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192 Journal of Nematology, Volume 9, No. 3, July 1977
soluble proteins of Ditylenchus dipsaci and
D. triformis. J. Nematol. 3:79-84.
10. KRUSBERG, L. R. 1960. Hydrolytic and
respiratory enzymes of species of Ditylenchus
and Pratylenchus. Phytopathology 50:9-22.
11. KRUSBERG, L. R. 196l. Studies of the
culturing and parasitism of plant parasitic
nematodes, in particular Ditylenchus dipsaci
and Aphelenchoides ritzemabosi on alfalfa
tissues. Nematologica 6:181-200.
12. KRUSBERG, L. R. 1964. Investigations oil
l)itylenchus dipsaci polygalacturonase and its
relation to parasitism of this nematode on
alfalfa (Medicago sativa). Nemalologica 10:72
(Abstr.).
13. KRUSBERG, L. R. 1967. Pectinases in
Ditylenchus dipsaci. Nematologica 13:443-451.
14. LOWRY, O. H., N. J. ROSEBROUGH, A. L.
FARR, and R. J. RANDALL. 1951. Protein
measurement with the Folin phenol reagent.
J. Biol. Chem. 193:265-275.
15. MILLER, G. L. 1959. Use of dinitrosalicylic acid
reagent for determination of reducing sugar.
Anal. Chem. 31:426-428.
16. MUSE, B. D., L. D. MOORE, R. R. MUSE, and
A. S. WILLIAMS. 1970. Pectolytic and
celhtlolytic enzymes of two populations of
Ditylenchus dipsaci on "Wando" pea (Pisum
sativum L.). J. Nematol. 2:118-124.
17. NEUKOM, H. 1960. 12bet Farbreaktionen yon
Uronsaiiren mit Thiobarbitursaiire. Chimia
14:165-167.
18. PRIDHAM, J. B. 1956. Determination of sugars
on paper chromatograms with P-anisidine
hydrochloride. Anal. Chem. 28:1967-1968.
19. RIEDEL, R. M., and W. F. MAI. 1971.
Pectinases in aqueous extracts of Ditylenchus
dipsaci. J. Nematol. 3:28-38.
20. RIEDEL, R. M., and W. F. MAI. 1971. A com-
parison of pectinases from Ditylenchus dipsaci
and Allium cepa callus tissue. J. Nematol.
3:174-178.
21. SEINHORST, J. w. 1959. The host range of
Ditylenchus dipsaci and methods for its
investigation. Pages 44-49, in J. F. Southey,
ed. Plant Nematology. Minist. Agric. Fish.
Food Tech. Bull. 7. HMSO, London.
22. SMITH, W. K. 1958. A survey of the production
of pectic enzymes by plant pathogenic and
other bacteria. J. Gen. Microbiol. 18:33-41.
23. STEPHENS, G. J., and R. K. S. WOOD. 1975.
Killing of protoplasts by soft-rot bacteria.
Physiol. Plant Pathol. 5:165-181.
24. TRACEY, M. V. 1958. Cellulase and chitinase in
plant nematodes. Nematologica 3:179-183.
Effects of Hydrolytic Enzymes on Plant-parasitic Nematodes
P. M. MILLEW and D. C. SANDS 2
Abstract: Proteases, lipase, and chitinase killed Tylenchorhynchus dubius in vitro and in soil.
Tylenchorhynchus dubius was more susceptible to the enzymes than Pratylenchus penetrans.
Papain was the most effective protease, and other enzymes were less effective. Heating enzymes
to 80 C for 10 rain greatly reduced nematicidal effectiveness. Scanning electron micrographs
showed that papain and chitinase produced structural changes in the cuticle of T. dubius.
Lipase removed a thin outer layer. Papain removed material filling the striata, or furrow,
between the horizontal bands. When added to soil, chitinase, lipase, collagenase, and proteases
(papain and bromelain) decreased motility of T. dubius populations up to 75%. Bromelain was
the most active in soil against T. dubius, and collagenase was the most active in soil against P.
penetrans. Key Words: Tylenchorhynchus dubius, Pratylenchus penetrans, chitinase, papain,
collagenase, lipase, cuticle, soil, scanning electron microscope.
Organic matter added to soil decreases
populations of plant-parasitic nematodes
(6, 9, 10). Nematodes are presumably ex-
posed to different types of enzymes released
during the decomposition of the different
Received for publication 19 December 1975.
1Department of Plant Pathology and Botany, The Connecti-
cut Agricultural Experiment Station, P. O. Box 1106, New
Haven, Conn. 06504. We thank Margaret Finkbeiner for
technical assistance and Dr. Allen Pooley, Peabody Museum
and Yale University, for assistance with the scanning
electron-microscopy.
2 Present address: Department of Plant Pathology, Montana
State University, Bozeman, NIT 59715.
types of organic matter in soil. In this
paper, we report the effects of several
enzymes from plant tissues on plant-
parasitic nematodes. The enzymes were used
singly or in combination, in vitro and in
soil. The influence of papain and collage-
nase on nematodes parasitic on animals
has already been reported (1, 2).
MATERIALS AND METHODS
Tylenchorhynchus dubius was extracted
by sugar flotation and a tissue technique
(7) from a bluegrass turf rhizosphere.
Pratylenchus penetrans was extracted from
potato rhizosphere by sugar flotation.
Enzymes tested were the following com-
mercial preparations: wheat-germ lipase,
steapsin (lipase), and erepsin (protease)
from Nutritional Biochemicals Corpora-
tion; collagenase and crystalline papain
from Sigma Biochemicals; chitinase and
pronase (protease) from Calbiochem; and
bromelain from Dole Division of Castle
and Cooke. In the first test, enzymes were
dissolved in deionized water at 0.8 mg/ml.
Two ml of a standardized nematode
suspension containing 84 T. dubius/ml
were mixed with 2 ml of enzyme solution
in small plastic cups at room temperature
(22 C). Treatments were replicated four
times and tests were repeated twice. Live,
motile nematodes were counted after 18,
36, and 48 h. This procedure was used in
the succeeding tests.
In additional tests, as in the previous
tests, nematode suspensions were combined
with enzyme solutions to give concentra-
tions of 0.01, 0.1, and 1 mg/ml of papain
and wheat-germ lipase and 0.001, 0.01, and
0.I mg/ml of chitinase in 0.05 N potassium
phosphate buffer at pH 6.8. Motility counts
were made after 24 and 48 h. For a control,
enzyme preparations in double strength
buffer were used so that, when they were
added to an equal volume of nematode
suspension after they were cooled, they
would have the same enzyme concentration
as the unheated nematode suspension. They
were heated at 80 C for 10 rain and cooled
prior to being added to nematodes. All
treatments were replicated 4 times, and the
test was repeated twice.
To determine whether the enzymes were
killing or merely immobilizing T. dubius, 1
ml of a nematode suspension, which was
obtained from a bluegrass sod and con-
tained 79 T. dubius, 25 Hoplolaimus
tylenchiformis, and 5 P. penetrans, was
mixed with enzyme solutions in water to
give concentrations of 1 mg of papain,
wheat-germ lipase, and collagenase, and 0.1
mg of chitinase/ml. After being incubated
for 48 h at 22 C, the active nematodes were
counted. Then the nematodes aml enzymes
were washed onto a 38-tLm sieve and rinsed
for 3 min with water to wash away the
enzymes. Then the nematodes were washed
Enzymes Effect Nematodes: Miller, Sands 193
from the sieve into glass petri dishes and
motile ones were counted after 24 and 96 h.
Activity of papain, collagenase, lipase
and chitinase (0.4 mg/ml) was tested in
0.1 N potassium phosphate buffer to deter-
mine influence of pH on enzyme activity
against nematodes. After the mixtures were
incubated for 16 h at 22 C, counts of motile
nematodes were made.
The effects of a protease inhibitor,
pentachloronitrobenzene (PCNB) (3), on
papain activity against T. dubius was tested
by adding 0.2 mg of PCNB (a.i.)/ml with
and without 0.4 mg of papain/ml. Counts
were made after 24 h.
To determine whether morphological
changes, visible with a light microscope,
occurred during immersion in enzymes, T.
dubius was incubated for 16 h at 22 C in
chitinase, 0.1 mg/ml of deionized water, or
water alone, and then stained in iodide-
potassium iodide in sulfuric acid (4).
Tylenchorhynchus dubius was also incu-
bated for 16 h at 22 C in a similar manner
in 1 mg of wheat-germ lipase/ml of water,
and then stained for lipids after the method
of Glick (4).
The influence of these enzymes on the
cuticle of T. dubius was determined with
the scanning electron microscope. After T.
dubius was incubated for 2.5 h at 22 C in
0.1 N potassium phosphate buffer (pH
6.8) containing 1 mg of lipase or papain or
0.1 mg of chitinase/ml, the nematodes were
pelleted by centrifugation at 1,000 g for 2
min. They were re-suspended in 35%
(w/vol.) sugar solution, centrifuged at 1,000
g for 2 min again, and then removed with a
pipette from the top of the solution. The
nematodes were placed on a 0.22-mu milli-
pore filter under vacuum to remove the
sucrose solution and washed repeatedly with
deionized water. Nematodes on the mem-
brane filters were immediately frozen at
--50 C for 2 h and vacuum-dried. Samples
were stored over dessicant at --5 C. Control
nematodes were handled in the same man-
ner but without enzymes. Sections of each
filter disc containing approximately 15-30
nematodes were mounted with graphite
DAG (Acheson Colloid Co.), shadow-cast
with gold in 360 ° rotation, and viewed at
5,000 X with a scanning electron microscope
(ETEC Corp. California, Autoscan SEM).
The first five nematodes with clearly visible
194 Journal of Nematology, Volume 9, No. 3,
surface features were photographed at mid-
section.
Soil infested with P. penetrans and T.
dubius was mixed with either papain,
chitinase, collagenase, wheat-germ lipase, or
bromelain at rates of 4 and 40 mg/kg.
Treatments were replicated 4 times and
tests were repeated twice. After 3 weeks,
nematodes were extracted from 100 gm of
soil by sugar flotation and motile nema-
todes were counted.
RESULTS
In nonbuffered solutions, papain,
chitinase, and wheat-germ lipase killed all
T. dubius in 48 h (Table 1). Although not
given in Table 1, P. penetrans and H.
tylenchiformis were more resistant than T.
dubius to papain; 37% of P. penetrans and
28% of H. tylenchiformis were still motile
after 48 h while T. dubius was completely
immotile.
Toxicity o~ papain and lipase against
T. dubius decreased with dilution of the
enzyme, but chitinase was equally toxic at
high and low concentrations (Table 2).
Heating destroyed activity of chitinase and
lipase and most of the activity of papain for
24 h, but chitinase and lipase solutions
showed some activity after 48 h.
After 24 h, all T. dubius in collagenase
TABLE 1. Influence of some hydrolytic enzymes
on motility of Tylenchorhynchus dubius in vitro
after 18, 36, and 48 h.
Percent of nematodes
motile
Enzyme (0.4 mg/ml) 18 h 36 h 48 h
Water alone 92 a r 85 a 88 a
Wheat-germ lipase 86 a 31 bc 0 d
Papain 25 c 8 d 9 d
Chitinase 67 b 48 b 0 d
Collagenase 82 c 46 b 22 c
Steapsin 80 a .~5 b 53 h
Pronase 90 a 99 a 53 b
Promelain 88 a 88 a 88
a
Erepsin 89 a 87 a 72 a
Mixture of all enzymes' 53 b 25 c 23 c
YFigures followed by same letter not significantly
different from each other, according to Duncan's
Multiple Range Test (P=0.05).
zAll seven enzymes were each used at 0.4 mg/ml in
mixture and gave a total enzyme content of 2.8
mg/ml,
July 1977
TABLE 2. Effects of concentration and heat
inactivation on toxicity of enzymes to Tylen-
chorhynchus dubius in vitro.
Percent of
nematode
Concentration motileY
Enzyme x (mg/ml) 24 h 48 h
None -- 100 a 95 a
Chitinase 0.001 78 b 38 c
0.01 74 b 35 c
0.1 81 ab
35 c
0.01 heated" 100 a 77 ab
Papain 0.01 96 a 65 b
0.I 85 ab 19 c
1.0 0e 0d
1 heated 78 b 77 ah
Lipase
(wheat germ) 0.01 78 b 54 b
0.1 56 c 31 c
1.0 24 d 11 d
0.I heated 0 e 85 a
Xtn 0.1 N potassium phosphate buffer at pH 6.8.
rFigures followed by same letters not significantly
different from each other by Duncan's Multiple
Range Test (P=0.05).
"Enzyme preparation heated at 80 C for 10 min;
then cooled to room temperature before being
mixed with nematodes.
were immobile and over 80% of those in
papain and chitinase were also. As in the
previous tests, lipase was less effective, im-
mobilizing only 57% of T. dubius. H.
tylenchiformis and P. penetrans, as well as
the microbivorous nematodes, were barely
affected by the enzymes. After the enzymes
were washed away, T. dubius was still im-
motile after 96 h in collagenase, papain,
and chitinase. Of those which had been
treated with lipase, only 41% were im-
motile; thus a few regained motility. H.
tylenchiformis, P. penetrans, and the micro-
bivorous nematodes, appearing unaffected
by tile enzymes, were still motile and active.
Acidity had a great influence on enzyme
activity (Table 3). Collagenase and papain
were more active at pH 6 than at pH 5.
Papain was ineffective at pH 5. Chitinase
and lipase were effective at pH 5 and pH 6,
and also reduced the motility of P. pene-
trans 20-30% at pH 5 and 6.
PCNB reduced effectiveness of papain
against T. dubius. Papain reduced motility
of T. dubius 77 %; PCNB alone reduced it
27%; but PCNB plus papain reduced it
TABLE 3. Effects of pH on toxicity of four
hydrolytic enzymes to
Tylenchorhynchus dubius in
vitro.
Percent of
Enzyme r pH motile nematodes
Buffer alone 5 94 a
6 73 ab
Papain 5 91 a
6 36 cd
Collagenase 5 40 c
6 12d
Chitinase 5 25 d
6 39 c
Lipase 5 39 c
6 46 c
tin 0.1 N potassium phosphate buffer for 16 h at
22 C.
"Each figure average of four replicates with 33
nematodes added/replicate. Figures followed by
same letter not significantly different from each
other, according to Duncan's New Multiple Range
Test (P= 0.05).
only 24%, a reduction equal to that of
PCNB alone.
The toxicity of enzymes in soil did not
parallel their toxicity in aqueous solutions
(Table 4). Papain was the most toxic in
aqueous solutions against
T. dubius;
brome-
lain was the most toxic in the soil since 40
mg/kg of soil reduced
T. dubius
popula-
tions 76%. Papain, collagenase, lipase, and
chitinase were moderately toxic in soil at
one or more concentrations.
PratyIenchus
penetrans
was not affected by papain in
soil, and was affected most by 40 mg/kg of
collagenase in soil.
There were no visible structural changes
in the nematodes when they were examined
with the light microscope.
Incubating
T. dubius
in active enzyme
solutions for 2.5 h produced changes in the
cuticle that were observed with the scanning
electron microscope. The cuticle of the
nontreated nematode (Fig. l-A) shows
regnlar, transverse striations or bands that
are slightly indented and apparently rigid.
The entire nematode appears to be coated
with a very thin, slightly electron-opaque,
layer. Chitinase removed the tops of the
bands and caused lateral folding of T.
dubius
and loss of transverse rigidity (Fig.
l-B). Papain deepened striations and made
them appear as deeper furrows (Fig. l-C).
Enzymes Effect Nematodes:
Miller, Sands
195
TABLE 4. Toxicity of four hydrolytic enzymes
to
Pratylenchus penetrans
and
Tylenchorhynchus
dubius
populations in soil.
Treatment
No. of motile
nematodes/100
grn soilr
Concentration P. T.
(mg/kg of soil)
petetrans dubius
Water check -- 45 b 40 a
Papain 10 51 b 21 c
40 55 b 19
c
160 37 c 14
d
160 heated • 91 a 47 a
Chitinase 4 56 b 18 c
Collagenase 4 72 a 47 a
40 23 d 23 c
Lipase 4 38 c 21 c
Bromelain 40 38 c 9 d
YAverage of four replicates 3 weeks after addition
of buffered enzymes. Each figure followed by
same
letter not significantly different from each other,
according to Duncan's Multiple Range Test
(1" = 0.05).
• Enzyme heated to 80 C for 10 rain.
Lipase appeared to remove the thin outer
layer, mentioned before, on the untreated
nematode, but did not change the appear-
ance of the nematode cuticle enough to
show in photoga-aphs. Lipase also caused
some loss of rigidity of the transverse bands.
Chitinase and papain treatments for 2.5
h severely distorted many of the nematodes,
perhaps as many as 80 %, but micrographs
were not made of severely distorted nema-
todes.
DISCUSSION
The data presented in this paper
indicate that solutions of lipase, protease,
and chitinase hydrolytic enzymes are toxic
to nematodes but are more toxic to T.
dubius
than
P. penetrans
or
H. tylenchi-
[ormis.
Enzyme activity resulted in
modification of the nematodes cuticle, an
occurrence which was concomitant with
death.
These results show that the enzymes
actually killed, rather than temporarily
immobilized
T. dubius
and thus were
nematicidal. They were able to cause death
only in
T. dubius,
however,
H. tylenchi-
formis
and
P. penetrans
were much more
resistant to enzymes.
196
Journal of Nematology, Vohtme 9, No. 3, July 1977
FIG. I-(A-D). Scanning electron micrographs (5,000 X) of midsection of
Tylenchorhynchus dubius. A)
No enzyme treatment;
B)
Chitinase;
C)
Protease;
D)
Lipase.
Rich and Miller (11) previously ob-
served that PCNB applied to soil as a
control for fungi had no inhibitory effect
on nematodes, and in fact, the nematode
popultion was stimulated. Such an increase
in nematode populations might occur if the
PCNB inhibited proteases that were toxic to
P. penetrans
in soil in the same way that it
inactivated papain in this test, although
papain is not toxic to
P. penetrans.
Toxic effects of the enzymes were not
always the same in solution and in soil.
Factors which may have accounted for these
differences include enzyme degradation by
other enzymes and adsorption on soil
organic matter or particles and soil pH.
The different effects of enzyme treat-
ments on the three species of plant-parasitic
nematodes may have related to feeding
habit and taxonomic differences.
Pratylen-
chus penetrans
is a migratory endoparasite,
living primarily inside the roots, and is
thus exposed to a wide variety of enzymes.
Tylenchorhynchus dubius
is an ectoparasite,
exposed only to enzymes in the rhizosphere.
It is not surprising that
P. penetrans
might
be more resistant to enzymes or have a
different resistance than
T. dubius.
Saprophagous nematodes occasionally ob-
served in the soil samples were resistant to
the enzyme treatment, possibly because they
were constantly in an environment of decay-
ing organic matter and enzymes and only
those resistant to these enzymes survived.
Tflenchorhynchus dubius
was severely
injured, even when it was in contact with
enzymes for only 2.5 to 24 h. It is to be
expected that
T. dubius
populations might
decrease if they were exposed for several
days or weeks in soil with a high content
of decaying organic matter which released
several different types of enzymes, as well as
toxic products o[ decomposition.
LITERATURE CITED
1. BERGER, J., and C. F. ASENGIO. 1940.
Anthehnintic activity of crystalline papain.
Science 9 1:387-388.
2. DAWSON, B. 1960. Use of collagenase in the
characterization o[ pseudocoelomic mem-
branes of
Ascaris
lumbricoides. Nature 187:
799.
3. GLAZER, A. N., and E. L. SMITH. 1971.
Papain and other plant sulfhydryl proteolytic
enzymes. Pages 501-546
in
P. D. Boyer, ed.
The enzymes. Vol. 3, Academic Press, New
York.
4. GLICK, l), 1948, Techniques of histo- and
cytochemistry, lnterscience Publishers Inc.
1949. 4 p,
5. H1RSCHMANN, H. 1971. Comparative mor-
phology and anatomy. Pages 11-64
in
Zuckermann, B. M., W. F. Mai and R.
A.
Enzymes Effect Nematodes: Miller, Sands 197
Rohde, eds. Plant parasitic nematodes. Vol.
1, Academic Press, New York.
6. MANKAU, R. 1968. Effect of organic and
inorganic nitrogen on nematode populations
on turf. Plant Dis. Rep. 52:46-48.
7. MILLER, P. M. 1957, Cheap, disposable filters
for nematode surveys. Plant Dis. Rep. 41:
192-193.
8. MILLER, P. M. 1957. A method for the quick
separation of nematodes from soil samples.
Plant Dis. Rep. 41:194.
9. MILLER, P. M., G. S. TAYLOR, and S. E.
WIHRHEIM. 1968. Effects of cellulosic soil
amendments anti fertilizer on Heterodera
tabacum. Plant Dis. Rep. 52:441-445.
10. MILLER, P. M., D. C. SANDS, and S. RICH.
1973. Effect of industrial mycelial residue,
wood fiber waste, and chitin on plant para-
sitic nematodes attd some soil borne diseases.
Plant Dis. Rep. 57:438-442.
11. RICH, S., and P. M. MILLER. 1964. Verticil-
lium wilt of strawberries made worse by soil
fungicides that stimulate meadow nematode
populations. Plant Dis. Rep. 58:246-248.
12. SAYRE, R. M. 1971. Biotic influences in soil
environment. Pages 235-236
in
Zuckerman,
B. M., W. R. Mai and R. A. Rohde, eds.
Plant parasitic nematodes. Academic Press,
New York.
Species Differentiation in Caenorhabditis briggsae
and
Caenorhabditis elegans
P. A. FRIEDMAN, E. G. PLATZER, and J. E. EBY ~
Abslract:
Identification of five laboratory strains (1-5) of putative
Caenorhabditis briggsae
was
undertaken. Exantinatiou of the male bnrsal ray arrangement, mating tests with males of
Caenorhabditis elegans,
malate dehydrogenase zymograms, and SDS polyacrylamide electro-
phoresis demonstrated that strain 4 was
C. briggsae
and the others were
C. elegans. Key Words:
bursal ray arrangement, mating test, malate dehydrogenase, SDS polyacryamide electrophoresis,
morphology, taxonomy.
"Fwo free-living nematodes, Caenorhab-
ditis briggsae (Dougherty and Nigon, 1949)
Dougherty, 1953 and C. eIegans (Maupas,
1900) Dougherty, 1953 have become im-
portant model systems for biological studies
and have been used extensively for bio-
chemical (14), nutritional (13), genetic (2,
6), nettrobiological (17),
and aging (14, 18)
research. The basic biology o[ both nema-
todes was described by Nigon and
Received for publication 25 October 1976.
1 Department of
Nematology,
University of California, River-
side, CA 92502. We thank Dr. R. L. Russell and Mr. Carl
.[t~hnson, Division Biolngy, Cal Tech, for assistance in ob-
taining males of
C. briggsae,
strains 1 and 2, and C.
elegans.
Dougherty (12). These species provide a
unique opportunity to study the biology of
closely related nematode species in culture.
We have been investigating drug action
and nutritional physiology in two strains
o[ Caenorhabditis that supposedly were
representative of C. briggsae and C. elegans.
However, the nutritional requirements of
the C. briggsae strain did not correspond to
those reported by Hansen and Buecher (7).
To resolve this discrepancy, other labora-
tory strains of C. briggsae were obtained
for the comparative studies presented
herein.
The specific morphological separation
