Nicolay, R.

Sikora, R.A.

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Notes brèves 
IMPROVED  TECHNIQUES  FOR  THE  DETECTION  OF  NEMATOPHAGOUS 
FUNGI  AND  THEIR  ACTMTY  AGAINST  TARGET  NEMATODES 
Rainer NICOLAY* and  Richard A. SIKORA* 
Fungal  parasites  are  important in natural  regulation 
of plant parasitic nematode populations. Methods for 
the  isolation  and  observation f endoparasitic  and  nema- 
tode  trapping  fungi  have  been  published  (Duddington, 
1955; Cooke, 1961; Barron, 1969), with some applicable 
for  quantitative or semiquantitative  evaluation of anta- 
gonistic activity (Klemmer & Nakano, 1964; Eren & 
Pramer, 1965; Mankau, 1975; Heintz, 1978). 
Al1 techniques however have certain disadvantages. 
Because the detection of  slow developing  endoparasites 
is often masked by fast growing trapping fungi it is 
important  to  test  for  the two groups separately. 
Two  standard techniques and  the improvements 
made  are  outlined in this  paper. 
Baermann-funnel  technique 
The Baermann-funnel allows active nematodes pass- 
age through  a  filter.  Specimens  parasitized by endopara- 
sitic fungi  and  not fully inactivated pass through the 
filter  and  parasitism is usually  detected by transfer to 
agar  plates. 
Whereas,  numerous  examinations have been  made by 
adding nematodes as prey to the agar plates, direct 
confrontation  with  the  fungi in soil has  been avoided. 
Since the differentiation between nematode species in 
field soil following  parasitism is difficult an  attempt was 
made to replace the naturally  present soil populations 
with a target  nematode  marked by staining with chry- 
soidin. This  stain was recommanded by Doliwa 
(1955-1956) and  Kampfe (1956) as a viability stain. 
The technique allows direct  deteciion of specific 
antagonists of the nematode in question. 
METHOD : 
1. The Baermann-funnel is replaced by a  dish  (Oosten- 
brink,  1954) that  supports 100  g of  soil which reduces 
variability obtained  with  other  techniques  using  small 
amounts of soil. 
2. Field soil is placed on  the dish on two milk filters over 
water and active  nematodes  extracted  for  four days 
at room  temperature. 
3. 
4. 
5. 
6. 
7. 
8. 
The extracted  nematodes  are  discarded  from the dish 
and 5 O00 nematodes of the  target species in 10 ml 
of water  spread evenly over the field soil on  the filters. 
Heavy soils should  be  dried for  one  or two days before 
application of the target  nematode.  Drying  reduces 
exposure of endoparasitic fungi with adhesive conidia 
to excessive moisture  that  could  reduce  attachment 
to  the nematode  cuticle. 
The dishes are not filled with water during the 
48 hr incubation stage. The nematodes are stained 
with  chrysoidin  for 24 hr prior to addition. 
The dishes  are then filled  with  water  for  nematode 
extraction. 
24 hr later the nematode suspension in  the  dish is 
poured ont0 a 20 pm aperture sieve, washed with 
water, and transferred into a 15 ml graduated cen- 
trifuge  tube. 
The suspension is centrifuged  for 5 mn at  2 500 rpm. 
The supernatant is removed with  a  pipette and  the 
remaining 0.3 ml mixed with 0.3 ml solution con- 
taining 100 'ppm streptomycin  sulphate + 200 ppm 
penicillin. 
The resulting suspension is spread evenly over 
1.5 O/O water  agar in a  9 cm diam. Pew dish. 
The dishes are kept at room temperature, examin- 
ation  for  parasitized  nematodes is made immediately 
after  addition and  at two day  intervals for  ten days. 
Differential  centrifugation  technique 
The differential centrifugation technique in use, separ- 
ates the heavier spores of the nematode-trapping fungi 
from  the lighter  spores of endoparasites. The soil frac- 
tion containing the latter is poured  ont0 a water agar 
Petri  dish and spread  with  a glass rod over 50 O/O of the 
dish  surface.  Nematodes  are  added in a  water  suspen- 
sion. This suspension  and the water from  the soil extract 
often spreads soil particles over the Petri dish surface 
making  observations  difficult. 
In addition, fungi  such as  Chytridiomycetes, Oomyce- 
tes and Zygomycetes are rarely detected  (Barron, 1977). 
Furthermore,  the  amount of soil poured  ont0  the agar 
R. Nicolay & R. A. Sikora 
surface is small which increases variation  and  requires 
the  use of many  replicates. 
These problems can be reduced by adding larger 
amounts of soi1 to grooves cut out of the agar. The 
sediment of the  first  centrifugation  step  that is routinely 
discarded can also be used for examination of nema- 
tode-trapping  fungi. 
METHOD : 
1. 100  g of soil are  suspended in 150 ml of water. 
2. The suspension is poured  ont0  a 250 pm  aperture 
sieve and washed with as little water as possible 
through  the sieve into  a beaker. 
3. The filtrate is agitated  for 10 s, then  poured  ont0  a 
100  pm  aperture sieve and washed through  the sieve 
into a  beaker. 
4. The resulting  suspension is centrifuged at 650  g  for 
2 mn (Diirschner, 1983). 
5. The supernatant is transferred to clean centrifuge 
tubes while the sediment  containing  the  spores of 
nematode-trapping fungi is mixed with 1 ml of 
water, 1-1.5 ml of the soil suspension is pipetted  into 
a 5-6 mm wide groove cut  into  the  middle of a  water 
agar  Petri  dish  (9 cm diam.) by means of two joined 
scalpels. 
6. 3 000 chrysoidin stained nematodes are added as 
bait  in 0.6 ml of water. 
7. The supernatant of the first centrifugation step 
containing the spores of endoparasitic fungi is 
centrifuged  again at 4 060 g for 30 mn. 
8. The sediment is resuspended  and  pipetted  into the 
grooves in water  agar.  Endoparasitic fungi  are best 
observed by cutting  out a cross-shaped groove 
which  increases  nematode  contact  with  the  fungal 
spores. In addition  this allows a 2 ml  soil suspension 
to  be used  per  plate. 
9. 3 O00 nematodes are pipetted onto the soil in  the 
grooves for  the detection of endoparasites. Nema- 
tode  penetration  into  the  agar is alleviated by adding 
a  small amount of  silver  Sand to  the grooves. 
10. The plates  are  kept at room  temperature  with 
examination for parasitized specimens made im- 
mediately after addition and at two day intervals 
for two weeks. 
The examination of trapping  fungi  requires  more 
time  and  might take up to six  weeks  if examination 
is made  for  fungi with adhesive knobs  or  branches. 
Evaluation is also possible by  transferring the agar 
plates to Oostenbrink dishes after the incubation 
period and counting the stained nematodes that 
have not  been  trapped. 
Conclusions 
The methods described allow detection of fungal 
antagonists in soil samples from  the field on a target 
nematode without special infectivity tests. They are 
efficient  and  time  saving and give an overview of the 
spectrum of nematophagous fungi attacking mobile 
nematodes in field soil. Quantitative examinations are 
also possible. The stain allows marking of viable target 
nematodes,  increases  accuracy, and allows the use of a 
selected  indicator  species for specific  fungal  antagonism. 
The stain can be used for al1 nematode species. 
Cg:imum stain  concentrations Vary and  must be  pre- 
determined. A 24 hr staining period and a chrysoidin 
concentration of 1:20 O00 is recommended  for Hetero- 
dera species, Neoaplectana carpocapsae, Neoaplectana 
bibionis and Heterorhabditis species, whereas, a 
concentration of 1 : 50 O00 is sufficient for free living 
nematodes such as Panagrellus redivivus and Caenor- 
habditis elegans. 
REFERENCES 
BARRON, G. L. (1969).  Isolation  and  maintenance  of  endopara- 
sitic  nematophagous  Hyphomycetes. Can. J. Bot., 47 : 
BARRON, G. L. (1977). T h e  nematode-destroyingfungi. Topics 
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Guelph/Ontario,  Canada,  140  p. 
COOKE, R. C.  (1961).  Agar  disk  method for the  direct  obser- 
vation  of  nematode-trapping fungi in the soil. Nature, 191 : 
DOLIWA, U. (1955-1956).  Experimentelle  Untersuchungen  an 
Kartoffelnematoden (Heterodera  rostochiensis Wollenwe- 
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DUDDINGTON, C. L. (1955).  Notes  on the technique of hand- 
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D~RSCHNER, U. (1983). Pilzliche Endoparasiten an bewegli- 
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EREN, J. & PRAMER, D. (1965). The most  probable  number of 
nematode-trapping  fungi in soil. Soi1 Science, 99 : 285. 
HEINTZ, C. E. (1978). Assessing the predacity of nematode 
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WPFE, L. (1956). Zur Venvendbarkeit  von  Chrysoidin  als 
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11411-1412. 
Accepté  pour  publication le 20juillet 1987. 
116 


Improved techniques for the detection of nematophagous fungi and their activity against target nematodes

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