FEMS Microbiology Ecology 31 (1985) 293-299 293 Published by Elsevier

FEC 00037

Microsclerotial germination of Verticillium dahliae as affected by rape rhizosphere

(Fluorescein diacetate: Brassica napus L.)

Stefan Olsson and 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. SUMMARY

The germination of nylon net-trapped micro- sclerotia of Verticillium dahliae pathogenic to rape (Brassica napus) was assessed in different systems by fluorescence microscopy using fluorescein di- acetate. The influence of the culture's age and the size of the microsclerotia on germination per- centages was assessed in water, mineral salts solu- tion and mineral salts solution plus sucrose for 3 V. dahliae isolates. Large microsclerotia germ- inated better than smaller ones. The microsclerotia of 2 isolates showed decreased germination per- centages with culture age over a 4-11-week period. Microsclerotial germination percentages were al- ways 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 mm 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 devel- ops around the root. This gradient is influenced by factors such as soil humidity and uptake by micro- organisms [2]. Fungal propagules stimulated to germinate by root exudates may form a 'germina- tion gradient' around the root that may influence the relation between the disease index and the inoculum density for a given hos t -pa thogen-soi 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 in- fecting 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 germina- tion 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

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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, fluo- rescein diacetate, was used.

3. MATERIALS 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 microsclerotia- and conidia-forming patterns. Isolate S forms mi- crosclerotia in abundance and conidia in relatively large numbers, whereas P2 produces fewer micro- sclerotia 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 in- cubated without shaking for 4 11 weeks at 20°C in the dark. For isolate S, flasks were also in- cubated 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., DIN 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 microsclerot ia /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 suc- tion 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 solu- tion [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 mm for each net. About 120 microsclerotia per net were counted. Five nets were used for each treat- ment. 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 mois-

tened sand. Three nets (40-~m) with trapped mi- crosclerotia (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

The Swedish Seed Association SvalOf, Sweden) were germinated in a plastic Petri dish on mois- tened 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 micro- scopic 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 examina- tion 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 per- formed on two different occasions. The first time, the rhizospheres of 4 plants, and the second time, those of 2 plants were investigated.

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) supple- mented with 50/.tl of 0.1% (w/v) FDA 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].

4. RESULTS

4.1. Estimation of germinated microsclerotia The same technique was used to visualize

germinating microsclerotia in both the liquid sys- tem and in the sand culture system. With the FDA-staining technique, germinated micro- sclerotia had green-glowing hyphae while un- germinated microsclerotia could be seen by chang- ing to transmitted light. On the nylon nets, there- fore, newly emerging hyphae were easily dis- tinguished from old hyphal fragments attached to

1 1 1 i i / ~ ~ ~ ~ ROOT POSITION

/ \

NET DISKS r- ~ ---

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

50

ISOLATE Vd71/36 NET PORE SIZE 40urn % ..... 100

A SE I

5 0

ISOLATE Vd71/36 NET PORE SIZE 80urn

B SE I

e

o ;, 7 1I w e e k s o ,,. 4 "I 1-1 w e e k s

O~ ISOLATE P2 NET PORE SIZE 40um O~ ISOLATE P2 NET PORE SIZE 80um 1 O0 1 O0

50 50

C SE ,

f

- 8

D SE I

. . . . . . . . - i - _ J

0 4 7 11 w e e k s 0 ~, ~, 7 11 w e e k s

O~ ISOLATE S NET PORE SIZE 40um % ISOLATE S NET PORE SIZE 80urn 100 100

50

E SE I

e

50

F sE I

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.

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 germina- tion of 27%. The microsclerotial germination per- centages 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 con- stant 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 germina- tion 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, al- though 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 germina- tion obtained far away from the root were prob- ably an effect of the limited fungistasis in the sand system, since these germination percentages were

297

%

70

.~ 60

E

O= 50 g t=

40 o

2 0

10

0

i , i

.5 10 1"5 mm d is tance f rom root ax is

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 DD- water and mineral salts solution (Fig. 2).

5. DISCUSSION

Fungal propagules can sometimes be con- sidered 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 im- portant, 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 micro- sclerotia germinated soon after the root tips passed, and that a steep germination gradient from the root rapidly developed. This was probably an ef- fect 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-in- dependent as has been reported earlier for this fungus. Filonow and Lockwood [9] found V. dahliae microsclerotia to be independent of exo- genous nutrients for germination, and even after 45 days of diffusive stress, they had lost only a little of their nutrient independency. This dif- ference 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 forma- tion pattern. For Vd 71/36 microsclerotial differ- entiation 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 tech- nique, it is also possible to record the positions of the propagules in relation to a root or other sources of germination-stimulating substances.

A C K N O W L E D G E M E N T S

We thank Dr. Bengt SOderstrOm, Dr. Erland Baath and Dr. Conny Liljenberg for valuable dis- cussions. This study was supported by grants from

the Swedish Natural Science Research Council and the Swedish Council for Forestry and Agri- cultural Research.

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