrytis cinerea were kindly provided by Dr Tsubaki,
Institute of Biological Sciences, Tsukuba University, Japan. Fusarium monilijbrmt)and Helminthosporium oryzae
were obtained from Toyama Agricultural Experimental Station, Japan. All fungi were maintained on agar slants
of yeast/malt medium [yeast extract 0.4% (w/v), malt extract 1 % (w/v) and glucose 0.4% (w/v)].
Cultures for testing elicitor activity were obtained by inoculating the stock culture on agar slants of Czapek-Dox
medium [sucrose 3% (w/v), N a N 0 3 0.3% (w/v), KzHPOj 0.1% (w/v), MgS0,.7HI0 0.05:/, (w/v), KCl 0.05%
(w/v) and FeSO,. 7H,O 0.001 % (wlv)]. After incubation for 7 d at 25 "C, spores were collected in sterile deionized
water, and 1 ml samples (approx. lo6 spores) were used to inoculate 100 ml Czapek-Dox medium in 250 ml flasks.
After 72 h incubation on a reciprocal shaker (72 strokes min-I) at 25 "C, these cultures were used for testing the
presence of substances with elicitor activity.
0001-2766 0 1986 SGM
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 03 May 2017 22:30:38
772
M . A M I N , F . K U R O S A K I A N D A . NISHI
Preparation ojculture3ltrates. mycelial walls arld myceliul extracts. Culture filtrates were separated frotn mycelia
by suction filtration. The filtrates were dialysed against several changes of water (4"C), sterilized by membrane
filtration (Millipore filter GS 0.22 pm) and kept at - 30 "C until use. Mycelial walls were prepared as described by
Yoshikawa et al. (198 1). Cell-free mycelial extract was obtained by centrifugation of the homogenized mycelia.
The clear extract was collected and kept at - 30 "C until use.
Production of 6-methosymellein in carrot suspensioii culture. Carrot cells derived from a root of carrot (Duucus
carota L. cv. Kintoki) were cultured in the synthetic medium of Murashige & Skoog (1962) according to Okamura
et ul. (1975). The substance to be tested was added to a 7-d-old culture of carrot cells (late exponential phase). The
cells were harvested after 24 h by suction filtration for determination of their 6-methoxymellein content.
Production of' 6-mefho.~ymelluinin carrot roofs. Carrot roots were surface sterilized by soaking in ethanol for
15 min and discs (5 mm thick, 20 mm diameter) were prepared using a sterile cork borer. The test substance was
placed on the cut surface of the discs. In some experiments, the cut surface was covered by a heat-sterilized glass
microfibre filter (Whatman, 25 mm diameter) beforr: inoculation. After 24 h incubation at 27 "C in sterile Petri
dishes, the discs were homogenized with sand and their 6-methoxymellein contents were determined.
Determination of' 6-metho~xymellein.Procedures f x extraction and purification of 6-methoxymellein from
cultured carrot cells and carrot roots were described previously (Kurosaki & Nishi, 1983). Extracts were developed
on TLC plates (Merck, silica gel 60 F 254) with benzene/methanol (100:2, v/v), and 6-methoxymellein content
was determined as described by a scanning method in which the absorbance of the sample was measured at
265 nm and that of the reference at 400 nm using a ddal-wavelength chromatoscanner (Kurosaki & Nishi, 1983).
Assuy of' elicitor activity. Elicitor activity was determined by the production of 6-methoxymellein in cultured
carrot cells or carrot root discs. The activity was expressed as pg 6-methoxymellein per 100 ml culture or per root
disc. Heat-labile elicitors were sterilized by membrane filtration, and heat-stable elicitors by autoclaving (1 20 "C
for 15 min) before addition to cultured carrot or carrot root discs.
Pectinolytic activity in fungal culture jiltrates. This was assayed from the rate of production of new reducing
termini resulting from the hydrolysis of polygalacturonic acid (Sigma, grade 11) at 30 "C. The reaction mixtures
contained 0.5 ml culture filtrate and 9.5 ml 0.251'4 (w/v) polygalacturonic acid sodium salt in 0.2 M-acetate buffer
(pH 5.2). The reaction was stopped by mixing 0.2 11-11 reaction mixture with 0.2 ml alkaline copper reagent and
reducing ends were measured by the modified Nelson procedure (Somoygi, 1952). Controls were run by adding the
alkaline copper reagent to the reaction mixture betore incubation.
Carrot pectic polysaccharides. Cell wall materials 'were isolated from suspensions of cultured carrot cells and
purified according to Asamizu & Nishi (1979). Pectic polysaccharides were extracted with 0.5% (w/v) ammonium
oxalate at 90 "C. The extract was dialysed against distilled water and lyophilized. The lyophilized materials were
dissolved in acetate buffer (pH 5.2) to give a final concentration of 0.1% (w/v), then autoclaved and stored at
-30 "C until use.
RESULTS
Elicitation of 6-methoxymellein production by C . globosum in carrot roots
The cut surfaces of carrot root discs were inoculated, either directly or after being covered by
glass microfibre filters, with 0.2 ml of a suspttnsion of either growing C. globosum mycelia (72 h
culture) or purified fungal walls. After 24 h incubation at 27"C, the accumulated 6methoxymellein was determined (Table 1). Carrot roots infected directly or indirectly with the
growing mycelia accumulated significant aniounts of 6-methoxymellein. The glass fibre paper
prevented direct contact of the mycelia with the root discs: microscopic examination of root
surfaces after incubation showed no sign of penetration. The wall preparations did not elicit an
appreciable increase in 6-methoxymellein.
Elicitor activity of the culture Jiltrates of C . globosum
Autoclaved and unautoclaved culture filtrates (1 ml), mycelial extract (1 ml) or purified fungal
walls (0.1 g in 10 ml deionized water) was ,3dded to 100 ml carrot cell culture to test elicitor
activity. The results (Table 2) indicated that the culture filtrates of C.globosum elicited 6methoxymellein production in carrot cell culture but that the activity was destroyed by
autoclaving. Carrot cells treated with mycelial walls or cell extract contained less 6methoxymellein than the control indicating that these fractions did not show elicitor activity.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 03 May 2017 22:30:38
Elicitation of' phytoalexin production
773
Table 1. Elicitor activity of C.globosum in carrot roots
The results are means of three separate experiments, ksu.
Elicitor activity
Treatment
(pg 6-methoxymellein per disc)
Uninoculated medium
Direct contact with mycelia
Indirect contact with mycelia'
Direct contact with mycelial wall
Indirect contact with mycelial wall*
10.5
0-01
12.3
0.69
9.2 k 0.18
1.5
0.33
11.3 _+ 0.20
* The cut surface of
the carrot root disc was covered with glass microfibre filter paper before inoculation.
Table 2. Elicitor actiziit)?oj'C. globosum in carrot cell culture
The results are the means of three separate experiments, f s u .
Elicitor activity
(pg b-methoxymellein per 100 ml culture)
,
Treatment
Uninoculated medium
Mycelial walls
Mycelial extract
Culture filtrates
Unautoclaved
8.6
6.5
3.8
18.6
k 1.40
k 1.20
f 0.52
f 1.80
1
Autoclaved
(1 20 "C for 15 min)
8.8 k 1.31
3.8 f 0-30
4.6
0.40
8.4 f 0.51
Releuse of' heat-stuble elicitor .from carrot cell homogenates
Boiled and unboiled culture filtrates of C. globosum were added to carrot cell homogenates and
incubated at 30 "C. At intervals, portions of the incubation mixture were withdrawn, autoclaved
and added to carrot cell culture to test elicitor activity (Fig. 1). The elicitor activity of the
incubation mixture was low at time 0 but it increased with time and two peaks of elicitor activity
appeared during the incubation of cell homogenates with the unboiled culture filtrates. Boiled
culture filtrates did not enhance the elicitor activity of the homogenates during the incubation.
These results suggested that a heat-labile factor in the culture filtrates of C . globosum stimulated
the release of heat-stable secondary elicitors from carrot cells. Since we have shown that a
commercial pectinase elicits the production of phytoalexin in carrot cells (Kurosaki & Nishi,
1984), we did experiments to determine whether the heat-labile factor derived from the fungus
was a pectinase or related enzyme.
Releuse oj' heat-stable elicitor-.from carrot pectic polysaccharides and citrus pectin
Pectic polysaccharides of carrot and citrus pectin were digested by culture filtrates of C.
globosum and the hydrolysates were assayed for heat-stable elicitor (Fig. 2). The pectic fraction
of carrot elicited 6-methoxymellein accumulation ; elicitor activity was increased by digestion
with unboiled culture filtrates of C. globosum for up to 2 h, but further digestion resulted in
complete loss of elicitor activity. Unhydrolysed citrus pectin did not elicit 6-methoxymellein
production, while incubation of citrus pectin with the fungal culture filtrates resulted in multiple
peaks of heat-stable elicitor activity.
Coincidence oj elicitor and pectinolytic acticities in the .fiingal culture filtrates
Preliminary experiments to characterize the fungal elicitor showed that the elicitor activity was
destroyed by heat or incubation with sodium periodate. This suggested that the elicitor might be
a glycoprotein in which both peptide (heat-sensitive) and sugar moieties (periodate-inactivated)
are essential for elicitor activity. Teinperature- and periodate-sensitivity were therefore used to
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 03 May 2017 22:30:38
774
M . A M I N , F . K I J R O S A K I A N D A . NISHI
I
0
1
I
2
3
4
5
Incubation time (h)
1
1
6
7
Fig. 1. Release of heat-stable elicitor from carrot cell homogenates. Cultured carrot cells (7 d old, 4 g
fresh weight) were suspended in 10 ml acetate buffer (pH 52), killed by autoclaving and homogenized
by sonication. A 10 ml portion of the homogenate was mixed with an equal amount of filter-sterilized
culture filtrate of C.globosum and incubated at 30 "C. At intervals, 2 ml samples were withdrawn,
chilled in an ice bath and centrifuged. The supernatants were autoclaved to destroy the heat-labile
elicitor and assayed for heat-stable elicitor activity as described in Methods. Control experiments were
run with fungal culture filtrates boiled at 100 "C for 15 min. 0,
Boiled fungal filtrates; 0 , unboiled
fungal filtrates. Each point represents the mean of two separate experiments, the results of which
agreed to within 10%.
0
1
2
3
4
0
1
2
3
4
Incubation time (h)
Fig. 2. Release of heat-stable elicitors from carrot pectic polysaccharides and citrus pectin.
Experimental procedures were the same as for Fig. 1 except that carrot pectic fraction and citrus pectin
(each 0.1%, w/v) were used instead of carrot cell homogenates. (a) Elicitor released from carrot pectic
fraction with boiled (0)
and unboiled ( 0 )C. globosum culture filtrates and (b) elicitor released from
citrus pectin with boiled (0)
and unboiled ( 0 )culture filtrates. Each point represents the mean of two
separate experiments, the results of which agreed to within 10%.
correlate elicitor and pectinolytic activities. The results in Fig. 3(a) show that the elicitor and
pectinolytic activities of C. globosum culture filtrates exhibited similar profiles of temperature
sensitivity except at 40 "C. Incubation at 613 "C for 15 min caused loss of 7 0 4 0 % of both the
activities (Fig. 3 b). When culture filtrates were incubated with sodium periodate (final
concentration, 0.014 M) at 30 "C, the elicitor activity decreased progressively in parallel with the
pectinolytic activity during the incubat ion (Fig. 4).
Elicitor activity in the culture filtrates of other fungi
Pectinolytic enzymes have been detected in many fungi and bacteria (Rexova-Benkova,
1976). We tested the culture filtrates of threl? other fungi for the presence of heat-labile elicitor
and the ability to release heat-stable elicitor from citrus pectin (Table 3). The results, indicating
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 03 May 2017 22:30:38
Elicitation o j phytoalexin production
775
120
100
h
8
80
W
.-wx 60
2
40
20
n
"
0
20 40 60 80 100 120 0
Temperature ( O C)
5
10
15
Time (min)
20
Fig. 3. Effect of temperature on elicitor and pectinolytic activity of C. gfobosum culture filtrates. C.
globosum was grown in Czapek-Dox medium for 72 h at 25 "C. The culture filtrate (20 ml) was dialysed,
lyophilized and redissolved in 10 mlO.2 M-acetate buffer (pH 5.2). and 1 ml portions were incubated at
temperatures of 0 to 120 "C for 15 min (a) or at 60 "C for 0 to 20 min (6).After incubation, the solutions
were chilled in an ice bath and a 0.5 ml portion was used for the assay of elicitor activity (a);the other
0.5 ml was assayed for pectinolytic activity (0).
The activities are expressed as percentages of the value
obtained when the incubation was done at 0°C. Each point represents the mean of two separate
experiments, the results of which agreed to within 10%.
100
-
80
h
8
A
.-+
2
60
40
20
0
0
1
2
3
4
5
6
7
Incubation time (h)
Fig. 4. Effect of periodate treatment on elicitor and pectinolytic activity of C. globosum culture filtrates.
Culture filtrate was prepared as described for Fig. 3, and 1 ml portions were incubated with 20 pl0.7 Msodium periodate at 30°C. At the indicated times, 0.1 ml was withdrawn, mixed with 2p1 ethylene
glycol and diluted with 0.9 mlO.2 M-acetate buffer (pH 5.2). A 0.5 ml portion was used for the assay of
elicitor (a)and the other 0.5 ml was assayed for pectinolytic activity (A).
Controls (open symbols) were
run as above, but with ethylene glycol added before the addition of periodate. The activities are
expressed as percentages of the value obtained immediately after the addition of periodate (0 time).
Each point represents the mean of two separate experiments, the results of which agreed to within 10%.
the heat sensitivity of the elicitor in the culture filtrates and the release of heat-stable elicitor
from citrus pectin after 30 min digestion, supported the idea that pectinolytic activity of these
fungi served as an elicitor of phytoalexin accumulation in carrot.
DISCUSSION
The results presented in this paper strongly suggest that phytoalexin accumulation in carrot
suspension culture is triggered by extracellular pectinolytic enzymes released from various fungi.
The results in Table 1 indicate that direct contact of carrot tissue with fungal mycelia is not
necessary for elicitation of phytoalexin production and hence that the elicitor activity resulted
from substance(s) diffusing either from the fungus or the carrot root. The accumulation of 6methoxymellein in carrot suspension cultures after the addition of C. globosum culture filtrates
and the loss of elicitor ability by autoclaving indicated that the elicitor is a heat-labile substance
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 03 May 2017 22:30:38
776
M . AMIN, F. KUROSAKI A N D A . NISHI
Table 3. Elicitor activity in !he culture filtrates of various jungi
(a)Culture filtrates of fungi (1 ml) were obtained after cultivation for 72 h in Czapek-Dox medium and
added to a carrot cell suspension after being hcated (100 "C, 15 min) or unheated. (b) Culture filtrate
(1 ml) was mixed with 1 ml 0-1% citrus pectin in acetate buffer (pH 5-2) and incubated for 30 min at
30 "C. After the treatment, the samples were autoclaved and added to a carrot cell suspension. Control
experiments were run with heat-inactivated (100 "C, 15 min) fungal culture filtrate. The results in both
( a )and (b)are means of three experiments, +set. The 6-methoxymellein contents of carrot cell cultures
supplied with 0.2 M-acetate buffer alone and with 0.1 % citrus pectin in 0.2 M-acetate buffer were 8.7 .+
0.5 and 5.2 f 0.7 pg per 100 ml culture, respectively.
Elicitor activity (pg 6-methoxymellein per 100 ml culture)
( a ) Culture filtrates of fungi
Fungus
C. globosum
B . cinerea
F . monillforme
H . oryzae
rpL.--
v
Heated at 100°C
for 15 min
Unheated
8.6 k 0.2
11.3 k 0.9
7.5
0.5
16.5 k 0.5
z2.2
35.0
33.8
37.5
k
k
2
k
2.2
0.7
3.7
1.5
(b) Citrus pectin incubated
with culture filtrates of fungi
7
Heated at 100°C
for 15 min
4.8 k 0.6
10.5 h 1.3
9.5 k 0.5
16.7
1.7
Unheated
30.2
48.8
50.0
52.0
k 2.2
k 3.8
k 3.0
k 4.0
released into the culture filtrates of the fungus (Table 2). The coincidence of the heat sensitivity
(Fig. 3) and periodate inactivation (Fig. 4) profiles of elicitor and pectinolytic activity in the
culture filtrates of C. globosum suggests that pectinolytic activity in the culture filtrates is
essential for elicitor activity. Culture filtrate 5 of C. globosum catalysed the release of heat-stable
elicitors from carrot cell homogenates, pectic: fraction of carrot cell wall and citrus pectin (Figs 1
and 2). These results suggest that the heat-stable elicitors may be hydrolysis products of the
pectic fraction of plant cell wall released by the activity of pectinolytic enzymes in the culture
filtrates.
We observed multiple peaks of elicitor activity when carrot cell homogenates and citrus
pectin were incubated with fungal culture filtrates. Pectinolytic enzymes that catalyse the
release of active elicitors may also inactivate them during further incubation (Bruce & West,
1982; Davis et al., 1984). The pectinase-released elicitors are probably not a single molecular
species but rather heterogeneous cellular fragments containing oligogalacturonides as essential
moieties. Jin & West (1984) have shown that galacturonide oligomers of appropriate size are
responsible for the elicitor activity in casbene synthesis; oligomers smaller than the nonamer
showed little or no elicitor activity. Nothnagel et al. (1983) reported that several distinct elicitors
for glycinol production are released from both cell walls of soybean and citrus pectin by partial
acid hydrolysis. The fact that the pectic fraction of carrot had elicitor activity before digestion
by the culture filtrates (Fig. 2) may be attributable to the extraction procedure, which involves
pronase treatment. Pronase has been shown to release heat-stable elicitor from carrot cell
homogenates (Kurosaki & Nishi, 1984). The difference in response of cultured carrot cells to the
hydrolysates of carrot pectic fraction and citrus pectin may be partly related to differences in
their chemical composition. For instance, the pectin preparation of carrot contained 20 %
galacturonic acid (Asamizu & Nishi, 1979) while citrus pectin contains 75% uronic acid.
Our present findings are analogous to those obtained by Lee & West (198 1 a, b) and Davis et nl.
(1984). They demonstrated that pectin-degrading enzymes from R . stolonifer and E. carotovora
are elicitors of phytoalexins in castor bean and soybean, respectively. It has also been shown that
these enzymes can release heat-stable elicitor from plant cell wall and citrus pectin. These
observations, together with the present results, suggest that phytoalexin production in many
plant species is triggered by pectinolytic endcymesof infecting micro-organisms, and the plant
cells recognize the partial degradation products of cell wall polymer released by the enzymes as
the endogenous elicitor. Unlike soybean, however, carrot cells did not respond to fungal wall
preparations, which are a potent elicitor of phytoalexin production in leguminous plants (West,
1981).
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 03 May 2017 22:30:38
Elicitation of phytoalexin production
777
REFERENCES
ASAMIZU,T. & NISHI,A . (1979). Biosynthesis of cell
wall polysaccharides in cultured carrot cells. PIanta
146, 49-54.
BRUCE, R. J . & WEST, C. A . (1982). Elicitation of
casbene synthetase activity in castor bean. Plant
Physiology 69, 1181-1 188.
DAVIS,K . R., LYON, G. D., DARVILI.,
A . G. &
ALBERSHEIM,
P. (1984). Host--pathogen interactions.
XXV. Endopolygalacturonic acid lyase from Erwinia
carotovoru elicits phytoalexin accumulation by releasing plant cell wall fragments. Plant Physiology
74, 52-60.
GRISEBACH,H. & EBEL, J . (1978). Phytoalexins,
chemical defense substances of higher plants'?
Angewandte Chemie International (Edition in English)
17, 635 647.
JIN, D. F. & WEST, C. A . (1984). Characteristics of
galacturonic acid oligomers as elicitors of casbene
synthetase activity in castor bean seedlings. Plant
Physiology 74, 989 -993.
KEEN,N . , PARTIDGE,
J . & ZAKI,A . (1972). Pathogenproduced elicitor of a chemical defense mechanism
in soybean monogenically resistant to Phytophthora
megasperma var. sojue. Ph~~topathologjJ
62, 768.
KuC, J . (1972). Compounds accumulating in plants
after infection. In Microbial Toxins P'ZII, pp. 21 1 247. Edited by K . Solomon. New York: Academic
Press.
KUROSAKI,F. & NISHI, A. (1983). Isolation and
antimicrobial activity of the phytoalexin 6-methoxymellein from cultured carrot cells. Phyrochemnrry 22,
669-672.
KUROSAKI,F. & NISHI, A . (1984). Elicitation of
phytoalexin production in cultured carrot cells.
Physiological Plant Pathology 24, 169 1 76.
LEE, S. C. & WEST,C. A . (1981a). Polygalacturonase
from Rhizopus stolonijer, an elicitor of casbene
synthetase activity in castor bean (Ricinus communis
L.) seedlings. Plant Physiology 67, 633-639.
LEE, S. C. & WEST, C. A . (1981b). Properties of
Rhizopus stolonijer polygalacturonase, an elicitor of
casbene synthetase in castor bean (Ricinus communis
L.) seedlings. Plant Physiology 67, 640-645.
MURASHIGE,
T. & SKOOC,F. (1962). A revised medium
for rapid growth and bioassays with tobacco tissue
cultures. Physiologia plantarum 15, 473-497.
NOTHNAGEL,
E. A., MCNEIL,M., ALBERSHEZM,
P. &
DELL,A . (1983). Host--pathogen interactions. XXII.
A galacturonic acid oligosaccharide from plant cell
walls elicits phytoalexins. Plant Physiology 71, 916926.
OKAMURA,
S., SUEKI,K . & NISHI,A. (1975). Physiological changes of carrot cells in suspension culture,
during growth and senescence. Physiologia plantarum
33, 251-255.
REXOVA-BENKOVA,
L. (1976). Pectic enzymes. Adaances in Carbohjvdmte Chemistrji and Biochemisrrj.
153, 375-380.
SOMOGYI,M. (1952). Notes on sugar determination.
Journal of Biological Chemistrj. 195, 19-23.
WEST,C. A. (1981). Fungal elicitors of the phytoalexin
response in higher plants. Naturwissensclrafien 68,
447-457.
YOSHIKAWA,
M., MATAMA,M . & MASAGO,H . (198 1 ).
Release of a soluble phytoalexin elicitor from
mycelial walls of Phytophthora megasperma var. sojae
by soybean tissues. Plant Physiology 67, 1032-1035.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 03 May 2017 22:30:38