The Effect of Temperature and Moisture on the Survival of
Heterodera glycines in the Absence of a Host
D. A. SLACK, R. D. RIGGS and M. L. HAMBLEN 1
Abstract: Soybean-cyst nematode larvae survived in water up to 630 days, depending on incubation
temperature. Most larvae were killed when ice crystals formed in water, and all died after 1 day at 40 C. At
temperatures of O, 4, 8 and 12 C, larvae survived for the duration of the experiments (630 days). From 16 to
36 C, survival was inversely correlated with temperature. In naturally infested soil, nematode survival was
similax but more extended and related to moisture level. Larvae survived 7-19 months in flooded soil, 29-38
months in dry soil, and for 90 months in soil maintained near its field capacity.
Heterodera glyeOzes is an obligate parasite
with a broad host range (6, 9, 10, l l, 12, 14,
15). Like most cyst-forming nematodes, it is
able to survive for extended periods in the
absence of a host. Temperature and moisture
affect survival of free larvae or contents of cyst
bodies. Eggs in 11. rostochiensis cysts survived
several months in dry soil at room humidity
(8). Larval emergence from tt. major cysts was
considerable after 3 months' storage in air-dry
soil, but ceased after 6 months (I). When H.
major cysts were removed from the soil and
dried, larval emergence ceased in a matter of
hours (5, 19). Lewis and Mat (7)showed that
m o i s t u r e level changes the e f f e c t of
temperature on survival ofH. rostochiensis eggs
and larvae within cysts, and that alternating
temperature had a more adverse effect than
c o n s t a n t temperature. H. schachtii larvae
survived in water for 6 months, and, in soil
w i t h o u t plants in the greenhouse, larvae
survived for 12 months (3). H. schaehtii
survived 3 months in fallow surface soil in
which the temperature went as high as 52 C
(17). Drying reduced the viability of the
c o n t e n t s o f H. schaehtii cysts, but the
reduction appeared to be a function of rate of
drying rather than dryness (18).
The results of studies on the effect of
temperature, moisture, soil type, cyst condition,
aeration and other factors on various stages of
the life cycle and survival of Heterodera
glycines have been reported (2, 4, 6, 13, 16,
19). This is a report of studies on the effect of a
range of temperatures and various moisture
levels on the survival of H. glycines eggs and
larvae in the absence of a host plant.
R e c e i v e d for p u b l i c a t i o n 14 F e b r u a r y 1 9 7 2 .
1 Department
o f Plant P a t h o l o g y ,
Arkansas, F a y e t t e v i l l e 7 2 7 0 1 .
University
or
MATERIALS AND METHODS
In order to study the effect of temperature
on the survival of larvae, aliquots of freshly
hatched larvae were placed in syracuse watch
glasses held at various temperatures from - 4 to
40 C. Water was added periodically to maintain
a water level of 1-2 ram. The samples were
checked at intervals, and various tests used to
determine whether or not the larvae were alive
and active. The first test was to touch them
with a probe, and if this produced no
movement, a few larvae were cut to determine
whether the body contents flowed. Soybean
seedlings were inoculated at intervals and later
processed for the recovery of mature females to
determine whether the larvae were capable of
penetration and development.
We checked survival of nematodes in soil as
influenced by temperature and soil moisture by
placing large samples of infested soil in plastic
bags at the various temperatures. One series of
samples was kept moist throughout the test
period, another set was allowed to dry to
air-dryness. A sample was taken at intervals,
processed by roiling and sieving and the residue
on the sieve placed on a funnel to check for
larval recovery. When the number of larvae
recovered from the funnels dropped to a low
level, samples were planted to soybean, Glycine
max L. 'Lee', to determine whether surviving
larvae would penetrate roots and develop.
The influence of four moisture levels on
survival of H. glycines in the soil was tested in
the greenhouse using natuarally infested soil of
two types, sandy and clay, potted in 7.6-cm
pots and maintained at the following moisture
levels: (i) air dry (no water added); (it) watered
daily to keep at field capacity: (iii) saturated by
setting pots in a basin of water: and (iv)
submerged in water at all times. Samples were
taken at intervals for a bioassay of nematode
survival. A Baermann funnel assay at the
263
264 Journal of Nematology, Vol. 4, No. 4, October 1972
beginning of the test indicated a population
of
500 viable eggs and largae/100 cc of soil.
TABLE 2. The effect of t e mpe ra t ure and soil moisture
on survival of tleterodera glycines in soil.
RESULTS
Months survival in
Moist soil a
Dry soil
Heterodera glycines larvae in water became
inactive after a short period of incubation, but
could be induced to move by a touch with a
probe. Larvae in water remained infective for
only 1 day at - 4 and 40 C (Table 1), although
they survived for somewhat longer. From 0 to
12 C, larvae survived for approximately 630
days, at which time samples were depleted.
Survival at 16-36 C was inversely correlated
w i t h the temperature. When cysts were
removed from the soil and incubated in water
at 40 C, larvae were recovered after 2 days but
not after 1 week (131.
Temperature had a similar effect on the
survival of a natural population in the soil, but
nematodes survived for a nmch longer period
( T a b l e 2). Freezing did not immediately
destroy the nematodes, and at 40 C in dry soil a
positive bioassay was obtained at 50 days. At 4,
8, 12, 16 and 20 C, nematodes survived the
entire test period (6-8 years). Above 20 C, the
survival time decreased as the temperature
increased. At 20 C, and below survival was
better in the moist soil. From 24 to 36 C,
survival was better in the dry soft.
Te mp
Baermann
to)
funnel
-4
4
8
12
16
20
24
28
32
36
40
49 b,c
96
84
84
84
84
26
22
22
6
4/15
Last
movementa
-4
0
4
8
12
16
20
24
28
32
36
40
26 b
630 c
630
630
630
190
190
66
44
30
12
1/4
Days to:
Last
infection
1
630
630
630
630
530
310
120
60
44
15
l 1/6
Last
turgid
60
630
630
630
630
570
420
28O
210
150
35
7
aLaxvae became inactive after a short period, b u t
could be induced to move by a t o u c h with a pulp
canal file. When m o v e m e n t was no longer obtained , a
biological check and a turgidity test was used to
d e t e r m i n e survival.
bMost larvae died i m m e d i a t e l y upon the f o r m a t i o n of
ice crystals.
CSamples at O, 4, 8 and 12 C were depleted at 630
days.
funnel
Bioassay
96
84
84
84
84
23
17
17
6
4/15
84
72
72
72
72
30
30
30
16
4/15
84
72
72
72
72
30
30
11
11
1 2/3
a s o i l was ke pt in plastic bags, and moisture a dde d
periodically: the dry soil was allowed to dry and
water was never added.
bA t - 4 C, few larvae were found t h r o u g h o u t the test.
CAt 4, 8, 12, 16 and 20 C, larvae were recovered and
t he bioassay was positive w he n the supply of infested
soil was depleted.
Survival ofH. glycines definitely was related
to moisture level (Table 3). Infection was
obtained after 7-19 months under flooded
c o n d i t i o n s , and 29-38 months under air dry
conditions. In saturated soft, infection occurred
for as long as 63 months and for 90 months in
soil watered daily.
T ABLE 1. Survival of Heterodera glycines larvae in
water at various temperatures.
Temp
(C)
Baermann
Bioassay
DISCUSSION
At 36-40 C and below 20 C, a delayed
r e a d i n g at r o o m t e m p e r a t u r e following
treatment was often required. The turgidity
test, in which the nematode was punctured to
determine whether the contents were still under
pressure, was not necessarily an accurate
evaluation of survival but did indicate a stage in
TABLE 3. Survival of tteterodera glycines at various
moisture levels under greenhouse conditions.
Moisture
c ondi t i ons
Flooded
Saturated
Watered daily
Air dry
Number of m o n t h s survived in
Test 1
Test 2
Sandy soil
Clay soil
Sandy soil
7
29
90 a
29
aNegative after 96 months.
19
38
90
38
11
63
40
35
Heterodera glycines: Slack et al.
the degeneration of the larvae. The bioassay
was the most practical test in that it assessed
the infectivity of the larvae; turgidity was a
rough estimate of this capability.
Endo (2) reported that H. glycines larvae
survived only 30 min at 100 F (37.8 C). At 40
C (104 F), larvae remained infective for 28 hr
and appparently were alive somewhat longer in
our tests (Table 1). Ichinohe (6) reported that
eggs remained viable in cysts held at - 4 0 C for
as long as 7 months, and Slack and Hamblen
(13) obtained larval emergence from cysts
maintained at - 2 4 C for 18 months. However,
larvae are sensitive to freezing; infectivity failed
to occur after 1 day when ice crystals formed in
the water surrounding them, even though
movement and turgidity were recorded for a
longer period.
H. glycines survived longer in the soil than
in w a t e r , but the effect o f temperature
presented a similar pattern. The fact that
infection occurred in soil stored air dry at 40 C
for 50 days whereas no larvae were recovered
from a funnel may indicate that the presence o f
host roots had a stimulating effect on the
near-dead larvae. On the other hand, the host
may provide a hatching stimulus for eggs which
would not otherwise hatch. Previous studies
indicated that 11. glycines did not develop in
the host at 34 C and these studies showed that
nemas survived at 36 C for 6-1 l months (Slack
et al., unpublished data). This indicates that the
lack o f development in host tissues at 34 C is
not due to death o f the nemas. A change in
host physiology at 34 C might occur which
prevents nema development or this temperature
may induce a type o f dormancy in the nemas.
Infection occurred over a period of 30 months
at 24 and 28 C, and in some field soils the
temperature at a depth o f 6 inches may n o t
exceed 28 C. Since there are periods when the
s o i l at t h i s d e p t h h a s a much lower
temperature, this may help to account for the
fact that H. glycines can survive in the field in
the absence o f a host for several years. The
longer survival time at 24, 28, 32 and 36 C in
dry soil as compared to moist soil was probably
related to egg hatching. At the low moisture
level the egg hatch would probably have been
low or none. l a r v a e in eggs should survive
longer than larvae free in the soil because they
do not desiccate as readily in eggs.
Drying or flooding o f the soil did n o t
readily kill the nematodes, but soil type
influenced the time of survival. The survival
265
period was longer in a clay type soil similar to
t h a t o n w h i c h r i c e m i g h t b e grown.
Unpublished field observations have indicated
that the H. glycines population is not reduced
appreciably during a rice crop, even though rice
is not a host. Lack o f egg hatching under low
oxygen levels may account for the survival of
H. glycines under flooded conditions in the
field and greenhouse. In dry soil the nematodes
survived for 3 years, which indicates they could
survive in soil balls on implements, boots, shoes
or other possible carriers to be dropped off in
other fields. Under normal water conditions the
nematodes were able to survive almost 8 years
in the absence o f a host, implying that very
long rotations would be required to reduce the
population to a minimum in the soil. Even in
the absence o f soybeans, certain c o m m o n
weeds are hosts and could serve to maintain a
source o f the nema for reestablishment o f the
population when planted to a susceptible crop.
LITERATURE CITED
1.DUGGAN, J. J. 1960. Effect of soil drying on the
viability of Heterodera major cysts. Nature
(London). 185 : 554-555.
2.ENDO, B. Y. 1962. Lethal time-temperature
r e l a t i o n s f o r H e t e r o d e r a glycines.
Phytopathology 52: 992-997.
3.GOLDEN, A. M. and THELMA SHAFER. 1960.
Survival of emerged larvae of the sugar-beet
nematode (Heterodera schachtii) in water and
in soil. Nematologiea 5:32-36.
4.HAMBLEN, M. L. and D. A. SLACK. 1959.
Factors influencing the emergence of larvae
from cysts of Heterodera glycines lchinohe.
Cyst development, condition, and variability.
Playtopathology 49: 317 (A bstr.).
5.HESLING, J. J. 1956. Some observations on
Heterodera major. Nematologica 1:56-63.
6.ICHINOHE, M. 1955. Studies on the morphology
and ecology of the soybean nematode,
Heterodera glycines, in Japan (in Japanese with
English summary). Rep. Hokkaido Nat. Agr.
Exp. Sta. 48:1-65.
7. LEWIS, F. J. and W. F. MAI. 1957. Survival of
encysted eggs and larvae of the golden
n e m a t o d e to alternating temperatures.
Phytopathology 47:527 (Abstr.).
8.LEWIS, F. J. and W. F. MAI. 1960. Survival of
encysted and free larvae of the golden
nematode in relation to temperature and
relative humidity. Proc. Hehninthol. Soc. Wash.
27:80-85.
9. RIGGS, R. D. and M. L. HAMBLEN. 1962.
Soybean-cyst nematode host studies in the
family Leguminosae. AIk. Agr. Exp. Sta. Rep.
Ser. 110:1-18.
10.RIGGS, R. D. and M. L. HAMBLEN. 1966.
Additional weed hosts of Heterodera glycines.
Plant Dis. Rep. 50:15-16.
266 Journal o f Nematology, VoL 4, No. 4, October 1972
I1.RIGGS, R. D. and M. L. HAMBLEN. 1966.
Further studies on the host range of the
soybean-cyst nematode. Ark. Agr. Exp. Sta.
Bull. 718:1-20.
12.SKOTLAND, C. B. 1957. Biological studies of the
soybean cyst nematode. Phytopathology
47:623-625.
13.SLACK, D. A. and M. L. IIAMBLEN. 1961. The
effect of various factors on larval emergence
f r o m c y s t s of Heterodera glycines.
Phytopathology 51:350-355.
14.SMART, G. C., JR. 1964. Additional hosts of the
soybean cyst nematode, Heterodera glycines,
including hosts in two additional plant families.
Plant Dis. Rep. 48:388-390.
15.SMART, G. C., JR. 1964. Physiological strains and
one additional host of the soybean cyst
nematode, Heterodera glycines. Plant Dis. Rep.
48:542-543.
16.SMART, G. C., JR., and B. A. WRIGHT. 1962.
Survival of the cysts of Heterodera glycines
adhering to stored sweetpotato, peanut and
peanut hay. Va. J. Sci. 13:219-220 (Abstr.).
17.TttOMASON, I. J. and D. FIFE. 1962. The effect
of temperature on development and survival of
Heterodera schachtii Schm. Nematologica
7:139-145.
18.VIGLIERCHIO, D. R. 1961. Effects of storage
environment on 'in vitro' hatching of larvae
from cysts of Heterodera schachtii Schmidt
1871. Phytopathology 51:623-625.
19.WlNSLOW, R. D. 1955. The hatching responses of
some root eelworms of the genus Heterodera.
Ann. Appl. Biol. 43:19-36.
A Method for Estimating Numbers of Eggs of Meloidogyne spp. in Soil I
D. W. BYRD, JR., HOWARD FERRIS and C. J. NUSBAUM 2
Abstract: A procedure for extracting eggs of Meloidogyne spp. from soil was developed by modifying and
combining certain existing techniques. Egg masses were elutriated from the soft, gelatinous matrices of the
egg masses were dissolved, and the dispersed eggs were stained to facilitate counting. Data on egg population
densities thus obtained facilitate the study of population dynamics of Meloidogyne spp. and the analysis of
root-knot epidemics. Key Words: egg extraction, population dynamics, root knot.
S t u d i e s o f p o p u l a t i o n d y n a m i c s of
root-knot nematodes, Meloidogyne spp., have
been hindered by the lack of a suitable
technique for extracting eggs from the soil (5).
P o p u l a t i o n density estimates based upon
e x t r a c t i o n o f free-living larvae may be
i n a c c u r a t e i f a large proportion of the
individuals is in the egg stage. Dickson and
Struble (3) developed a sieving and staining
technique for extracting egg masses of M.
incognita from soft. We used this technique
Received for publication 24 February 1972.
I P a p e r N o . 3 6 9 5 o f t h e J o u r n a l Series o f t h e N o r t h
Carolina State University Agricultural Experiment
S t a t i o n , R a l e i g h . T h e u s e o f t r a d e n a m e s in t h i s
publication does not imply endorsement by the
North Carolina Agricultural Experiment Station of
the product named, nor criticism of similar ones not
mentioned.
2Research
T e c h n i c i a n , E. G. Moss F e l l o w , a n d
Professor,
respectively,
Department
of Plant
Pathology, North Carolina State University, Raleigh,
North Carolina 27607. Present address of second
author: Department of Nematology, University of
California, Riverside, California 92502. The authors
e x p r e s s a p p r e c i a t i o n t o W. M. Williams f o r t a k i n g t h e
photographs.
with limited success on numerous samples
t a k e n from Meloidogyne-infested fields in
North Carolina. In general, the procedure was
too time-consuming to satisfy our needs.
In 1970, we undertook the development of
a procedure for extracting Meloidogyne eggs
from soil by modifying and combining certain
existing techniques. It is based upon the
premise that the egg masses remain intact in the
soil, either free or attached to host roots or
root fragments (2). Much attention was given to
the evaluation and improvement of each step as
our studies progressed. The major steps are as
follows.
A. Extraction o f egg masses.
1. A soil sample is mixed with a 15 × 25 cm
sample splitter with 12-ram separator slots
(Model SS 50, W. S. Tyler Co., Mentor, Ohio).
The divided sample is then composited. The
splitter is cleaned after each sample by a jet of
compressed air.
2. Egg masses, either attached to root
fragments or free in the soil, are extracted from
a 500-cc aliquant of the soil sample either by an
elutriator (Fig. IA) or by decantation and