Meyer, J.A.

Renard, Jean-Luc

Horizon / Pleins textes

Tram. Br.  mycol. Soc. 53 (3), 455-461 (1969) 
Printed in Great Britain 
SEROLOGICAL STUDY OF 
SAPROPHYTIC STRAINS OF FUSARIUM OXYSPORUM 
AND F. OXYSPORUM F. SP. ELAEIDIS 
BY J. L. RENARD ANDFA.  M E Y E 4  
Laboratories of Phytopathology, Orstom B.P. 20  Abidjan, 
Ivor_y Coast and Universip of Louvain, 42, De Croylaan Heverlee, Belgium 
(With 2 Text-figures) , 
Strains of F. oxysjorum Schlecht. f. sp. elaeidis Toovey and of F. oxysjorum 
Schlecht. isolated from soil are analysed serologically by the double diffusion 
technique in agar gel and by immunoelectrophoresis. The great variability 
in the antigenic constitution of extracts of strains within a phenotype or within 
a pathogenic forma renders the distinction of the different types impossible 
with serological techniques. All the strains studied, isolated from a restricted 
geographical area, seem to belong to one serological group. 
I n  the course of the investigation on the development of the population 
of F. ogsporum Schlecht. in the soil in relation to the vascular wilt disease 
of the oil palm (Elaeis guineensis Jacq.), three different wild types were 
isolated (Renard, 1967): type FOI rare and limited to the savannah, 
type F 0 4  found in all the soils analysed and type FOS isolated abundantly 
from the palm soils. Pathogenic strains FO isolated from diseased trees 
are of the FOS phenotype. FO and FOS are sporodochial types while 
FO I and F 0 4  are mycelial types differing in their pigmentation and the 
abundance of aerial mycelium. 
The symptoms of vascular wilt may appear from the third year, at 
random, in plantations established on savannah soils. 
It is thus interesting to know if there exists any relationship between 
the different types of Fusarium, from the saprophytic types of the savannah 
soil and the pathogenic form isolated from the oil palm. Meyer & Renard 
(1969)~ employing protein and esterase spectra obtained by polyacrylamide 
gel electrophoresis, have shown great biochemical heterogenicity within 
pathogenic formae even from restricted geographical areas. This technique 
seems thus inadequate for showing any relationship between our different 
isolates or for differentiating pathogenic forms from saprophytic ones. 
Madhosingh (I 964), Morton & Dukes ( I  966) and Kalyanasundaram, 
Desai & Sirsi (1967) were able to distinguish by serological techniques 
species within the genus Fusarium, and formae speciales and races within 
F. ogsporum. We therefore used immunochemical techniques to approach 
the problem of the relationship between the different types of F. ogsporum 
as cited above. 
456 Transactions British Mycological Society 
MATERIAL AND METHODS 
The origin of strains, their phenotypes and their corresponding antisera 
are given in Table I. 
All the strains were isolated from the region of Dabou (Ivory Coast). 
In  some experiments we included F. oxysporum Schlecht. f. sp. melonis 
Snyder & Hansen (M15) and F. solani (Mart.) Sacc. (FS). 
Table I. Origin, ope and corresponding antisera of the strains 
of Fusarium oxysporum studied 
Corresponding 
Strains TYP= antisera 
RI* 
Rio* 
RZI* 
R23* 
R25* 
RIQt  
RI43 
R16$ 
FO 
FO 
FO 
A 
E 
F 
FO G 
FÖ 
FOS 
FO I 
F04  
H 
B 
C 
D 
* F. oxysporum Schlecht. f. sp. elaeidis Toovey. 
f F. oxysporum Schlecht. from palm tree plantation soil. 
$ F. oxysborum SchIecht. from savannah soil. 
The isolates were grown at 28 O C  in the dark, in 500 ml Erlenmeyer 
flasks with ~ o o m l  of Richards medium. After g days incubation, the 
cultures were filtered and the mycelium washed and homogenized for 
3 min with O ' I M  phosphate buffer (pH 7.1) in a Waring blendor. This 
homogenate was ground in a Braun cell disintegrator in which it was 
mixed with 0-2-0-3 mm diam glass beads in the proportion of I : I (v/w). 
The containers were shaken mechanically for 4min, being placed in a 
metal cylinder whose axis described a circular movement at a speed of 
4,000 rev./min. whilst cooled by a stream of carbon dioxide. 
The antisera were obtained by intramuscular injection of the homo- 
genate emulsified into an equal volume of Freund's incomplete adjuvant. 
The injections were given twice a week and continued for five consecutive 
weeks. The sera were collected 8 days after the final injection. They were 
lyophilized or conserved at - 25O. 
The antigens used in the serological tests were obtained by centrifuging 
the homogenate at  3800 g ;  the soluble proteins in the supernatant were 
precipitated with saturated ammonium sulphate, centrifuged at 20 000 g,  
dialysed overnight against veronal buffer 0 . 0 2 5 ~  (pH 8.6) then resus- 
pended in the same buffer. The protein content was estimated by the 
method of Lowry, Rosebrough, Farr & Randall (1951). A final con- 
centration of 4500 to 6000 pg of proteins per ml was used in the serological 
tests. 
The agar double-diffusion method was used to compare antigen- 
antibody reactions following the standard Ouchterlony method. The 
diameter of the holes was 5 mm and the distance between the holes from 
Serology. J.  L. Renard and J. A. Meyer 457 
centre to centre IO mm. The agar plates were maintained at 25' and the 
results observed after 20 and 40 h reaction. 
Immunoelectrophoretic analysis was done on 25 x 75 mm glass slides 
covered with 4 ml of a I % agarose reinst (Serva) solution in veronal 
buffer, 0 . 0 2 5 ~  (pH 8.6), during go min at 4.5 V/cm. The development of 
I patterns by diffusion of antiserum was continued up to 48 h at room 
temperature. 
Comparison of antigen-antibody reactions was based on the number, 
position and intensity of the precipitation arcs. 
RESULTS 
Double da&ion in agar gel 
Antiserum A reacts with its homologue antigen R I, giving at least ten 
precipitation lines, but this result is not constant throughout. With dif- 
ferent extracts of R I  or with extracts of other strains of the same pheno- 
type the number of lines varies. In  cross reaction of antiserum A against 
the antigens FOS, FO I and F 0 4 ,  different number of lines are detected; 
between different strains of the same phenotype, the number of lines is 
not constant. 
Table 2. Maximum number o f  precipitation lines obtained in agar plates with 
antiserum A reacting agaimt antigens o f  F. oxysporum saprophytic gpes (FOS, 
F O  I, FO4), F. oxysporumf. sp. elaeidis (FO), F. oxysporumf. sp. melonis 
  MI^), and F. solani (FS)  
Precipitation lines 
TypeS.and - A > 
species a b c d e f g h i j 
FO + + + + + + + + + +  
FOS + . + + + + + + +  
FOI i . + + + +  
~ 0 4  . . + + + + + . + +  
. + .  . + . + +  g15 . . + .  . + + + + +  
The isolates from the Ivory Coast have five antigens in common; 
F. oxysjorum f. sp. melonis shares four antigens with them and F. solani only 
three. 
Within the forma elaeidis (FO type), the differences are as great as 
those existing between the different types studied. They are not only 
represented by different numbers of lines but also by differences in their 
intensity and clarity which suggests that the concentration for individual 
antigens is highly variable within a strain as well as between strains of 
the same phenotype. 
These results are partially confirmed by the absorption experiments. 
The mixing of antiserum A and its antigen FO in equal proportion is not 
sufficient to absorb the antiserum completely, but when one part of anti- 
serum is mixed with three parts of antigen, the antiserum is completely 
absorbed by FO and FOS; when absorbed with FO4, it reacts against 
FOS and FOY giving two precipitation lines, and when absorbed with 
458 Transactions British Mycological Society 
FO I three arcs with FO antigen, two with FOS and one with FO I and 
FO4. Figure I shows the result of one experiment. 
The macromolecular fraction of FO and FOS extracts as defined by 
Gooding (1966) for Fomes annosus, reacts with the antiserum A giving four 
arcs, while those of FO I and F 0 4  give only three arcs respectively. 
Fol" O 
I -  
- 
:FOI O 
ll 
Fig. I .  Diagrams of serological reactions with antiserum A absorbed with extracts of 
the different phenotypes or diluted with physiological saline in different proportions. 
a-b-c-d, one part antiserum mixed with one part antigenic extract; e-f-g-h, one part 
antiserum mixed with. three parts antigenic extract; i, one part antiserum mixed with 
one part physiological saline; j, one part antiserum mixed with three parts physiological 
salie; k, semm A undiluted. 
V I  ----AL------  V I I 
- 11 
4- 
Fig. 2. Schematic reconstitution of the immunoelectrophoretic analysis of antiserum A 
against FO antigens. 
Immunoelectrophoresis 
. The immunoelectrophoresis of FO antigens against antiserum A gives 
16 precipitation lines which may be grouped into five zones (Fig. 2 ) .  
Towards the anode there exist three zones of higher protein concentration. 
The most, distant one contains the lines o, I, 2, 3 and 4; lines 5 and 6 
Serology. J. L. Renard àndJ. A. Meyer 459 
correspond to a zone of average migration, these lines are close to each 
other and wide spread; lines 7, 8, 12 and 13 belong to the group of slow 
electrophoretic migration. Lines 9, IO, 14 and 15 have sometimes no 
migration or a very slight one towards the cathode. Lastly, line I I migrates 
typically towards the cathode. Lines o, 3, 7, 12, 13 and 15 are always 
faint and it is among them that the greatest variability exists. This is 
apparent in different extracts from the same isolate as well as between 
extracts of isolates of different phenotypes. 
All the antisera share at least the antibodies o, I, 2, 4, 5, 6, 9, IO, I I, 15 
(Table 3) corresponding to the presence of common molecular constituents 
in the antigenic extracts (Table 4). 
Table 3. Constitution of the antisera revealed by the cross-reaction 'of each serum 
against the dayerent antigens from saprophytic strains of F. oxysporum and strains 
of F. oxysporumf. sp, elaeidis 
1 Total 
Precipitation lines number 
Antisera o I 2 3 4 5 6 7 8 g I O  I I  12 13 14 15 bodies 
A + + + + + + + + + + + + + + . +  1.5 
E + + +  + + + .  . + + + .  . . +  1 0  
G + + + + + + + . + + + + . . i +  13 
H + + + . + + +  + + + .  + + 1 1  
B + + + . + + + $ & + + + . i . +  13 
G + + + . + + +  + + + .  . + I O  
D + + + . + + + $ i + + .  . . * $ +  13 
I r of anti- 
F + + + i + + + . + + + + . .  + I 2  
Table 4. Maximum number of antigenic constituents detected . 
in the dafferent phenotypes o f  F. oxysporum - I  
Total- 
Precipitation lines number 
L of Pheno- - 
types o I 2 3 4 5 6 7 8 g I O  I I  12 13 14 15 antigens 
FO + + + + + + + + + + + + + + - i - +  16 
F O S + + + . + + + + + + + + . +  + I3 
F O I + + + . + + +  + + +  . i +  I I  
F 0 4 + + + . + + + / $ + + + $ . + +  1 4 '  
Table 5. Number of precipitation arcs detected in cross-reactions between the antisera 
and the extracts of the afferent strains each representing a phenotype of 
F. oxysporum 
Phenotypes 
Antisera R I  (FO) ~ Rr3 (FOS) R14 (FOI) R16 (FÒ4) 
A E 
F 
G 
H 
B .  
G 
D 
15 
I O  
I 2  
I 2  
IO 
12 
7 
9 
, I O  
5 
5 
5 
7 
I í  
7 
8 
6 
5 
7 
i 
8 
I O  
I O  
8 
8 
8 
8 
9 
9 
11 
The italicized values represent the number of lines developed by an antiserum against its 
homologue antigen. 
460 Transactions British Mycological Society 
Table 3 shows how heterogeneous are the antisera of the different strains 
of F. oqwporum f. sp. elaeidis, the number of arcs developed against the 
antigens of this type varying from IO to 15. 
In  Table 5 are collected all the results of the cross-reactions of the 
different antisera against the antigens of four strains, each representing a 
phenotype. The results show that the number of precipitation lines is not 
necessarily the highest when an antiserum reacts against its homologue 
antigen. 
DISCUSSION 
Immunoelectrophoresis allows a better separation and the detection of 
a greater number of antigens than the double diffusion in agar plates. 
However, the complex constitution of antigenic extracts, the heterogenicity 
of the antisera inherent to the differential animal response and the very 
low titre of the antisera (no reaction occurs with any dilution above I :8) 
nevertheless limit the amelioration expected from this method. 
Within a strain, electropherograms are not exactly identical from one 
culture to another one, the variations bearing as well on the number of 
arcs as on the intensity of their specific precipitate. Between the strains 
the same variability is encountered and is not greater between the isolates 
of different types than within a type. 
Isolates FOY FOS and F 0 4  possess I 2 molecular constituents in common. 
The differences between the isolates are situated at the level of the lines 
3, 7, 8, 12, 13, 14. They may be of qualitative or of quantitative nature, 
i.e. that the absence of a line does not mean necessarily that the molecular 
constituents are absent, but only at a level not detectable. This variability 
does not allow the separation of the types on the base of these observations, 
and does not confirm the gross cultural differences among the types. Be- 
tween the isolates FOI and the others, the serological relation seems to 
be more distant but not stable enough to differentiate them only on this 
basis. 
Data reported in this paper reveal no major qualitative difference be- 
tween saprophytic and pathogenic strains and between strains of different 
wild types. We may therefore regard all our isolates as belonging to the 
same serological group. Other authors have, however, distinguished taxo- 
nomic entities with serological techniques. Suerth & Varney (1968) claimed 
to differentiate several races of Phytophthora fragariae and Cheung & 
Marshall (1967) to characterise isolates of Trametes spp. In  the latter case 
differences were obtained only with one antiserum and a limited number 
of cultures. In  the same way, Madhosingh (1964)~ Kalyanasundaram et al. 
(I 967) and Morton & Dukes ( I  966) could differentiate respectively species 
of Fusarium and pathogenic forms and races of F. oxysporum but these 
authors limited their investigations to one strain per taxa. 
Meyer & Renard (1969) have shown with protein spectra obtained by 
electrophoresis on polyacrylamide gel that within pathogenic formae there 
exist great differences between the strains although separate entities can 
,be distinguished. The discriminating power of the protein spectra revealed 
with amido-black seems thus to be much better than that of the spectra 
Serology. J. L. Renard andJ. A. Meyer 461 
, obtained by immunoelectrophoresis. These authors obtained up to 25 pro- 
I tein bands on acrylamide gels instead of I 6 antigenic compounds obtained 
by immunoelectrophoresis. 
I 
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(Acceped for publication 25 July 1969) 


Serological study of saprophytic strains of Fusarium oxysporum and F. oxysporum F. sp. elaeidis

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