FEMS Microbiology Ecology 31 (1985) 293-299
Published by Elsevier
293
FEC 00037
Microsclerotial germination of Verticillium dahliae as affected by
rape rhizosphere
(Fluorescein diacetate: Brassica napus L.)
Stefan O l s s o n a n d Birgit N o r d b r i n g - H e r t z
Department of Microbial Ecology, Unit,ersity of Lund, Ecology Building, Helgonavi~gen 5, S-223 62 Lund. Sweden
Received 2 April 1985
Revision received 15 June 1985
Accepted 12 July 1985
1. S U M M A R Y
The germination of nylon net-trapped microsclerotia of Verticillium dahliae pathogenic to rape
(Brassica napus) was assessed in different systems
by fluorescence microscopy using fluorescein diacetate. The influence of the culture's age and the
size of the microsclerotia on germination percentages was assessed in water, mineral salts solution and mineral salts solution plus sucrose for 3
V. dahliae isolates. Large microsclerotia germinated better than smaller ones. The microsclerotia
of 2 isolates showed decreased germination percentages with culture age over a 4-11-week period.
Microsclerotial germination percentages were always higher in mineral salts solution plus sucrose
than in mineral salts solution alone or water. In a
sand culture system with the intact rape plant,
microsclerotial germination percentages were high
close to the root and decreased in a steep gradient
to background levels within 5 m m from the root.
2. I N T R O D U C T I O N
The effect of plant roots on the germination of
fungal propagules has attracted much interest, as
it is the first event in the infection cycle of a
soil-borne plant pathogen. Root exudate, in a
broad sense, is the trigger of fungal propagule
germination and active substances in the exudate
have been characterized [1]. The root exudates
diffuse into the soil and an exudate gradient develops around the root. This gradient is influenced by
factors such as soil humidity and uptake by microorganisms [2]. Fungal propagules stimulated to
germinate by root exudates may form a 'germination gradient' around the root that may influence
the relation between the disease index and the
inoculum density for a given h o s t - p a t h o g e n - s o i l
situation [3,4].
V. dahliae is a soil-borne pathogen that survives
in soil by means of microsclerotia. The fungus
infects and causes disease in a wide range of
dicotyledonous hosts [5] but does not seem to
cause disease in monocotyledons, even when infecting the root cortex [6-8]. Several investigations
have dealt with the factors affecting V. dahliae
microsclerotial germination [9,10] and, in some
cases, the effects of root exudates on the germination have been estimated [11].
Within an ongoing study of the biological and
chemical interactions in the rhizosphere of the
rape plant (B. napus), the ecology and infection
biology of V. dahliae has been included. In this
work, microsclerotial germination, as affected by
0168-6496/85/$03.30 © 1985 Federation of European Microbiological Societies
294
the culture age and size of microsclerotia, was
determined in different solutions. In addition, the
germination of microsclerotia in the rhizosphere of
the host plant was assessed. In both cases a
germination assay using a fluorescent probe, fluorescein diacetate, was used.
3. M A T E R I A L S A N D M E T H O D S
3.1. Isolates
Three isolates of V. dahliae Kleb. were used (S,
P2 and Vd 71/36). All three were isolated from
rape plants (B. napus L.) from different localities
in southern Sweden. The choice of the isolates was
based on the differences in their microsclerotiaand conidia-forming patterns. Isolate S forms microsclerotia in abundance and conidia in relatively
large numbers, whereas P2 produces fewer microsclerotia but conidia in abundance. Vd 71/36
forms lower numbers of both microsclerotia and
conidia than the other two isolates.
3.2. Production of microsclerotia
Stock cultures of V. dahliae were kept on
Czapek-Dox Agar (Czapek-Dox sol., Oxoid; 1.5%
Bacto agar, Difco) in test tubes. Pieces of the dark
mycelial mats were used as the inoculum for liquid
cultures in 100-ml Erlenmeyer flasks containing 20
ml of Czapek-Dox solution. The flasks were incubated without shaking for 4 11 weeks at 20°C
in the dark. For isolate S, flasks were also incubated for 31 weeks.
3.3. Preparation of the microsclerotial suspension
A piece of the dark mycelial mat from a liquid
culture was transferred to a 50-ml screw-cap tube
containing 3-mm diameter glass beads. Distilled
deionized water (DD-water) was added and the
tube was placed in a wrist-action shaker (Griffin
and George, U.K.) at full speed for 3 min. The
microsclerotia were then washed out with several
portions of DD-water. The suspension was filtered
through a nylon net (pore size 250/xm) fitted in a
Millipore filter holder (Sterifil Holder, 47 mm) in
order to get rid of the mycelia and large aggregates
of microsclerotia.
3.4. Trapping of microsclerotia in nylon nets
The method for trapping microsclerotia in nylon
nets was a modification of a technique previously
described [12]. Nylon nets of 40-/.tm or 80-~m pore
size (Swiss Silk Bolting Cloth Mfg. Co. Ltd., D I N
130-40, 17P-80) were cut in 25-mm diameter disks
and washed thoroughly in boiling DD-water and
dried. A net disk was placed in a Millipore disk
filter holder (Glass Microanalysis, S.S. Support, 25
mm). Enough microsclerotial suspension (3 20 ml)
to trap 10-20 microsclerotia/mm 2 in the pores
was added to the 40-~m nets. About 1-3 times
more of the microsclerotial suspension was used
for the 80-btm nets. The filter apparatus was set up
so that the suspension could not pass through the
nylon net unless suction was applied. When suction was applied, the microsclerotial suspension
filtered through the net with great force and the
microsclerotia were trapped in the net pores. After
washing twice with 15 ml of DD-water, the net
was removed and loose microsclerotia were scraped
off in DD-water, leaving only thoroughly trapped
microsclerotia in the net pores.
3.5. Germination assay in liquid systems
The nets with microsclerotia were placed in
9-cm plastic Petri dishes (5 nets/dish) containing
20 ml of test solution. Three different solutions
were used: DD-water, Pfeffers mineral salts solution [13] and Pfeffers solution supplemented with
1.5% sucrose (w/v). The dishes were incubated for
40-48 h at 20°C before staining and microscopic
examination. The total number of microsclerotia
and germinated microsclerotia on the 40-~m nets
were counted in 15 square fields of 0.71 x 0.71
m m for each net. About 120 microsclerotia per net
were counted. Five nets were used for each treatment. For the 80-btm nets, 15 microsclerotia on
each net were scored for germination. Three nets
were used for each treatment. This assay was
carried out for all three isolates of V. dahliae.
3.6. Germination assay in the rhizosphere
A 9-cm plastic Petri dish was filled with moistened sand. Three nets (40-~m) with trapped microsclerotia (isolate S) were placed between the
sand and the lid, with the net's upper surface
facing the sand (Fig. 1A). Rape seeds (Brink, from
295
3. 7. Staining and microscopy
The net-trapped microsclerotia were stained
with a modification of the FDA staining technique
for active fungal hyphae [14,15]. The nets were
transferred to a 5-cm Petri dish with 10 ml of 60
mM potassium phosphate buffer (pH 7.5) supplemented with 50/.tl of 0.1% (w/v) F D A in acetone.
After staining for 5 rain, the nets were washed in
10 ml of buffer. The nets were mounted in the
buffer on a microscopic slide, before microscopic
examination. Except for using a 10 × objective and
12.5 x oculars in the microscope, the equipment
was according to S6derstr~3m [14].
The Swedish Seed Association SvalOf, Sweden)
were germinated in a plastic Petri dish on moistened filter paper (20°C in the dark). A germinated
seed with the root emerging approx. 2 mm was
planted in the sand through a hole in the dish. The
dish was placed with the hole upwards and slightly
tilted towards the lid in a growth cabinet (Fi-totron
600H, Fisons) for 2 - 4 days (16 h day, 20°C d a y /
15°C night, RH 80%, 10000 lux). During that time
the root grew along the lid and across one of the
nets. The root and the net positions were marked
on the lid and the markings transferred to a microscopic slide (Fig. 1B). The dish was opened and
the intersections between the root and the net were
marked. The net was then immediately stained and
put on the marked microscopic slide for examination under the microscope. Microsclerotial
germination was assessed about 1 day after the
root tip had grown past the microsclerotia on the
net. The total number of microsclerotia and the
number of those that had germinated were counted
at different distances perpendicular to the root
and within 1 3 cm of the root tip (Fig. 1B).
Rhizosphere germination experiments were performed on two different occasions. The first time,
the rhizospheres of 4 plants, and the second time,
those of 2 plants were investigated.
4. RESULTS
4.1. Estimation of germinated microsclerotia
The same technique was used to visualize
germinating microsclerotia in both the liquid system and in the sand culture system. With the
FDA-staining technique, germinated microsclerotia had green-glowing hyphae while ungerminated microsclerotia could be seen by changing to transmitted light. On the nylon nets, therefore, newly emerging hyphae were easily distinguished from old hyphal fragments attached to
111i i / ~ ~ ~ ~
/
NET DISKSr-
~
ROOT POSITION
\
--A
B
Fig. 1. Microslerotial germination assay in rape root rhizosphere. (A) Rape seedling and net discs in a sand-filled Petri dish. (B) Net
disc with V. dahliae microsclerotia (dotted field) just after the root tip passed the disc. x, Fields of view where germination was
assessed.
296
%
lO(1
ISOLATE Vd71/36
A
NET PORE SIZE 40urn
.....
%
100
ISOLATE Vd71/36
SE I
B
50
NET PORE SIZE 80urn
SE I
50
e
o
O~
1O0
;,
ISOLATE
7
P2
1I w e e k s
NET PORE SIZE
C
40um
o
O~
1O0
,,.4
ISOLATE
"I
P2
SE ,
NET PORE SIZE 80um
D
50
1-1 w e e k s
_ J
SE I
........-i
50
f
0
O~
100
4
ISOLATE S
-
8
7
11 w e e k s
NET PORE SIZE
E
40um
SE
0
%
100
~, ~,
ISOLATE
S
NET PORE SIZE
F
I
50
7
11 w e e k s
80urn
sE I
50
e
0
~. 4
7
11 ~'3"1 w e e k s
0
~,
.~
11 ~'31 w e e k s
Fig. 2. Influence of culture age and size of microsclerotia on microsclerotial germination percentages of three V. dahliae isolates,
Vd71/36, P2 and S in three solutions. O, DD-water; , t mineral salts solution; II, mineral salts solution with 1.5% sucrose.
297
%
the microsclerotia, since the old and presumably
dead hyphae were not FDA-active.
4.2. The effects of culture age and microsclerotial
size on microsclerotial germination
The large microsclerotia (trapped in the pores
of the 80-/~m nets) had a mean germination of
51%, and the smaller microsclerotia (trapped in
the pores of the 40-/~m nets) had a mean germination of 27%. The microsclerotial germination percentages decreased with the age of the culture for 2
of the isolates (Vd 71/36 and P2, Fig. 2A-C).
Isolate P2 seemed to be less affected by the culture
age, since the germination percentage on the 80-~m
nets, in Pfeffers solution with sucrose, was constant over the 4-11-week period (Fig. 2D). The
microsclerotial germination percentages of isolate
S were little affected by the age of the culture, and
microsclerotia from 31-week-old cultures showed
no decrease in germination percentages compared
to microsclerotia from 4-week-old cultures (Fig.
2E, F).
The germination percentages in the solutions
tested were always lower in DD-water or mineral
salts solution than in mineral salts solution with
1.5% sucrose. DD-water gave the lowest germination percentages, except in one case (Fig. 2D).
The 40-~m nets trapped about 10 times as
many microsclerotia as the 80-~m nets, from the
same microsclerotial suspension. Therefore, although large microsclerotia germinated better than
smaller ones, the 40-#m nets were used in all
subsequent experiments.
4.3. Microsclerotial germination in the rhizosphere
The microsclerotia germinated in decreasing
numbers with increasing distance from the root
axis (Fig. 3). The germination gradient was rather
steep, and decrased to background level (about
30%) within 5 mm of the root axis. While still
within the reach of the root hairs (about 2.5 mm),
the germination percentages were < 45%, i.e., far
below half the difference between the background
level and the level obtained close to the root. The
relatively high levels of microsclerotial germination obtained far away from the root were probably an effect of the limited fungistasis in the sand
system, since these germination percentages were
70
.~ 60
E
O=50
g
t=
40
o
i, i
20
10
0
.5
10
distance
from
1"5
mm
root axis
Fig. 3. Percentage of germination of V. dahliae microsclerotia
on nylon nets plotted against distance from root axis. Bars
indicate SE.
close to those obtained in the experiments in DDwater and mineral salts solution (Fig. 2).
5. DISCUSSION
Fungal propagules can sometimes be considered to be stationary in the soil while the root
grows into the reach of these propagules. Root
growth rates are often more than 1 c m / d a y [16]
and few fungi grow that fast [17-19]. This makes
the timing of the propagule germination important, especially for fungi like V. dahliae, which
grow more slowly than the root extension rate and
preferably attack the susceptible region near the
root tip [20]. The first few centimetres of the root
is a region with a high exudation rate [16] and it
would be advantageous for the fungi to be able to
germinate quickly after being triggered by the
presence of exudates coming from the growing
root tip. V. dahliae frequently attacks through the
root cap and the elongation zone [5,8,21], and its
radial growth rate is .just a few m m / d a y [22,
298
Olsson, unpublished]. The ability to germinate
within a relatively short time could therefore be
most important for the entire infection process.
This study has shown that V. dahliae microsclerotia germinated soon after the root tips passed,
and that a steep germination gradient from the
root rapidly developed. This was probably an effect of root exudation, since rape root exudates
stimulate germination (Olsson, unpublished).
The V. dahliae microsclerotia used in this study
germinated better in mineral salts solution with
sucrose than in mineral salts solution alone or in
water, indicating that they were not as nutrient-independent as has been reported earlier for this
fungus. Filonow and Lockwood [9] found V.
dahliae microsclerotia to be independent of exogenous nutrients for germination, and even after
45 days of diffusive stress, they had lost only a
little of their nutrient independency. This difference in nutrient dependency could be due to
isolate differences, since isolates from rape seem to
differ considerably from isolates obtained from
other plants [23].
The germination percentages of the isolate Vd
71/36 decreased with the age of the culture. This
could be due to its different microsclerotia formation pattern. For Vd 71/36 microsclerotial differentiation started later and the microsclerotia
formed were less melanised when compared to the
other isolates. This could possibly influence the
quality of the microsclerotia and thus their
germination potential.
The trapping of microsclerotia or other fungal
propagules in the pores of nylon nets combined
with FDA-staining makes it possible to study
propagule germination with the same 'probe' in
different environments from pure systems to soil
and thus to make comparisons. With this technique, it is also possible to record the positions of
the propagules in relation to a root or other sources
of germination-stimulating substances.
ACKNOWLEDGEMENTS
We thank Dr. Bengt SOderstrOm, Dr. Erland
Baath and Dr. Conny Liljenberg for valuable discussions. This study was supported by grants from
the Swedish Natural Science Research Council
and the Swedish Council for Forestry and Agricultural Research.
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