Supplement to the Journal of Nematology 29(4S):742-745. 1997.
© The Society of Nematologists 1997.
Tannic Acid Effects on Hatching of Heterodera glycines
in Vitro
S. CHEN, 2 D. W. DICKSON,3 AND T. E. HEWLETT3
Abstract: Effects of tannic acid on hatching of Heteroderaglycineseggs were determined in vitro using
three batches of eggs obtained from greenhouse cultures in Florida or from naturally infested field soil
in Minnesota. A quadratic model fits the percentage egg hatch. Hatch increased with increasing tannic
acid concentrations from 0 to about 39 mg/liter, then declined with further increases in concentration.
Tannic acid did not induce hatching of dormant eggs obtained from the field,
Key words: cyst, dormancy, egg, hatch, Heteroderaglycines, soybean cyst nematode, tannic acid.
M a n y factors i n f l u e n c e h a t c h i n g o f n e m a t o d e eggs, i n c l u d i n g t e m p e r a t u r e , soil aeration, m o i s t u r e , p H , e x u d a t e s f r o m h o s t
plants a n d o t h e r o r g a n i c c o m p o u n d s , a n d
i n o r g a n i c ions (Clarke a n d Perry, 1977). Num e r o u s studies have b e e n d o n e o n h a t c h i n g
o f the s o y b e a n cyst n e m a t o d e , Heterodera glycines, that i n c l u d e b o t h inhibitors a n d stimulants o f hatching. Generally, exudates f r o m
h o s t plants stimulate h a t c h i n g o f the soyb e a n cyst n e m a t o d e (Caballero et al., 1986;
O k a d a , 1971a, 1971b; S c h m i t t a n d Riggs,
1991; Takasugi et al., 1974; Tefft a n d B o n e ,
1985; Tefft et al., 1982; T s u t s u m i a n d Sakurai, 1966), whereas n o n h o s t s d o n o t stimulate h a t c h i n g (Schmitt a n d Riggs, 1991; Tsutsumi a n d Sakurai, 1966). A t t e m p t s have
b e e n m a d e to find n a t u r a l or synthesized
p r o d u c t s that m a y be applied in fields to
stimulate n e m a t o d e h a t c h i n g a n d r e d u c e
p o p u l a t i o n densities w h e n the h o s t crops
are absent. Five herbicides a n d zinc fertilizers were tested f o r their potential to stimulate h a t c h i n g , b u t n o n e were effective at
practical c o n c e n t r a t i o n s ( B e h m et al., 1995;
W o n g et al., 1993). I n p r e l i m i n a r y studies,
tannic acid attracted r o o t - k n o t n e m a t o d e s
b u t n o t s o y b e a n cyst n e m a t o d e (Hewlett et
Received for publication 16 April 1997.
i MinnesotaAgricultural Experiment StationJournal Series
No. 971055001.Part of the research was done at the University
of Florida, Research supported in part by USDAGrant No.
58-319R-1-009and the Minnesota SoybeanResearch and Promotion Council.
2 University of Minnesota, Southern Experiment Station,
35838 120th Street, Waseca,MN 56093-4521.
E-mail: [email protected]
s Entomology and NematologyDepartment, Institute of
Food and Agricultural Sciences, Universityof Florida, Gainesville, FL 32611-0620.
E-mail: [email protected]
742
al., 1995). This c o m p o u n d r e d u c e d infection o f t o m a t o by the r o o t - k n o t n e m a t o d e ,
Meloidogyne arenaria, in the g r e e n h o u s e a n d
m i c r o p l o t s ( u n p u b l i s h e d ; H e w l e t t et al.,
1995). T a n n i c acid a n d its a n a l o g u e s are
a b u n d a n t in m a n y p l a n t products, a n d tannins have b e e n r e p o r t e d to have antimicrobial properties (Scalbert, 1991). O u r objectives were to evaluate effects o f tannic acid
o n h a t c h i n g o f the s o y b e a n cyst n e m a t o d e
a n d to d e t e r m i n e the possibility o f using the
c o m p o u n d to i n d u c e n e m a t o d e s to hatch.
MATERIALS AND METHODS
Test 1: T h e source o f n e m a t o d e s used f o r
this test was a p o p u l a t i o n o f H. glycines race
3 that h a d b e e n m a i n t a i n e d f o r 4 years o n
the susceptible soybean (Glycine max) cultivar C o b b in a g r e e n h o u s e at the University
o f Florida.
Soybean seeds were sown in pots o n 11
J a n u a r y 1995. Fifty-seven days after sowing,
females a n d cysts were extracted f r o m roots
a n d soil by a p r o c e d u r e described previously
( C h e n et al., 1996). T h e females a n d cysts
were c r u s h e d in a tissue g r i n d e r to release
eggs. T h e eggs were separated f r o m debris
by c e n t r i f u g i n g t h e m in a 0% to 55% (w/v)
sucrose linear gradient. T h e b a n d containing eggs a n d second-stage juveniles (J2) was
transferred to a 38-tam-pore sieve a n d rinsed
with sufficient water to s e p a r a t e J2 f r o m
eggs. T h e eggs r e t a i n e d o n the sieve were
transferred o n t o a n o t h e r 38-1am-pore sieve
that h a d b e e n autoclaved, rinsed with sterile
water, a n d treated with a solution containing 100 m g o f streptomycin, 50 m g o f chlortetracycline, a n d 30 m g o f quinolinol p e r
liter for 24 h o u r s at 10 °C. T h e eggs were
H a t c h i n g Effects o f Tannic Acid o n H. glycines: Chen et at. 743
rinsed with sterile water a n d treated again
with 0.5% chlorhexidine diacetate salt for 15
minutes, a n d t h e n rinsed with sterile water.
Eggs were s u s p e n d e d in sterile deionized water at a concentration o f 3,000 e g g s / m l . O n e
milliliter o f the egg suspension was placed
on each 1-cm-diam. sieve with 35-pm openings. T h e sieves with eggs were placed in a
24-well tissue culture plate.
Tannic acid was tested at concentrations
of 0, 39, 156, 625, 2,500, a n d 10,000 m g /
liter in sterile deionized water. A solution o f
4 m M ZnC12, a hatching stimulant o f the
soybean cyst n e m a t o d e (Tefft et al., 1982),
was used to d e t e r m i n e viability a n d degree
of d o r m a n c y o f the n e m a t o d e eggs. T h e solutions were passed t h r o u g h a 0.45-pm filter
to r e m o v e possible c o n t a m i n a n t s such as
bacteria a n d fungal spores. Sufficient solution was a d d e d into each tissue culture well
so as to reach the b o t t o m o f the sieves. Four
replicates o f each concentration were used.
T h e eggs were i n c u b a t e d in the solutions at
23 to 24 °C. T h e solutions were replaced by
freshly p r e p a r e d solutions on days 3 a n d 7.
T h e n u m b e r s o f n e m a t o d e s h a t c h e d by days
3, 7, a n d 14 were c o u n t e d , a n d p e r c e n t
h a t c h was calculated. T h e data was subjected
to analysis by regressing m e a n p e r c e n t hatch
of the four replicates against concentration
using a quadratic model.
Test 2: T h e same n e m a t o d e p o p u l a t i o n
used in the Test 1 was cultured in the greenhouse f r o m 9 March to 20 May 1995. All the
p r o c e d u r e s were the same as Test 1. However, tannic acid concentrations o f 0, 0.15,
0.6, 1.2, 2.4, 4.9, 9.8, 39, 156, 625, 2,500, a n d
10,000 m g / l i t e r in sterile deionized water
were used.
Test 3: T h e source o f n e m a t o d e s used for
this test was a p o p u l a t i o n of H. glycines race
3 collected f r o m soybean rhizosphere in a
naturally infested field site in Minnesota o n
12 O c t o b e r 1995. T h e soil was stored at
r o o m t e m p e r a t u r e (23 to 24 °C) until 7 Nov e m b e r 1995, w h e n the cysts were extracted.
Eggs a n d juveniles were released f r o m the
cysts by a modified mechanical m e t h o d (Niblack et al., 1993) a n d separated f r o m debris
by centrifugation in a 35% (w/v) sucrose
solution. T h e eggs a n d J2 were transferred
o n t o a 38-pm-pore sieve a n d rinsed with sufficient water to remove J2. T h e eggs were
stored in a solution containing 100 m g of
streptomycin, 50 m g o f chlortetracycline,
a n d 30 m g o f q u i n o l i n o l / l i t e r water at 4 °C
for 3 weeks before they were tested. T h e
concentrations o f tannic acid were the same
as in Test 2, a n d all o t h e r p r o c e d u r e s were
the same as in Test 1.
RESULTS AND DISCUSSION
In all tests, low concentrations o f tannic
acid increased n e m a t o d e hatching. P e r c e n t
hatch increased with an increase of tannic
acid concentration u p to 35 to 40 m g / l i t e r
a n d t h e n declined with increasing concent r a t i o n (Fig. 1A-C). W h e n c o m p a r e d at
similar c o n c e n t r a t i o n s , the p e r c e n t e g g
hatch differed a m o n g the three tests. In Test
1 (Fig. 1A), w h e n eggs were i n c u b a t e d in 39
m g / l i t e r tannic acid solution for 14 days,
32% of the eggs h a t c h e d (60% increase o f
hatch c o m p a r e d to hatch in water). In contrast, eggs in tannic acid at 10,000 m g / l i t e r
(1% w/v) h a d only 6.8% cumulative h a t c h
at day 14 (61% decrease c o m p a r e d to h a t c h
in water). H a t c h in ZnC12 at 4 m M increased
by 15% c o m p a r e d to hatch in water at day
14, which indicated that the eggs were n o t
d o r m a n t . In Test 2, 42% o f eggs h a t c h e d in
the 39 m g / l i t e r tannic acid solution at day
14, which r e p r e s e n t e d a 13% increase comp a r e d to h a t c h in water. Cumulative egg
hatch decreased 56% in the 10,000 r a g / l i t e r
tannic acid solution at day 14 c o m p a r e d to
hatch in water. I n c u b a t i o n in ZnCI~ did n o t
increase hatch of eggs c o m p a r e d to water in
Test 2. H a t c h o f n e m a t o d e eggs collected
f r o m the field site in Minnesota was very low.
In water, only 0.9% of the eggs h a d h a t c h e d
by day 14, a n d in tannic acid solution cumulative h a t c h r e a c h e d a m a x i m u m o f only
1.7% at 39 m g / l i t e r by day 14. H a t c h decreased to 0.5% in the 10,000 m g / l i t e r tannic acid solution at day 14, or 55% of hatch
in water. W h e n these eggs were incubated in
4 m M ZnC12, 77% o f the eggs h a t c h e d by day
14, indicating that egg viability was high b u t
that the eggs were d o r m a n t .
T h e m e c h a n i s m involved in i n c r e a s e d
744
Supplement to the Journal of Nematology, Volume 29, No. 4S, December 1997
40
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FIG. 1. The effect of different concentration of tannic acid on hatching of Heterodera glycines. A quadratic
model, Y= a ~- bx + cx2, was used to predict percentage hatch in different concentrations of tannic acid solutions
with incubation periods of 3, 7, and 14 days. (3d, 7d, 14d). A) Test 1, using nematode eggs obtained from a
greenhouse culture in Florida during 11 January t h r o u g h 9 March 1995. Curve 3 d: .2 = 0.95, P = 0.01. Curve 7 d:
.2 = 0.98, P = 0.003. Curve 14 d: .2 = 0.88, P = 0.04. B) Test 2, using nematode eggs obtained from a greenhouse
culture in Florida during 9 March t h r o u g h 20 May 1995. Curve 3 d: r~ = 0.21, P = 0.35. Curve 7 d: r2 = 0.91, P <
0.0001. Curve 14 d: .2 = 0.90, P < 0.001. C) Test 3, using n e m a t o d e eggs collected from a Minnesota soybean field
12 October 1995. Curve 3 d: .2 = 0.75, P = 0.004. Curve 7 d: ,2 = 0.77, P = 0.003. Curve 14 d: *2 = 0.81, P < 0.001.
H a t c h i n g Effects o f T a n n i c Acid o n H. glycines: Chen et al.
h a t c h o f t h e s o y b e a n cyst n e m a t o d e i n low
c o n c e n t r a t i o n s o f t a n n i c acid s o l u t i o n s is
n o t u n d e r s t o o d . T h e results o f T e s t 3 i n d i cate t h a t t a n n i c acid d i d n o t b r e a k the dorm a n c y o f the eggs. Nevertheless, c o n c e n t r a t i o n o f 3 5 - 4 0 m g / l i t e r o f t a n n i c acid will
i n c r e a s e h a t c h i n g o f n o n d o r m a n t eggs.
H i g h c o n c e n t r a t i o n s o f t a n n i c acid i n h i b i t
h a t c h i n g . F u r t h e r tests are n e e d e d to d e t e r m i n e if t h e J2 w i t h i n the egg is killed o r j u s t
i n h i b i t e d . Several r e s e a r c h e r s have r e p o r t e d
t h a t t h e c o n c e n t r a t i o n effect o n e g g h a t c h i n g is c r i t i c a l ( C l a r k e a n d P e r r y , 1977;
Clarke a n d S h e p h e r d , 1967; O k a d a , 1971b).
S o m e n e m a t i c i d e s , such as a l d i c a r b a n d carb o f n r a n , s t i m u l a t e h a t c h i n g at low c o n c e n t r a t i o n s b u t i n h i b i t h a t c h i n g at h i g h c o n c e n t r a t i o n s ( H o u g h a n d T h o m a s o n , 1975;
Steele, 1983; Steele a n d H o d g e s , 1975). T h e
s t i m u l a t o r y effects o f low c o n c e n t r a t i o n s o f
nematicides on nematode hatching may be
a t t r i b u t e d to i n c r e a s e d activity o f j u v e n i l e s
i n t h e egg ( H o u g h a n d T h o m a s o n , 1975;
Steele a n d H o d g e s , 1975). T a n n i c acid m a y
act i n a s i m i l a r way.
B e c a u s e t a n n i c a c i d c a n n o t b r e a k dorm a n c y o f the n e m a t o d e eggs, we t h i n k t h a t
it m a y b e u n r e a l i s t i c to use e i t h e r t a n n i c acid
or p l a n t p r o d u c t s c o n t a i n i n g t a n n i c acid to
i n d u c e the s o y b e a n cyst n e m a t o d e to h a t c h
a n d r e d u c e n e m a t o d e p o p u l a t i o n i n fields
d u r i n g p e r i o d s w i t h o u t h o s t plants.
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