CALIFORNIA STATE UNIVERSITY, NORTHRIDGE
FATTY ACID CONTENT OF MEMBRANES OF PHYTOPIITHORA
CINNAMOMI IN CHOLESTEROL SUP_PLEMENTED MEDIA
A thesis submitted in partial satisfaction of the
requirements for the degree of Master of Science in
Biology
by
Richard Grant Ballard
January, 1978
The Thesis of Richard Grant Ballard is approved:
-
California State University, Northridge
ii
ACRNOWLEDGEMENTS
I would like to express my great appreciation to Dr. Donald
Bianchi for his .great patience with a slow learner and to my many
friends who were called upon frequently to bolster frequently
sagging spirits.
iii
TABLE OF CONTENTS
PAGE
ACKNOWLEDGEMENT
----------------------------------------------
LIST OF TABLES AND FIGURES
--------------------~--------------
Hi
v
vii
ABSTRACT
-INTRODUCTION
1
5
MATERIALS AND METHODS
RESULTS ------------------------------------------------------ 13
DISCUSSION
55
REFERENCES
62
iv
LIST OF TABLES AND FIGURES
TABLE
PAGE
I
Growth Rate of P. cinnamomi witn Increasing Concentration of Cholest;rol ------------------------------------- 14
II
Gas Chromatographic Analysis of Fatty.Acid Extract.
Showing Peak Identification ----------------------------- 20
III
Fatty Acid Content of P. cinnarnomi. Vegetative Mycelia
Levels Expressed as Fractions or Percent of Total Fatty
Acid ---------------------------------------------------- 24
IV
Fatty Acids of the Polar Lipid Fraction of Vegetative
P. cinnamorni Mycelia Expressed aa Fraction of Total
Fatty Acid Content in Polar Lipid Fraction -------------- 31
V The Effects of Cholesterol on Differentiation of
Sporangia in P. cinnarnomi ----------------------·-------- 43
VI
Fatty Acids of the Polar Lipid Fraction of Induced
P. cinnarnomi Mycelia Expressed as Fraction of Total
Fatty Acid Conten~ in Polar Lipid Fraction -------------- 44
FIGURE
1
Growth Kinetics of P. cinnamomi at 25°C With Various
Concentrations of Cholesterol Supplement ---------------- 15
2 Equivalent Chain Lengths of Fatty Acid Methyl Esters
on DEGS Column at Conditions Specified in Text ---------- 21
3 Total Fatty Acids Percent Compositional Changes of
Myristic Acid (14:0) ------------------------------------ 25
4
Total Fatty Acids Percent Compositional Changes of
Palmitic Acid-- ( 16 :0) ------------------------------------ 27
5 Total·Fatty Acids Percent Compositional Changes of
Oleic Acid (18:1) --------------------------------------- 29
6
Total Fatty Acids Percent Compositional Changes of
Linoleic Acid (18:2) ------------------------------------ 33
7 Polar Lipids of Vegetative Mycelia Percent Compositional Changes of Myristic Acid (14:0) Component -------- 35
v
FIGURE
PAGE
8
Polar Lipids cf Vegetative Mycelia Percent Compositional Changes of Palmitic Acid (16:0) --·--------------- 37
9
Polar Lipids of Vegetative Mycelia Percent Compo.sitional Changes of Linoleic Acid (18:2) ------------------ 39
10 Polar Lipids of Induced Mycelia Compositional Changes
of Myristic Acid (14:0) ------------------~~------------- 45
11 Polar Lipids of Induced Mycelia Compositional Changes
of Palmitic Acid (16:0) --------------~------------------ 47
12 Polar Lipids of Induced Mycelia Compositional Changes ·
of Stearic Acid (18:0) ---------------------------------- 49
13 Polar Lipids of Induced Mycelia Compositional Changes
of Linoleic Acid (18:1) --------------------------------- 51
14 Polar Lipids of Induced Mycelia Compositional Changes
of Linoleic Acid {18:2) --------------------------------- 53
vi
ABSTRACT
FATTY ACID CONTENT OF MEMBRANES OF PHYTOPHTHORA
CL"mAMONI IN CHOLESTEROL SUPPLEMENTED MEDIA
by
Richard Grant Ballard
Master of Science in Biology
January, 1978
Phytophthora cinnamomi Rands, a root pathogen of worldwide
importance, is a member of a family of fungi that lacks the ability
to synthesize sterols.
Sterols are considered important as hor-
mones, membrane structure stabilizers, and steroid precursors.
~he
effect of cholesterol on growth, asexual reproduction, total
fatty acid content and fatty acid content of polar lipids of vegetative and asexually reproducing cultures was studied.
Cholesterol
supplementation was shown to enhance growth and sporulation.
Levels of total fatty acids, myristic acid (14:0) and linoleic acid
(18:2) were shown to increase while palmitic acid (16:0) decreased
with increasing amounts of cholesterol supplementation.
No recog-
nizable trends were observed in the fatty acid content of the polar
lipid fraction of vegetative or sporulating mycelia, though
definite rearrangements occurred with increasing levels of cholesterol supplementation.
Sporulation response also increased as
concentration of cholesterol supplementation increased.
vii
INTRODUCTION
Phytophthora cinnamomi Rands is a root pathogen of worldwide
importance (Zentmyer and Erwin, 1970).
The ~ungus infects the
roots of such diverse and economically important plants as avocado,
pineapple, pear, pine, eucalyptus as well as camellia, heather,
azalea, rhododendron.
also on conifers.
It causes root
ro~
not only on hardwoods but
It is a major cause of seedling root rots and
damping-off disease.
It occurs in soils with poor drainage, the
excess moisture is necessary for pathogenesis.
As roots rot, leaves
of the host become light colored and wilt; this occurs even if the
soil is moist.
Trees decline over a period
~f
years (Westco.tt,
1971).
Phytophthora and Pythium are interesting because of their inability to synthesize -sterols (Hendrix, 1964, 1965, 1970), (Chee and
~urner,
1966), (Haskins, et al, 1964).
The lack of a sterol syn-
thesizing mechanism is rare in plants and animals.
Insects and
certain protozoans are the only other eucaryotes that lack this
abilitynormally (Hendrix, 1970).
The Pythiaceae are unique because
they can grow vegetatively without sterols, but they require sterols
for differentiation of reproductive structures.
receive sterols
~s
a supplement in their diet.
Insects must
Protozoa manufacture
a sterol .analogue (Ferguson, et al, 1975).
It has been shown convincingly that addition of sterols to the
medium not only increases growth rate of hyphae, but also permits·
asexual and sexual reproduction in previously sterile mycelia of the
Pythiaceae (Hendrix, 1964, 1965), (Haskins, et al, 1964), (Chee and
1
2
Turner, 1966), (Seitsma and Haskins, -1967), (Hendrix, et al, 1969).
Schlosser and Gottlieb (1968) reported an increase in the growth
rate of several species of Pythium by 65-1001. if cholesterol was
added to the growth medium.
They found the rate of metabolism in-
creases in sterol supplemented mycelia, and produces an increase in
phosphorylated compounds (ATP).
They were able to show that one of
the cellular sites of action of sterolsis the cellular (Plasma)
membrane.
Optimum synthetic capacity is reached only when the
sterols become incorporated into the membrane structure.
Schlosser, et al, (1969) used labelled acetate- 14c, a usual
precursor to sterol, plus other labelled intermediates, and found
that the inability of the Pythiaceae to synthesl~e sterols is
generally lacking, at no point in the known synthetic pathway for
cholesterol does synthesis begin.
This group of fungi compensated
for this inability by developing a very efficient system for the
absorption and interconversion of sterols.
Hendrix (1975) deter-
mined that the minimum sterol concentration for oospore production
in Phytophthora sp. to be about 10-6M. with maximum activity at
.
concentrat1ons
of about 10 -4M •
The primary location of sterols in fungi has been shown to be
in the plasma membrane (Olsen, 19·73).
Sietsma and Haskins (1968)
and Olsen (1973) found a correlation between the amount of sterols
in the mycelia and the inhibitory effect of polyene antibiotics.
Pythiaceae, in the absence of sterols, were shown to be insensitive
to polyene antibiotics, but when grown in the presence of sterols,
growth was inhibited.
Child, Defago, and Haskins (1969) showed
3
that when unsupplemented mycelia of the
Pythiac~ae
are placed in
disti-lled water, certain constituents (nucleotides, nitrogen,
protein) were prone to leak out.
duced this tendency significantly.
The addition of cholesterol reT.his stabilization was reversed
when polyene antibiotics were added to the substrate.
Polyene
antibiotics are thought to combine with cholesterol in membranes
and as a consequence produce a hole in the membrane.
At the present time there is interest by many_workers on the
effect of cholesterol on the plasma membrane.
Specifically the
interest is in the changes that occur on introduction of this compound since it causes such pronounced changes as increased metabolic
activity and morphological differentiation of sexual and asexual
reproductive structures.
Brushaber, et al, (1972) working with Pythium sp. found no
significant differences in fatty acid composition with cholesterol
supplementation.
They found, however, that the proportion of
phosphatidyl serine relative to other phospholipids is reduced by
one half in mycelia grown with cholesterol.
Major phospholipids
reported were phosphatidyl ethanolamine, phosphatidyl serine, and
phosphatidyl choline.
Hendrix and Rouser (1976) working with
Phytophthora parasitica
~
nicotianae found that when cholesterol
was present or absent from the medium phospholipid content of
Phytophthora did not vary appreciably.
In
~rtificial
systems., cholesterol has been shown to restrict
the motion of phospholipid hydrocarbon chains so they are less fluid
than in the liquid crystalline phase of pure phospholipid systems
4
(Rothman and Engelman, 1972).
Finean (1973) suggested that the most
significant feature from a structural viewpoint may be the average
level of unsaturation in the lipid phase as
~
whole.
Studies have
indicated that cholesterol can be accomodated in phospholipid up to
a limiting proportion.
This proportion is determined largely by
the degree of unsaturation of fatty acid chains in the phospholipid.
Ferguson, et al, (1975) found ergosterol supplementation in
Tetrahymena pyriformis altered the proportions of the fatty acids,
although not all lipid classes were affected to the same extent.
They noted changes in the shortening of fatty acyl chain length in
acids of normal series and a lowering in
deg~ee
of unsaturation.
Each class of polar lipid has a distinctive fatty acid composition.
The work of Ferguson and associates with Tetrahymena suggests there
may be significant changes in the fatty acid composition of membranes when sterol supplemented in the medium replaces the analogue
tetrahymanol.
This study attempts to correlate cholesterol supplementation
with changes in growth rate and sporulation response and postulated
fatty acid quantitative compositional changes in Total and Polar
vegetative lipids; and also fatty acid composition of polar lipids
of asexually induced mycelia were compared.
METHODS AND MATERIALS
Phytophthora cinnamomi strain SB-216-1 was obtained from the
American Type Culture Collection of
Baltimor~,
Maryland.
Mainten-
ance cultures were grown on agar plates containing a defined medium.
Transfers were made every two weeks.
Inoculum was prepared by frag-
menting mycelia growing on the surface of the agar plates into fine
pieces with an inoculating loop, and immediately suspending these
pieces in sterile water.
Portions of the
·-
inoculat~on
suspension
were then transferred aseptically to the sterilized flasks of broth.
For experimental studies, cultures were grown in 500 ml of liquid
defined medium contained in 3000 ml Fernbach flasks.
inoculated with about 2 ml of suspension.
Each flask was
This usually provided
each flask with about 50 pieces of mycelia, providing at least 50
centers of growth per culture.
All cultures were incubated at 25°C
in still culture in a Scientific Products incubator equipped with
fluorescent lights providing about 200 ft. candles illumination to
the cultures.
Sporangia! Induction
Sporangia were induced by the method developed by Chen and
Zentmyer (1970) and modified by Hwang, et al, (1975).
Fragmented
pieces of agar with mycelia from 2-3 day old cultures were transferred to sterilized discs of washed, uncoated cellophane about 70mm
in diameter laid over fresh defined agar medium.
Cultures were
incubated for 24-48 hours at 25°C in the lighted incubator.
The
cellophane membranes with mycelia were then removed from the medium
5
6
and placed in glass petri dishes containing 50 ml of defined broth
diluted 1:3 with distilled water (.5% Sucrose) and incubated for 24
The broth was removed and 2~ ml of mineral salt
hours at 25°C.
solution of Chen and Zentmyer (1970) was added, immediately drained
and quickly suspended again in the mineral salt solution.
0
-
tion was at 25 C with continuous fluorescent light.
Incuba-
When sporangia
began to form (after about 9 hours) the mineral salts solution was
removed and the mycelia were incubated an additional 12 hours in
moist conditionse
Defined Growth Medium
The composition of the defined medium was modified from that
of Chee and Newhook (1966):
1.0 gram
o.s
0.5
Fe-EDTA
Oe6 ml (from stock solution to give final
concentration of 6 ppm of Fe)
Trace elements
1.0 ml (from stock solution to give final
concentration of 0.5 ppm of B (H Bo ),
3 3
0.5 ppm of Mn (Mnso • 4H o>, 0.5 ppm Zn
4
2
(Znso • 7H o>, 0.02 ppm Mo (NH )Mo o •
4
7 24
4
2
4H
o).
2
I.-Glutamine
2.5 grams
Thiamine
0.5 mg
7
0.5 grams {dissolved separately and added
last to avoid precipitation)
Deionized water to make a final volume of 1 liter
The pH of the medium was adjusted with lN NaOH to 6.3 - 6.5. 10
grams of sucrose was dissolved in a small volume of water, autoclaved separately, and added to the medium as it was cooling.
The
medium was autoclaved at 15 lb. pressure at 121°C for 15 minutes.
When cholesterol was supplemented it was suspended.in N,N-dimethyl
formamide (DMF) at desired concentrations and added to the medium
at a DMF concentration of 0.2% to the total volume of the medium
before autoclaving.
Nutritional tests (Chee and Newhook, 1966)
showed Calcium to have no significant effect on growth and sporulation of
!•
cinnamomi.
In broth cultures NaN0
3
was substituted for
Ca(N0 ) with no adverse effects. Cholesterol was added to the
3 2
6
medium in several concentrations, 2 x 10- 7 , 2 x 10- , 2 x 10-5 ,
2 x 10
-4
M., the highest of which approached saturation.
Chen and Zentmyer (1970) mineral salt solution for induction
of sporulation:
Ca(N0 )
3 2
O.OlM
KNOJ
0.005M
MgS0
4
0.004M
Deionized water to 1 liter
FeEDTA
1 ml per liter added after autoclaving
_ through millipore filter
8
Harvesting
Mycelia were usually grown for about five days in defined
broth.
At the fifth day following inoculati_on, cultures are in the
final stage of logarithmic
growth~
Mycelia were harvested using
suction and a buchner funnel, the harvested mycelial mats were
washed immediately with about 1 liter of distilled water, transferred into culture tubes (25 x 105mm) and quickly frozen at -20°C.
The frozen mycelial mats were lyophilized within a week after harvesting.
0
The temperature of the tubes was reduced to about -80 C
before lyophilization.
After about 18 hours, the tubes with dry
samples were removed from the Virtis Freeze Dryer, sealed with
paraffilm, and stored at room temperature in a desiccator with anhydrous Caso •
4
Extraction of total fatty acids
Approximately l.gram of
~yophilized
tissue was weighed out and
transferred to a test tube (15 x 150mm) with a teflon lined screw
cap5
Saponification was accomplished by adding 10 ml KOH in aqueous
methanol solution, (Sg KOH, 50 ml DI water, 50 ml methanol), N was
2
added into the tubes and the tubes then placed at 100°C in a Fisher
Isotemp Incubator for 12 - 14 hours (overnight).
The tubes were
cooled .quickly under running water and KOH solution was added to reestablish the original volume.
The contents of the screw cap tube
were centrifuged in a plastic centrifuge tube (30 x lOOmm) at 5000
rpm for 25 minutes to sediment cell debris.
Unsaponified material
was removed and discarded by extracting it from the resulting super-
9
natant with 2 x 15 ml portions of hexane (epiphase drawn off through
the top of the separatory funnel with a pipet).
The methanol hypo-
phase was acidified to about pH 1.5 with concentrated H so •
2 4
The
solution was centrifuged for 15 minutes at 5000 rpm to remove the
precipitate formed by addition of the acid.
F~tty
acids were ob-
tained by extracting the resultant supernatant with 3 x 15 ml
portions of hexane which was pooled in a 100 ml beaker.
Solvent
volume was reduced to 8-12 ml by evaporation in a stream of air in
the fume hood.
The fatty acid solution was transferred to a teflon
lined screw cap test tube (15 x 150mm) and the solvent was reduced
to 2-3 ml under a gentle stream of N •
2
Methylation of fatty acids
Fatty acids were methylated with 2 ml of
14~
Boron trifluoride
in Methanol by heating in a boiling water bath for 2-3 minutes.
After cooling, 4 ml of distilled water was added and the resulting
mixture was extracted 3x in 10 ml portions of hexane to recover
fatty acid methyl esters.
Extracts were pooled and transferred to
Teflon lined screw cap test tubes (15 x 45mm) and stored at -20°C.
Immediately prior to injection into the column of the Gas-liquid
chromatograph (GLC), solvent volume was reduced from 4 ml to approximately·O.l ml under N , sample size injected ranged from 2 to 10 ml.
2
Extraction and fractionation of total lipids
Lyophilized tissue was extracted 2x with chloroform-methanol
(2:lv/v) at room temperature with about 50 ml of solvent.
The ex-
tract was filtered through a sintered glass filter to remove debris.
10
Solvent volume was reduced to about 5 ml under the fume
remainder was reduced under N •
2
hood~
The
The dry crude lipid extract was
taken up with successive small volumes (2-5 .ml) of low phase solvent,
upper and lower phases were
obtaine~
from the mixture of chloroform-
methanol-water (200:100:75v/v/v), and percolated through a Sephadex
purification column (1 em id x 75 em) (W~thier, 1966) equipped with
a sintered glass bottom •. The column is packed 10 em high with
Sephadex G-25 beads.
The purpose of the column is. to remove non-
lipid contaminants that may have been extracted from the fungal
tissue with the lipids.
Purified lipids were eluted from the column
with sufficient low phase solvent to make a total of 25-30 ml of
effluent.
Eluate is concentrated to a small volume, then added to a
Silicic Acid column.
This column is prepared by adding 5 grams of
chloroform to make a slurry.
~olumn
The slurry is then poured into the
which is equipped with a sintered glass plug (2 em id x 40 em).
The column is washed with an additional 50 ml of chloroform.
The
purified lipid is added to the column in a small volume of low phase
solvent.
The column is developed with 100 ml volumes each of chloro-
form, acetone, and methanol.
After the collection of fractions,
solvent volume was reduced to about 5 ml.
At this time fraction
samples were subjected to thin layer chromatography using a hexanemethanol-acetic acid (80:20:lv/v/v) solvent to determine the efficiency of fractionation.
Spots were visualized by spraying the
plate with cupric acetate (3%) in orthophosphoric acid (Barber and
Mead, 1975), and charring at 100°C for 1-2 hours.
The polar lipid
samples were then saponified in 5% KOH in aqueous methanol overnight
11
0
at 100 C in the Isotemp oven.
The saponified polar lipids were acidified to pH 1.5 and centrifuged to remove the precipitate formed.
extracted 3x with 15 ml of hexane.
was reduced to about 5 ml.
of BF
3
The fatty acids were
After which the solvent volume
The samples were methylated with 2 ml
in methanol in a boiling water bath for 3-5 minutes.
Fatty
acid methyl esters were separated from this solution by adding 4 ml
of distilled water and extracting 3x in 10 ml of hexane.
The same
procedure for GLC injection as utilized for total fatty acids was
used for the polar lipid fatty acids.
Gas Chromatography
The Gas Chromatograph used for all experiments was a Beckman
GC-72-5 equipped with a six foot stainless steel column (O.D.
a
1/8
inch) packed with 151. Diethyl glycol succinate (DEGS) as the partition liquid and chromasorb WHP (mesh size 80/100) as support
material.
Sample detection and measurement was accomplished by a
flame ionization detector (FID) connected to a Beckman Model 1005
Recorder equipped with a DISC Integrator for measurement of peak
area.
Helium was used as a carrier gas with hydrogen and air for
the FID.
Gas flow and temperature settings of the chromatograph:
Column
165°C
Detector
265°
Inlet
Line
12
Air
300cc/minute
-Hydrogen
45cc/minute
He flow
95cc/minute
He left rotameter
20cc/minute
He right rotameter
20cc/minute
Retention times of known solutions of Methylated fatty acids
obtained from Sigffia Chemical Corporation were measured on the Gas
Chromatograph.
Retention time of peaks of unknoWn·samples were
compared with known samples utilizing a plotting device of Miwa,
et al, (1960), the Equivalent Chain Length Graph.
Known samples
were mixed with unknowns as a second test, increased peak height of
these mixtures compared with unknown peaks alone helped identify
location of unknown peaks.
RESULTS
The Effects of Cholesterol on Growth
Cholesterol was added to the defined
gr~wth
concentrations up to near saturation level.
daily from the second to the eighth day.
measure of growth.
Figure 1.
Cultures were harvested
The
is described in the Materials and Methods.
medium in selected
~ethod
of harvesting
Dry weight was used as a
The results of this experiment are shown in
By the second day the cultures were in the log phase of
growth which continues until about the fourth day.
Initially, there
is no noticeable effect of the cholesterol on growth.
However, by
the end of the log phase, more growth is produced in the cultures
supplemented with cholesterol except at saturation levels.
Supple-
mentation of 0.385 mg/500 ml.(2 x 10-6M) cholesterol causes an increase in dry weight by a factor of about 1.5 which appears to remain so into the stationary phase of growth. Supplementation with
. 5
.
3.85 mg/500 ml (2 x 10- M) does not stimulate or inhibit growth
during log phase but causes an extended period of logarithmic growth
and by the ninth day has produced more growth than the cultures at
lower concentrations.
Rate of growth as well as total growth is
also affected by cholesterol supplementation.
grams/day are shown in Table I.
The growth
rate~
in
Again the growth rate increases
with increasing concentration of cholesterol except at near saturation levels (3.85 mg/500 mO.
Morphological Changes Induced by Cholesterol
The principal change observed in cultures supplemented with
13
14
TABLE I
Growth Rate of
!•
cinnamomi with
Increasing Concentration of Cholesterol
Supplement
Rate (g/day)
(mg)
o.o
mg
0.090
0.038
0.099
0.385
0.120
3.850
0.063
FIGURE 1
Growth Kinetics of~ cinnamomi at 25°C
With Various Concentrations of Cholesterol Supplement
16
..
•7
.s
, ,"'
I
I
I
I,
/
.3
I
4 •
,t
I
I
, , "'
,,'
./·
,.'
.
. ..;..--·-~
.
/.'
/'
/
,"'
/•'
.
.·
;?·
'
1 .
,,. I :
1
1
I ....
J
•
I
I
, r:
i
:r
I
-.
I
:1
I I)
.e...
M
-
•
1
I
'o.o
J
I
mg
0.038 mg
ll
I
.07
:1
0.385 mg
I
J
If
1:
.05
..........
3.850 mg
I'
J.:
.03
~------------~---+------~----------~----~~----------._----4
1
2
3
4
5
Time (Days)
6
7
8
9
17
chole~terol
was the greatly enhanced
production~£
aerial hyphae.
It was observed that in supplemented cultures hyphal gr-owth was
primarily above the surface of the agar.
I~
unsupplemented cultures,
hyphae grow on or below the surface and only a very small number of
aerial hyphae were produced.
When plates were exposed to fluctuating room conditions a certain rhythmicity was noted in the slower growing unsupplemented
cultures.
No rhythmicity was so noted in
suppleme~ted
cultures.
This may have been due to a greater light/dark or temperature sensitivity of the unsupplemented mycelia.
Microscopically it was observed that in cholesterol supplemented
cultures a greater number of chlamydospores were produced.
Chlamy-
dospores have thickened hyphal walls and occur in grapelike clusters
throughout the mycelial mat.
No sexual or asexual (oogonia or zoosporangia) reproductive
structures were produced in supplemented or unsupplemented cultures,
all mycelia remained in the vegetative state until induction conditions were introduced.
However, cultures grown on supplemented
media and then induced showed greater numbers of sporangia than cultures grown on unsupplemented media and then induced.
Fatty.Acid Analysis
The action of sterols such as cholesterol in fungi may function
as a substrate, or act as an inducer of metabolic or structural
changes.
Some of which may function as hormones.
Since growth was
increased by supplementation with cholesterol it could possibly act
18
as a substrate.
However, since it could act as an inducer as well,
its role in lipid synthesis was investigated further.
Cholesterol
is known to function structurally in membranes and has been shown
to alter the fatty acid composition of lipids, especially those of
the cell membrane.
Vegetative and asexual differentiated hyphae
were thus analyzed for the effect of cholesterol on total and polar
-fatty acids.
Identification of Fatty Acids
Before an analysis of the fatty acid component of lipids in the
mold could be made, a reference curve had to be determined for the
experimental conditions used in the gas chromatograph.
A reference
curve is established by injecting a mixture of several known, normal,
saturated fatty acid methyl esters into the_gas chromatograph and
plotting their retention times against their chain lengths (number
of carbon atoms in the acid).
Values for components of subsequent
samples put through under the same gas chromatographic operational
conditions were then read from the curve using these observed retention times.
Tentative characterization of major peaks was thus pos-
sible based on Equivalent Chain Length (ECL) and co-injection of
standards with unknowns.
Retention time was measured from the first
appearance of the solvent peak, which occurred immediately after injection of the sample.
Retention times of standards appeared consis-
tent with several injections.
When standard fatty acid methyl esters
were co-injected, the resultant known peaks appeared higher in magnitude than unknown peaks alone.
When these higher peaks coincided
19
with unknown peaks, tentative identification was possible ••
.
Figure 2 and Table II provide the information
on retenti,.on time
.
.
and chain length for identification of peaks in subsequent experimenta.
Gas Liquid Chromatography is useful because it permits rapid separation and identification of mixtures of·long chain fatty acids and
requires small amounts of material.
As with most chromatographic pro-
cedures, unequivocal identification of compounds
GLC alone is not
b~
possible, even when several different stationary phases (columns) are
employed.
Additional techniques (essentially microchemical) are
needed for confidence in identification.
As a consequence all identi-
fications made of fatty acid peaks are necessarily only tentative.
Total Fatty Acids
This study was made to investigate the changes in fatty acid
species with increasing cholesterol supplementation.
The mycelia were
grown on lipid-free media, supplemented with selected concentrations
of cholesterol.
cyc 1e.
The tissue was harvested about day 6 of the growth
Cu ltura 1 cond 1·t·1ons were a 1ways th e same:
25°C, still cul-
ture, light at 200 ft.-c, 500 ml broth forming a shallow medium in a
3 liter Fernbach flask.
Fatty acids were extracted from the tissues
and methylated as-described in the Materials and Methods.
These
techniques allow for the analysis of fatty acids with a carbon chain
length greater than 12.
Chain lengths of 12 or under tend to be
partly volatilized at room temperature and are infrequently found in
fungi.
TABLE II
Gas Chromatographic Analysis of Fatty
Acid Extract.
Peak Number
Retention Time
Showing Peak Identification.
1
2
3
4
5
6
2.6
4.2
4.9
6.5
7.1
8.2
14.0
16.0
16.3 17.8
18.0
18.6
7
8
9
10
11
12
10.4 12.2
14.0
17.3
22.5
31.7
19.4
20.6
21.2
22.4
23.8
'
ECL
14:0 16:0
Standards
------
- - - - - - - - - - · --
-~---
-
-
--------
-----
------
--··
--
---------
--
---
18:0
----~
18:1
~------·-
18:2
-~-~--
20.0
20:0
----
18:3
L----~~-
22:0
-~--·-
-- --
'-------
N
0
21
FIGURE 2
Equivalent Chain Lengths of Fatty Acid
Methyl Esters on DEGS Column at Conditions
Specified in Text
22
3
I ll
Q)
+J
='
c::
~
1
~
Q)
E
~
E-4
c::
0
~
+J
c::
Q)
+J
Q)
~
Chain Length (C-Atoms)
. 23
Table IV lists the total fatty acids extracted and the percent
of each component in the total extract.
The presence of cholesterol
did not induce any specific fatty acids nor did it completely inhibit
the synthesis of any specific fatty acid.
Seven of the fatty acids (16.3, 18:0, 20:0, 18:3, 22.0, 22.0,
and 22.8) are produced in very small amounts and were judged not to
change significantly with increasing concentrations of cholesterol.
Approximately 75% of the fatty acids are of four species (14:0, 16:0,
18:1, 18:2).
Oleic acid (18:1) does not change appreciably with in-
creasing cholesterol concentration.
However, palmitic acid (16:0)
decreases as the cholesterol concentration increases and myristic
acid (14:0) and linoleic acid (18:2) increase with supplementation of
cholesterol (Figures 3, 4, 5,. and 6).
Fatty Acids of Polar Lipids
To determine if cholesterol induces changes in the membrane
lipids, the fatty acids of the polar lipid fraction were extracted
from
cult~res
grown as described in the prior experment.
Without cholesterol supplementation, linolenic acid (18:3), 22.0,
and 22.2 are not detectable in the phospholipid fractions.
Linolenic
acid (18:3) and 22.2 are induced by cholesterol supplementation
(Table IV) but in ··low levels.
Most of the fatty acids of this polar
fraction are either palmitic acid (16:0) or linoleic acid (18:2).
When cholesterol is added to the growth medium, the level of palmitic
-
acid decreases slightly (Figure 8), while that of linoleic acid
appears to change depending upon the concentrations of supplement
TABLE III
Fatty Acid Content of
!•
cinnamomi.
Vegetative Mycelia Levels
Expressed as Fractions or Percent of Total Fatty Acid
Standards and ECL's
14:0
16:0
16.3
18:0
18:1
18:2
20:0
18:3
22.0
22.2
22.8
mg
.062
.276
.055
.017
.161
.247
.024
.032
.008
.103
.028
0.038 mg
.076
.272
.054
.017
.144
.245
.028
.025
.004
.085
.078
2x10-6M 0.385 mg
2xl0- 5M 3.85 mg
.081
.253
.061
.019
.142
.249
.034
.031
.045
.073
.059
.278
.060
.015
.173
.279
.022
.019
o.o
o.o
.056
.043
2xl0-~ 38.50 mg
.138
.164
.077
.011
.158
.337
.049
.022
.002
.. 027
.034
O.OM
2x10- 7M
o.o
.
I
N
.r::-
-25
FIGURE 3
Total Fatty Acids Percent
Compositional Changes of
Myristic Acid (14:0)
26
Percent of Fatty Acid
10
0
0
•
CIO
C't')
0
0
•
4-1
s::
Cl)
E
Cl)
~
Q.
§Cl)
11'1
CIO
C't')
0
•
..-4
...
0
Cl)
4-1
II)
Cl)
.....
0
.t::
0
11'1
CIO
•
C't')
""'0
t
0
11"1
•
CIO
C't')
\
20
30
27
FIGURE 4
Total Fatty Acids Percent
Compositional
C~anges
Palmitic Acid (16:0)
of
28
Percent of Fatty Acid
10
0
0
•
co
C"''
0
0
•
...,
.::
GJ
.....~
a.n
co
::J
0
Cl.
Cl.
{I)
C"''
•
.....0
....Gl
...,
10
.....Gl
a.n
co
,t::
C"''
0
0
•
'+of
0
co
~
0
a.n
co•
C"''
20
30
40
FIGURE 5
Total Fatty Acids Percent
Compositional Changes of
Oleic Acid (18:1)
30
Percent of Fatty Acid
10
0
0
•
co
('I'")
0
0
•
4.1
~
Gl
e
Gl
.-1
Cl.
Cl.
::s
Cl)
11"1
co
('I'")
0
•
.-1
0
H
Cl)
""'GlIll
.-1
0
.I:!
0
lf'\
cor
•
('I'")
~
0
;t
0
lf'\
•
co
('I'")
20
30
TABLE IV
Fatty Acids of the Polar Lipid Fraction of Vegetative
!•
cinnamomi Mycelia
Expressed as Fraction of Total Fatty Acid Content in Polar Lipid Fraction
Standards and ECL's
14:0
16:0
16.3
18:0
18:1
18:2
20:0
18:3
22.0
22.2
22.8
mg
.038
.419
.088
.025
.090
.241
T
o.o
o.o
o.o
.088
2x10- 7M 0.038 mg
2x10- 6M 0.385 mg
.143
.383
.071
.027
.104
.155
.015
.028
.034
.039
2x10- 5M 3.85 mg
.048
.384
.021
.022
.061
.295
.034
.014
o.o
o.o
.016
.092
O.OM
o.o
T • trace·
w
.....
32
(Figure 9).
Myristic acid (14:0) which is present in about the same
proportion as in the total fatty acid extract is also variable with
concentration of cholesterol supplement (Fig'l,lre 7).
The other fatty
acids do not change significantly with varying supplements of cholesterol (Table V).
The Effect of Cholesterol on Sporulation
Addition of cholesterol alone does not induce sporulation by
cinnamomi.
!•
What it does is enhance the response of the mycelia once
the induction procedure has been instituted.
This study was made to determine the degree of sporulation response due to cholesterol supplementation prior to induction.
Spor-
angia were induced using the technique described in Materials and
Methods.
Sporangia were counted by placing a Petri dish containing
the mycelial mat on the stage of a dissecting scope.
mycelial mat were selected at Fandom.
2
magnification (50.2 mm ).
Areas of the
Sporangia were counted at 25x
The numbers of sporangia counted at each
concentration appeared to vary from measurement to measurement, so
that many inductions and counts were made and an average was determined.
In part the variation in numbers of sporangia counted was due
to the sporangia produced in each culture not being uniformly placed.
Many more were produced in the outer margins of the cultures than
toward the middle.
Interestingly, when contaminating bacteria were
evident in some plates, these plates showed a reduced production of
sporangia as compared to others of identical concentrations; these
plates were not included in the computation of data.
33
FIGURE 6
Total Fatty Acids Percent
Compositional Changes of
Linoleic Acid (18:2)
34
Percen~
10
0
•
0
co
M
0
+J
•
0
c::
41
s41
.-1
p,.
p,.
11'1
::1
co
.-1
0
{I)
0
M
•
""
41
+J
Q)
41
.-1
0
.c:
11'1
,0
co
~
M
0
•
~
0
11'1
co•
M
20
of Fatty Acid
30
40
35
FIGURE 7
Polar Lipids of Vegetative Mycelia
Percent Compositional Changes of
Myristic Acid (14:0) Component
36
'
Percent of Fatty Acid
10
0
0
•
00
ttl
~
0
0
•
c::41
E
.....41
r:lo
r:lo
it'\
fJ)
='
ttl
.....
0
0
00
•
'41"'
~
II)
.....41
0
.c
it'\
0
00
~
ttl
0
•
~
0.
it'\ .
•
00
ttl
20
.
37
FIGURE 8
Polar Lipids of Vegetative Mycelia
Percent Compositional Changes of
Palmitic Acid (16:0)
38
Percent of Fatty Acid-
10
q
0
co
~•
0
~
t::
Ql
sQl
~
~
~
::1
II'\
co
M
•
0
tl)
~
0
J-1
Ql
~
Ill
Ql
~
0
.c:
u
1+-1
0
co
;&
II'\
co
•
M
20
30
40
39
FIGURE 9
Polar Lipids of Vegetative Mycelia
Percent Compositional Changes of
Linoleic Acid (18;2)
40
Percent of Fatty Acid
10
0
0
co
M
0
•
~
c::
~
.....41
Cl.
Cl.
::I
rJl
.....
Lt'l
co
M
0
•
....041
~
Ill
.....41
0
..r::
0
~
0
bO
:::E:
Lt'l
co
•
M
20
30
41
Sporangia! production improves with increasing concentration of
cholesterol in the medium (T&ble IV), except at saturating concentra.
-4
tiona (2xl0 M). The decline may be due to overgrowth of the culture
before induction of sporulation.
A few cultures supplemented with
2x10 -4M cholesterol actually produced up to 254 sporangia per field.
Sporangia produced were normal morphologically and functionally.
Sporangia were seen to dehisce and extrude viable zoospores.
Polar Induced Fatty Acids
This study was undertaken to determine if fatty acid content of
the polar lipids changes when vegetative mycelia were induced to
sporulate.
As described in the Materials and Methods, broth was re-
placed by Mineral Salts Solution, thereby inducing starvation conditiona.
As in Polar vegetative fatty acids, proportions of the major
80~
components of polar induced fatty acids are about the same.
of
the fatty acid content is limited to 14:0, 16:0, 18:1, 18:2.
Increasing concentration of cholesterol in. the medium apparently
does not effect increases in the level of linolenic (18:3) and C22.2
fatty acids as occurs in the polar vegetative lipids.
Palmitic acid
(16:0), 16.3, Linoleic acid (18:2), Arachidic acid (20:0) and 22.8
fatty acids (Table V and Figures 11 and 14) show random or no change
in levels with increasing concentration of cholesterol.
Myr~stic
acid (14:0) shows a slight but definite upward trend
with increasing concentration of cholesterol (Figure 10).
This same
trend is not shown in the Polar vegetative Cl4:0 levels.
18:1 shows
42
a reduction in proportion with addition of cholesterol supplement to
growth medium.
43
TABLE V
The Effects of Cholesterol on Differentiation
of Sporangia in
Supplement
cinnamomi
O.OM
Concentration
(O.Omg)
Average Number
1.07
Sporangia/Area
!•
2xl0- 6M 2xl0- 5M 2xl0- 4M
6.66
38.16
28.23
(
TABLE VI
Fatty Acids of the Polar Lipid Fraction of Induced
!•
cinnamomi Mycelia
Expressed as Fraction of Total Fatty Acid Content in Polar Lipid Fraction
14:0
16:0
16.3
18:0
18:1
18:2
20:0
18:3
22.0
22.2
22.8
.065
.375
.029
.042
.097
.241
.022
o.o
o.o
o.o
.117
2x10- 5M
.100
.457
.055
.032
.045
.239
o.o
.116
.386
.030
.030
.053
.239
.028
o.o
o.o
o.o
o. o
.073
2x10-~
o.o
o.o
Standards and ECL's
O.OM
2xl0- 6M
.103
:5:
45
FIGURE 10
Polar Lipids of Induced Mycelia Compositional
Changes of Myristic Acid (14:0)
46
w
00
•V1
0
3:
QQ
0
w
HI
V1
::r
0
.....
•00
0
"
"
.....
Gl
rt
11
0
•w
00
VI
0
Cl)
c::
"d
"d
.....
a
"
(I)
0
0
•
w
00
0
•0
oz
Ot
::I
rt
47
FIGURE 11
Polar Lipids of Induced Mycelia
Compositional Changes of
Palmitic Acid (16:0) ·
48
0
U'\
•
CIO
C"'\
U'\
~
•
C"'\
E
CIO
~
Cll
....Cll
~
~
:::l
Cl)
U'\
CIO
C"'\
0
•
....0
J.l
Cll
~
(I)
....0
Cll
.c:
.t.,)
CIO
C"'\
0
0
0
0
40
30
20
Percent of Fatty Acid
10
•
•
~
0
-£
49
FIGURE 12
Polar Lipids of Induced Mycelia
Composition Changes of
Stearic Acid (18:0)
50
0
Lrt
co•
C""'
0
0
2
Percent of Fatty Acid
10
•
(
51
FIGURE 13
Polar Lipids of Induced Mycelia
Compositional Changes of
Linoleic Acid (18:1)
52
Percent of Fatty Acid -
10
0
0
00
tf"'
0
+I
0
•
.::cu
ti
.-l
Clo
Clo
::s
"'
Cll
00
tf"'
.-l
0
0
•
""'cu
+I
Ill)
G)
.-l
0
.c
t.)
'f.C
0
00
"'
•
tf"'
~
0
"'•
00
tf"'
53 -
FIGURE 14
Polar Lipids of Induced Mycelia
Compositional Changes of
Linoleic Acid (18:2)
54
Percent of· Fatty Acid
10
0
0
00
C""'
0
0
•
.a
s::
a>
E
a>
....
Q.
§'
tl)
....
1.1"1
00
C""'
0
•
0
a>
""'
.a
Cl)
....0a>
1.1"1
00
0
C""'
..c::
•
~
0
~
0
1.1"1
•
00
C""'
20
30
DISCUSSION
The initial growth rates of the cholesterol unsupplemented, and
the 2xl0
-6 M and 2xl0 -5M chol-supplemented. cultures were found to be
about equal during lag and early log phases of growth.
However, during
later log phase growth, cholesterol supplements improved the growth
rate as concentrations increased.
Several workers have obtained simi-
lar results with species of Pythium and Phytophthora (Hendrix, 1964,
1965, 1969, Chee and Turner, 1966).
In contrast to these results,
Brushaber, et al, (1972) reported a reduction in dry weight yield of
Pythium for mycelia grown on supplemented media.
In a report by
Sietsma and Haskins (1968) it was shown that when cholesterol is supplied to mycelia of Pythium, o consumption decreased and lactic acid
2
and acetoin production increased.
Phytophthora is known to utilize the
standard Embden-Meyerhoff and Krebs cycle pathways and therefore with
the same amount of substrate less growth is expected and obtained with
anaerobic conditions than with aerobic.
It is important to consider
that in cholesterol supplemented cells with their stabilized membranes
and reduced leakiness, less energy is utilized to replace those nucleotides and amino acids that leak out and the energy thus saved in effect
can result in greater growth.
However, it is also possible, as shown
in Brushaber's 1972 study, that an oxygen deficit can be created by
rapidly respiring mycelia and effect lesser amounts of growth.
Since cholesterol is known to be primarily incorporated into membranes, it seems reasonable to conclude that the primary effect of
cholesterol on the cell is related to its association with tl1e plasma
membrane.
Cholesterol is known to improve the stability and permea-
55
56
bility of the membrane and reduce leakage.
It is_conceivable that the
improved condition of the membrane would allow an enhanced uptake of
nutrients from the medium and thus augment gr?wth.
Increased yield with increasing concentration of supplemental
cholesterol could result from cholesterol being utilized as substrate,
general enhancement of biosynthetic efficiency, or both.
Sietsma and
Haskins (1967), Hendrix, et al (1969) and Brushaber, et al (1972) have
shown that sterols are not utilized as an energy source in Pythium,
but for the most part are incorporated unchanged into membranes.
Sporulation
The cultures grown without cholesterol were found to produce a
small number of sporangia, this could be due to a small amount of
contaminating sterol in the agar.
With increased cholesterol supple-
mentation, an increasing differentiation response up to an apparent
"saturation" point was
observed~
The concentration of 2xl0 -4M is near
the saturation point of the dimethyl formamide to hold cholesterol in
solution. ·This same molarity was found to be significant in oospore
production in other Pythiaceous fungi (Hendrix, 1970).
Cholesterol
effects may be secondary in that improved transport into the cell
allows accumulation of fatty metabolites that enhance or provide metabolites ·for sporulation.
The effect may be primary in that the sporu-
lation response is strictly a membrane effect.
Cholesterol incorporation into the membrane changes its character
presumably by stabilization, to a degree such that sporulation is permitted.
That differentiation is a genetically mediated membrane
57
associated event is a concept widely accepted by developmental biolo•
gists.
It has been shown that for oospore induction a minimum concen-
6
tration of at least 10- M cholesterol is needed (Hendrix,
1970)~
Membrane Studies
Artificial membrane systems have been used to study the effect of
cholesterol on biomembranes.
It has been determined that cholesterol
when added to these systems, interdigitates with fatty acid components
of the membrane (Chapman, 1973).
Cholesterol's function in the mem-
brane is to stabilize the molecules by obstructing the tilting oscillations of fatty acid molecules.
The lipid bilayer condences increas-
ingly as more cholesterol is added.
As a consequence the bilayer
occupies a smaller area at its interface (DeGier,-et al, 1969).
Addition of cholesterol has been shown to cause a reduction of
cohesive forces between adjacent hydrocarbon chains.
Physical studies
suggest that cholesterol aligns parallel to fatty acid chains running
perpendicular to the surface of the membrane (Rothman and Engleman,
1972).
A 1:1 mixture of lecithin to cholesterol may be the optimum proportion of the two membrane components.
The 1:1 ratio is the maximum
achievable before precipitation of cholesterol occurs.
The maximum
number of cholesterol molecules normally bound to polar lipids can be
determined only with data for membranes maximally loaded with cholesterol (Rouser, et al, 1972).
In living systems a molar ratio of under
0.5 cholesterol to polar lipid is reported.
This suggests that choles-
terol does not bind with all polar lipids.
The presence of cholesterol
58
in biomembranes must be explained by its binding to polar lipids
rather than by its binding to proteins; this is a consequence of its
lipid nature (Jain, 1975).
The nature of the hydrocarbon chain is of considerable structural
importance to the lipid phase.
In most membrane systems a substantial
portion of fatty acid chains is unsaturated.
The average level of
unsaturation in the lipid phase may even be the most significant feature of the membrane from a structural point of view.
It has been
shown that cholesterol can be accomodated in phospholipids up to a
limiting proportion determined largely by the degree of unsaturation of
the fatty acid chains in the membrane (Finean, 1973).
When a sterol
requiring strain of bacteria is made to grow in the absence of cholesterol, its polar lipids become more saturated (Rottem, 1973).
Total Fatty Acid Content of P. cinnamomi
The fatty acid content of the vegetative mycelia of
f•
cinnamomi
was shown to be rearranged with increasing concentration of cholesterol
supplement.
Two components, a short chain saturated fatty acid
(myristic) and a comparatively long chain unsaturated fatty acid
(linoleic) increased by about
15~
total, while another saturated fatty
acid (palmitic) decreased by a similar percentage at 10 -4M.
These
rearrangements must be in response to environmental conditions external
to the mycelia.
Since changing cholesterol concentration is the only
variable, it must be concluded that the rearrangement is a direct consequence of the condition.
This rearrangement may be due to an increase in the level of
59
storage-products, triglycerides, as a result of improved metabolism.
Or it may be due to changes occurring in the membranes, which should
be reflected by similar changes occurring in
~he
fatty acid composition
of the polar lipid fraction.
Polar Fatty Acids of Vegetative Mycelia
With increasing concentrations of cholesterol supplied, definite
rearrangements of the major fatty acids of the polar lipid fraction
·occurs.
These rearrangements don't appear to reflect the trends noted
for the total fatty acids.
Palmitic acid levels in unsupplemented
culture decrease by 3.6%, and then stabilize with increased supplementation.
Myristic acid and linoleic acid, the other major components
change with each supplementation and show no trends.
It is noteworthy
that though definite changes are occurring in the percent composition
of these three components, their total proportions relative to the
other fatty acid components change by a very small amount.
The major-
ity of lipid rearrangements are occurring between the three components.
These fatty acid changes cannot be explained by current knowledge of
membrane lipids but could be a result of very complex events occurring
in the membrane lipids.
It is possible that what is known to occur in
one system may not function in another.
Rather than causing phospho-
lipid turnover to alter the fatty acid proportions, with short chain
and more saturated fatty acids replacing the longer unsaturated chains,
what may have happened is only a fluid rearrangement of the membrane
lipids.
Thus cholesterol would be accomodated or accepted into the
membrane without the consequent reduction of chain length or satura-
60
tion.
Fatty acid replacement would be random (it might be interesting
to repeat this aspect of the problem using a different methodology).
It is entirely possible that at some point in the treatment of the
lipids destruction or alteration occurred, thereby resulting in these
possibly anomalous results.
Polar Fatty Acids of Induced Mycelia
Of the three major fatty acid components, as concentration of
cholesterol increases, myristic acid shows a
defini~e
upward trend,
while linoleic acid remains stable, and palmitic acid fluctuates without trend.
These fatty acid results, though not showing the same
trends as the vegetative components is understandable.
The environmen-
tal conditions, both external and internal are very different.
Starva-
tion conditions have been imposed, causing the metabolite reserve to be
utilized.
The pH and ionic conditions of the medium have been altered
by"addition of the Mineral Salt solution.
The cultures are differentiating and utilizing stored lipids.
They are producing sporangia (and zoospores) rather than cell wall
material.
Bowman and Mumma (1967) have shown that the changes in lipid
composition in bacteria and fungi may result from slight changes in
environmental conditions and even stages of the growth cycle.
It is interesting to note that the total percentage of the three
major components, myristic, palmitic, and linoleic acids are roughly
the same in the induced mycelia as in the vegetative polar lipids.
The rearrangements that are occurring, within the group, appear to be
at the expense of each other.
61
Membrane lipids undergo changes with
supplem~ntation
of choles-
terol but do not reflect changes of the total fatty acid content.
can be said that changes caused by
cholestero~
stood and as a consequence are unpredictable.
It
are imperfectly underTechniques employed in
this study did not lend themselves to the determination of the degree
of cholesterol uptake by the fungus.
The sporulation and growth
studies suggest that increasing cholesterol-concentration controls
the amplitude of response and hence degree of uptake can therefore be
inferred.
REFERENCES
Barber, M.L. and J.F. Mead. 1975. Comparison of Lipids of Sea Urchin
Egg Ghosts Prepared before and after Fertilization. Wilhelm
Roux Archiv. 177:19-27~
Bowman, R.D. and R.O. Mumma. 1967. The Lipids of Pythium ultimum.
Biochem. Biophys. Acta. 144!501-510.
Brushaber, J.A., JaJ. Child, R.H. Haskins. 1972. Effects of Cholesterol on growth and Lipid Composition of Pythium sp. PRL2142.
Can. J. Microbiology 18:1059-1063.
Chapman, Ds 1973. Physical Chemistry of Phospholipids. In:
and Function of Phospholipids, G.B. Ansell ed. Elsevier
Scientific Publishing Co., New York.
Form
Chee, K.H. and N.A. Turner. 1966. Steroid Factor in Pea (Pisum
sativum L.) Influencing Growth and Sporulation of Phytophthora
cinnamomi Rands. New Zealand J. of Agric. Research 8:104-108.
Chen, D. and G.A. Zantmyer. 1970. Production of Sporangia by
Phytophthora cinnamomi in Axenic culture. Mycologia 62:397-402.
Child, J.J., G. Defago, R.H. Haskins. 1969. The Effect of Cholesterol
and Polyene Antibiotics on the Permeability of the Protoplasmic
Membrane of Pythium PRL 2142. Can. J. Microbial. 15:599-603.
DeGier, J., J.G. Mandersloot, L.L. Van Deenen. 1969. The Role of
,
Cholesterol in Lipid Membranes. Biochim. Biophys. Acta.
173:143-145.
Ferguson, K.A., F.M. Davis, R.L. Conner, J.R. Landry, F.B. Mallory.
1975. The Effect of Sterol Replacement in Vivo on the Fatty
Acid Composition of Tetrahymena. Jour. of Biological Chemistry
250:6998-7005.
Finean, J.B. 1973. Phospholipids in Biological Membranes and the
Study of Phospholipid-Protein Interactions. In: Form and
Function of Phospholipids. G.B. Ansell ed. Elsevier Publishing
Co., New York.
Haskins, RoH., A.P. Tulloch, R.G. Micetich. 1964. Steroids and
Stimulation of Sexual Reproduction of a Species of Pythium.
Can. J. Microbial. 10:187-195.
Hendrix, J.W. 1964. Sterol Induction of Reproduction and Stimulation
of Growth of Pythium and Phytophthora. Science 144:1028-1029.
62
63
Hendrix, J.W. 1965. Influence of Sterols on Growth and Reproduction
of Pythium and Phytophthora spp. Phytopathology 55:790-797.
Hendrix, J.W., S.M. Guttman, D.L. Wightman. 1969. Cation and Sterol
Effects on Growth of Phytophthora parasitica var. nicotinae.
Phytopathology 59:1620-1624.
Hendrix, J.W. 1970. Sterols in Growth and Reproduction of Fungi.
Ann. Rev. Phytopathology 8:111-130.
Hendrix, J.W., R.O. Bennett, E. Heftmann. 1970. Metabolism of Cholesterol by Pythium periplocum~ Microbios 2:11-15.
Hendrix, J.W. 1975. Differential Uptake and Metabolism of Sitosterol
and Cholesterol by Achlya, Pythium, and Phytophthora Species.
Can. J. Microbial. 21:735-737.
Hendrix, J.W. and G. Rouser. 1976. Polar Lipids of Phytophthora
parasitica var. nicotianae in Comparison with those of Selected
Other Fungi. Mycologia 68:354-361.
Hwang, S.C., W.H. Ko, M. Aragaki. 1975. A Simplified Method for
Sporangia! Production by Phytophthora cinnamomi. Mycologia
67:1233-1234.
Jain, M.K. 1975. Role of Cholesterol in Bicmembranes and Related
Systems. In: Current Topics in Membranes and Transport, ed.
F. Bronner and A. Kleinzeller. Academic Press, New York.
Miwa, T.K., K.L. Mikolajczak, F.R. Earle, I.A. Wolff. 1960. Gas
Chromatographic Characterization of Fatty Acids. J. Analytical
Chemistry 32:1739-1742.
Olsen, R.A. 1973. Triterpeneglycosides as Inhibitors of Fungal Growth
and Metabolism. 5. Role of the Sterol Contents of Some Fungi.
Physiol. Plant. 28:507-515.
Rothman, J.E. and D.M. Engelman. 1972e Molecular Mechanism for the
Interaction of Phospholipid with Cholesterol. Nature New
Biology 237:42-44.
Rottem, s., V. Cirillo, B. Kruyff, M. Shinitzky, s. Razin. 1973.
Cholesterol in Mycoplasma Membranes. Biochim. Biophys. Acta
323;495-508.
Rouser, G., G. Kritchevsky, A. Yamamoto, c. Baxter. 1972. Lipids in
the Nervous System of Different Species as a Function of Age.
Advances in Lipid Research 10:261-360.
64
Schlosser, E. and D. Gottlieb. 1968. The Effect-of Sterols on the
·
Metabolism of Pythium Species~ Archiv. fur Mikrobiologie
61:246-253.
Schlosser, E., P.D. Shaw, D. Gottlieb. 1969 •. Sterols in Species of
Pythium. Archiv. fur Mikrobiol. 66:147-153.
Sietsma, J.H. and R.H. Haskins. 1967. Further studies on Sterol
Stimulation of Sexual Reproduction in Pythium. Can. J.
Microbial. 13:361-367.
Sietsma, J.H. and R.H. Haskins. 1968. The Incorporation of Cholesterol .by Pythium sp. PRL 2142 and Some of Its Effects on Cell
Metabolism. Can. J. Biochem. 46:813-818.
Westcott, c. 1971. Plant Disease Handbook.
Co., New York.
Van Nostrand Reinhold
Wuthier, R.E. 1966. Purification of Lipids from Non-Lipid Contaminants on Sephadex Bead Columns. J. Lipid Research 7:558-561.
Zentmyer, G.A. and D.c. Erwin. 1970. Development and Reproduction of
Phytophthora. Phytopathology 60:1121-1127.