A survey of 552 soybean fields in 20 counties in Nebraska in 1986-88 revealed 35 fields infested with the soybean cyst nematode (SCN), Heterodera glycines. Identification was confirmed with a greenhouse bioassay, using \u27Lee 74\u27 soybean, and by the application of a DNA hybridization probe derived from SCN mitochondrial DNA. Most of the SCN-infested fields were located on the Missouri River floodplain and in the southeastern corner of the state

Powers, Thomas O.

Sandall, L. J.

Wysong, D. S

English

PubMed

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University of Nebraska - Lincoln 
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Papers in Plant Pathology Plant Pathology Department 
1989 
Distribution of Soybean Cyst Nematode in Nebraska 
Thomas O. Powers 
University of Nebraska-Lincoln, tpowers1@unl.edu 
L. J. Sandall 
University of Nebraska-Lincoln 
D. S Wysong 
University of Nebraska-Lincoln 
Follow this and additional works at: https://digitalcommons.unl.edu/plantpathpapers 
 Part of the Plant Pathology Commons 
Powers, Thomas O.; Sandall, L. J.; and Wysong, D. S, "Distribution of Soybean Cyst Nematode in Nebraska" 
(1989). Papers in Plant Pathology. 137. 
https://digitalcommons.unl.edu/plantpathpapers/137 
This Article is brought to you for free and open access by the Plant Pathology Department at 
DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Papers in Plant Pathology by 
an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. 
Supplement to Journal of Nematology 21, No. 4S:612-614. 1989. 
© The Society of Nematologists 1989. 
Distribution of Soybean Cyst Nematode in Nebraska 1 
T. O. POWERS, L. J .  SANDALL, AND D. S. WYSONG 2 
Abstract: A survey of 552 soybean fields in 20 counties in Nebraska in 1986-88 revealed 35 fields 
infested with the soybean cyst nematode (SCN), Heterodera glycines. Identification was confirmed 
with a greenhouse bioassay, using 'Lee 74' soybean, and by the application of a DNA hybridization 
probe derived from SCN mitochondrial DNA. Most of the SCN-infested fields were located on the 
Missouri River floodplain and in the southeastern corner of the state. 
Key words: Glycines max, Heterodera glycines, Nebraska, soybean, soybean cyst nematode, survey. 
Until 1986 Nebraska was the only major 
soybean (Glycines max L. Merrill) producing 
state without a known infestation of the 
soybean cyst nematode (SCN) Heterodera 
glycines Ichinohe (3). In the fall of 1986 a 
heavily infested field (>50 cyst/cm a soil) 
was identified on the Missouri River flood- 
plain in the southeast corner of the state 
(4). This level of infestation suggested a 
well-established population of long dura- 
tion and indicated that other SCN-infested 
fields might occur elsewhere in Nebraska. 
At that time a 2-year survey was initiated 
to determine the distribution of SCN in 
20 eastern Nebraska counties, where ap- 
proximately 52 % of Nebraska soybeans are 
grown. A greenhouse bioassay technique 
was used to enhance detection of field pop- 
ulations of low density. This paper reports 
the results of that survey. 
MATERIALS AND METHODS 
A preliminary sampling for SCN was 
conducted in eight fields along the Mis- 
souri River flood plain in the fall of 1986. 
Soil samples from 544 soybean fields were 
taken during July and August of the 1987 
and 1988 growing seasons (Fig. 1). In 1987 
sampling of 130 soybean fields was con- 
centrated in eight southeastern and east 
central Nebraska counties bordering the 
Received for publication 23 March 1989. 
Journal series No. 8458, Agricultural Research Division, 
University of Nebraska, Lincoln. 
2 Assistant Professor, Research Technologist, and Profes- 
sor, Department of Plant Pathology, University of Nebraska, 
Lincoln, NE 68583-0722. 
Appreciation isextended tothe Nebraska Soybean Devel- 
opment, Utilization, and Marketing Board and the American 
Soybean Association for partial support of this investigation, 
and to T. Harris, L. Hersh, M. Partridge, P. Pochop, and the 
Nebraska Department ofAgriculture for their participation 
in the 1988 field soil collection effort. 
612 
Missouri River. In 1988 sampling was ex- 
panded to include an additional 174 fields 
in the eight original counties plus 240 fields 
in the 12 neighboring counties to the west 
and north. Estimated field size ranged from 
3 to 100 ha. 
In each field, 25-50 soil cores, 2.5 cm 
d x 20 cm deep, from within the root 
zone were collected in a random pattern 
throughout the field and combined to make 
one 500-cm s composite sample. From each 
composite field sample, 100 cm s soil was 
mixed with approximately 1,500 cm 3 ster- 
ilized sand in 20-cm-d pots and planted with 
four 'Lee' soybeans. After 10 weeks of 
greenhouse growth at 25-28 C, potential 
cyst occurrence was determined by root 
examination and extraction from soil 
through flotation and isolation on a 250- 
tzm-pore sieve. 
If cysts were recovered in the green- 
house bioassay, the remaining soil from the 
original field sample was washed and care- 
fully examined for the presence of cysts. 
In some cases only a few brown cysts were 
recovered from both the bioassay tech- 
nique and by processing the original field 
sample. Since these could have been un- 
related cyst species, unable to reproduce 
on soybean but carried along in the soil 
sample during the bioassay, we examined 
the identity of these cysts through DNA 
hybridization. 
Cysts samples to be examined by DNA 
hybridization were handpicked into a mi- 
crofuge tube and ground in an MSB-pro- 
teinase K solution with a pellet pestle (1). 
The nematode lysate was incubated at 65 
C for 30 minutes, treated with 5 #1 RNAse 
(10 mg/ml) for 30 minutes at 37 C, and 
phenol extracted; the DNA was concen- 
Soybean Cyst Nematode  in Nebraska :  Powers et al. 613 
t in 1986 
J in 1987 
with soy- 
atode 
in 1988 
with soy- 
atode 
River 
t ra ted  by e thano l  p rec ip i ta t ion .  The  cel- 
lu lar  DNA was c leaved with the  res t r i c t ion  
enzyme HindI I I  and e lec t rophoret i ca l ly  
f rac t ionated  on 0.65% agarose  gels for  3 
hours  at 80 volts. The  DNA was t rans-  
fe r red  to n i t roce l lu lose  f i l ters and  hybr id -  
ized with an SCN mi tochondr ia l  DNA 
probe  (1). 
Host - race  tests (2,5) were  conducted  on 
one  popu la t ion  each f rom R ichardson  and  
Bur t  Count ies .  Both  the  R ichardson  and  
Bur t  County  f ield popu la t ions  were  located  
with in  1.6 km o f  the  Missour i  R iver ,  al- 
though the  Bur t  County  f ield was approx-  
imate ly  193 km upst ream f rom R ichardson  
County .  
Fig. 1. Soybean cyst nematode surveys in Ne- 
braska, 1986-88. Symbols do not indicate the actual 
number of fields sampled or infested. 
RESULTS AND DISCUSSION 
Th i r ty - f i ve  f ields in 1 1 count ies  sampled  
pos i t ive for  SCN (Tab le  1). Most  o f  the  
pos i t ive f ields were  located  wi th in  the  Mis- 
sour i  R iver  f loodp la in  (Fig. 1) and  had  soils 
cons is t ing o f  silt l oam or  silty clay loam 
texture .  Because many o f  these  f ields were  
located  with in  1.6 km o f  the  r iver ,  it is 
TABLE1. Soybean fields infested with soybean cyst nematode (SCN) identified by county in Nebraska 
1986-88. 
County 
1986-88 
1986 1987 1988 
Total Total 
Fields SCN-inf. Fields SCN-inf. Fields SCN-inf. Fields SCN-inf. 
sampled fields sampled fields sampled fields sampled fields 
Burr 20 2 20 3 40 5 
Cass 10 1 24 1 34 2 
Cedar 20 0 20 0 
Cuming 20 0 20 0 
Dakota 20 0 20 0 
Dixon 20 0 20 0 
Dodge 20 0 20 0 
Douglas 7 1 12 0 19 1 
Gage 20 1 20 1 
Johnson 20 1 20 1 
Lancaster 20 0 20 0 
Nemaha 20 2 25 4 45 6 
Otoe 20 1 24 0 44 1 
Pawnee 20 3 20 3 
Richardson 8 3 20 4 34 4 62 11 
Sarpy 13 2 15 1 28 3 
Saunders 20 0 20 0 
Thurston 20 0 20 0 
Washington 20 0 20 0 40 0 
Wayne 20 1 20 1 
Total 8 3 130 13 414 19 552 35 
614 Supplement to Journal of Nematology, Volume 21, October 1989 
possible that periodic f looding and migra- 
tory water fowl helped spread the nema- 
tode among locations. Not all positive fields, 
however, were confined to the floodplain. 
Isolated discoveries in Wayne, Douglas, 
Johnson,  Pawnee, and Gage Counties were 
geographically distant f rom other  known 
infested fields. 
Host-race tests conducted on SCN pop- 
ulations from two floodplain counties in- 
dicated that at least two races are present 
in Nebraska. The  Burr County population 
was determined to be race 4 (positive on 
all differentials), whereas the Richardson 
County population was found to be race 3 
(negative on all differentials). Neither  field 
had ever been planted with a SCN-resistant 
cultivar. This physiological variation in- 
dicates that genetic differentiation exists 
among these floodplain populations that is 
difficult to explain on the basis of  selection 
pressure imposed by resistant soybean cul- 
tivars. 
Approximately 5% of the field samples 
contained cysts that contained large num- 
bers of  apparently viable eggs but did not 
amplify under  greenhouse bioassay con- 
ditions. Typically 1-5 of  these cysts were 
recovered from the composite field sam- 
ple. None of  these cyst samples produced 
a positive hybridization signal, whereas tests 
of  cysts f rom samples that did amplify in 
the greenhouse bioassay showed distinct 
positive hybridization. This suggests that 
some fields contain a non-SCN cyst species 
that is unable to reproduce on soybean. No 
further  attempts were made to identify this 
species. 
Overall, the results of the survey indicate 
that SCN is not widespread throughout  the 
major soybean growing region of Nebras- 
ka, but is well established in some locations 
along the Missouri River floodplain and in 
the southeastern counties of  the state. 
LITERATURE CITED 
1. Besal, E. A., T. O. Powers, A. D. Radice, and L. 
J. Sandall. 1988. A DNA hybridization probe for the 
detection of soybean cyst nematode. Phytopathology 
78:1136-1139. 
2. Golden, A. M., J. M. Epps, R. D. Riggs, L. A. 
Duclos, J. A. Fox, and R. L. Bernard. 1970. Ter- 
minology and identity of infraspecific forms of the 
soybean cyst nematode (Heterodera glycines). Plant Dis- 
ease Reporter 54:544-546. 
3. Miller, L. I. 1985. Economic importance of cyst 
nematodes in North America. Pp. 373-385 in F. Lam- 
berti and C. E. Taylor, eds. Cyst nematodes. New 
York: Plenum Press. 
4. Powers, T. O., and D. S. Wysong. 1987. First 
report of soybean cyst nematode (Heterodera glycines) 
in Nebraska. Plant Disease 71:1146. 
5. Riggs, R. D., and D. P. Schmitt. 1988. Com- 
plete characterization f the race scheme for Heter- 
odera glycines. Journal of Nematology 20:392-395. 


Distribution of Soybean Cyst Nematode in Nebraska

DigitalCommons@University of Nebraska

University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Papers in Plant Pathology Plant Pathology Department 1989 Distribution of Soybean Cyst Nematode in Nebraska Thomas O. Powers University of Nebraska-Lincoln, tpowers1@unl.edu L. J. Sandall University of Nebraska-Lincoln D. S Wysong University of Nebraska-Lincoln Follow this and additional works at: https://digitalcommons.unl.edu/plantpathpapers  Part of the Plant Pathology Commons Powers, Thomas O.; Sandall, L. J.; and Wysong, D. S, "Distribution of Soybean Cyst Nematode in Nebraska" (1989). Papers in Plant Pathology. 137. https://digitalcommons.unl.edu/plantpathpapers/137 This Article is brought to you for free and open access by the Plant Pathology Department at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Papers in Plant Pathology by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Supplement to Journal of Nematology 21, No. 4S:612-614. 1989. © The Society of Nematologists 1989. Distribution of Soybean Cyst Nematode in Nebraska 1 T. O. POWERS, L. J .  SANDALL, AND D. S. WYSONG 2 Abstract: A survey of 552 soybean fields in 20 counties in Nebraska in 1986-88 revealed 35 fields infested with the soybean cyst nematode (SCN), Heterodera glycines. Identification was confirmed with a greenhouse bioassay, using 'Lee 74' soybean, and by the application of a DNA hybridization probe derived from SCN mitochondrial DNA. Most of the SCN-infested fields were located on the Missouri River floodplain and in the southeastern corner of the state. Key words: Glycines max, Heterodera glycines, Nebraska, soybean, soybean cyst nematode, survey. Until 1986 Nebraska was the only major soybean (Glycines max L. Merrill) producing state without a known infestation of the soybean cyst nematode (SCN) Heterodera glycines Ichinohe (3). In the fall of 1986 a heavily infested field (>50 cyst/cm a soil) was identified on the Missouri River flood- plain in the southeast corner of the state (4). This level of infestation suggested a well-established population of long dura- tion and indicated that other SCN-infested fields might occur elsewhere in Nebraska. At that time a 2-year survey was initiated to determine the distribution of SCN in 20 eastern Nebraska counties, where ap- proximately 52 % of Nebraska soybeans are grown. A greenhouse bioassay technique was used to enhance detection of field pop- ulations of low density. This paper reports the results of that survey. MATERIALS AND METHODS A preliminary sampling for SCN was conducted in eight fields along the Mis- souri River flood plain in the fall of 1986. Soil samples from 544 soybean fields were taken during July and August of the 1987 and 1988 growing seasons (Fig. 1). In 1987 sampling of 130 soybean fields was con- centrated in eight southeastern and east central Nebraska counties bordering the Received for publication 23 March 1989. Journal series No. 8458, Agricultural Research Division, University of Nebraska, Lincoln. 2 Assistant Professor, Research Technologist, and Profes- sor, Department of Plant Pathology, University of Nebraska, Lincoln, NE 68583-0722. Appreciation isextended tothe Nebraska Soybean Devel- opment, Utilization, and Marketing Board and the American Soybean Association for partial support of this investigation, and to T. Harris, L. Hersh, M. Partridge, P. Pochop, and the Nebraska Department ofAgriculture for their participation in the 1988 field soil collection effort. 612 Missouri River. In 1988 sampling was ex- panded to include an additional 174 fields in the eight original counties plus 240 fields in the 12 neighboring counties to the west and north. Estimated field size ranged from 3 to 100 ha. In each field, 25-50 soil cores, 2.5 cm d x 20 cm deep, from within the root zone were collected in a random pattern throughout the field and combined to make one 500-cm s composite sample. From each composite field sample, 100 cm s soil was mixed with approximately 1,500 cm 3 ster- ilized sand in 20-cm-d pots and planted with four 'Lee' soybeans. After 10 weeks of greenhouse growth at 25-28 C, potential cyst occurrence was determined by root examination and extraction from soil through flotation and isolation on a 250- tzm-pore sieve. If cysts were recovered in the green- house bioassay, the remaining soil from the original field sample was washed and care- fully examined for the presence of cysts. In some cases only a few brown cysts were recovered from both the bioassay tech- nique and by processing the original field sample. Since these could have been un- related cyst species, unable to reproduce on soybean but carried along in the soil sample during the bioassay, we examined the identity of these cysts through DNA hybridization. Cysts samples to be examined by DNA hybridization were handpicked into a mi- crofuge tube and ground in an MSB-pro- teinase K solution with a pellet pestle (1). The nematode lysate was incubated at 65 C for 30 minutes, treated with 5 #1 RNAse (10 mg/ml) for 30 minutes at 37 C, and phenol extracted; the DNA was concen- Soybean Cyst Nematode  in Nebraska :  Powers et al. 613 t in 1986 J in 1987 with soy- atode in 1988 with soy- atode River t ra ted  by e thano l  p rec ip i ta t ion .  The  cel- lu lar  DNA was c leaved with the  res t r i c t ion  enzyme HindI I I  and e lec t rophoret i ca l ly  f rac t ionated  on 0.65% agarose  gels for  3 hours  at 80 volts. The  DNA was t rans-  fe r red  to n i t roce l lu lose  f i l ters and  hybr id -  ized with an SCN mi tochondr ia l  DNA probe  (1). Host - race  tests (2,5) were  conducted  on one  popu la t ion  each f rom R ichardson  and  Bur t  Count ies .  Both  the  R ichardson  and  Bur t  County  f ield popu la t ions  were  located  with in  1.6 km o f  the  Missour i  R iver ,  al- though the  Bur t  County  f ield was approx-  imate ly  193 km upst ream f rom R ichardson  County .  Fig. 1. Soybean cyst nematode surveys in Ne- braska, 1986-88. Symbols do not indicate the actual number of fields sampled or infested. RESULTS AND DISCUSSION Th i r ty - f i ve  f ields in 1 1 count ies  sampled  pos i t ive for  SCN (Tab le  1). Most  o f  the  pos i t ive f ields were  located  wi th in  the  Mis- sour i  R iver  f loodp la in  (Fig. 1) and  had  soils cons is t ing o f  silt l oam or  silty clay loam texture .  Because many o f  these  f ields were  located  with in  1.6 km o f  the  r iver ,  it is TABLE1. Soybean fields infested with soybean cyst nematode (SCN) identified by county in Nebraska 1986-88. County 1986-88 1986 1987 1988 Total Total Fields SCN-inf. Fields SCN-inf. Fields SCN-inf. Fields SCN-inf. sampled fields sampled fields sampled fields sampled fields Burr 20 2 20 3 40 5 Cass 10 1 24 1 34 2 Cedar 20 0 20 0 Cuming 20 0 20 0 Dakota 20 0 20 0 Dixon 20 0 20 0 Dodge 20 0 20 0 Douglas 7 1 12 0 19 1 Gage 20 1 20 1 Johnson 20 1 20 1 Lancaster 20 0 20 0 Nemaha 20 2 25 4 45 6 Otoe 20 1 24 0 44 1 Pawnee 20 3 20 3 Richardson 8 3 20 4 34 4 62 11 Sarpy 13 2 15 1 28 3 Saunders 20 0 20 0 Thurston 20 0 20 0 Washington 20 0 20 0 40 0 Wayne 20 1 20 1 Total 8 3 130 13 414 19 552 35 614 Supplement to Journal of Nematology, Volume 21, October 1989 possible that periodic f looding and migra- tory water fowl helped spread the nema- tode among locations. Not all positive fields, however, were confined to the floodplain. Isolated discoveries in Wayne, Douglas, Johnson,  Pawnee, and Gage Counties were geographically distant f rom other  known infested fields. Host-race tests conducted on SCN pop- ulations from two floodplain counties in- dicated that at least two races are present in Nebraska. The  Burr County population was determined to be race 4 (positive on all differentials), whereas the Richardson County population was found to be race 3 (negative on all differentials). Neither  field had ever been planted with a SCN-resistant cultivar. This physiological variation in- dicates that genetic differentiation exists among these floodplain populations that is difficult to explain on the basis of  selection pressure imposed by resistant soybean cul- tivars. Approximately 5% of the field samples contained cysts that contained large num- bers of  apparently viable eggs but did not amplify under  greenhouse bioassay con- ditions. Typically 1-5 of  these cysts were recovered from the composite field sam- ple. None of  these cyst samples produced a positive hybridization signal, whereas tests of  cysts f rom samples that did amplify in the greenhouse bioassay showed distinct positive hybridization. This suggests that some fields contain a non-SCN cyst species that is unable to reproduce on soybean. No further  attempts were made to identify this species. Overall, the results of the survey indicate that SCN is not widespread throughout  the major soybean growing region of Nebras- ka, but is well established in some locations along the Missouri River floodplain and in the southeastern counties of  the state. LITERATURE CITED 1. Besal, E. A., T. O. Powers, A. D. Radice, and L. J. Sandall. 1988. A DNA hybridization probe for the detection of soybean cyst nematode. Phytopathology 78:1136-1139. 2. Golden, A. M., J. M. Epps, R. D. Riggs, L. A. Duclos, J. A. Fox, and R. L. Bernard. 1970. Ter- minology and identity of infraspecific forms of the soybean cyst nematode (Heterodera glycines). Plant Dis- ease Reporter 54:544-546. 3. Miller, L. I. 1985. Economic importance of cyst nematodes in North America. Pp. 373-385 in F. Lam- berti and C. E. Taylor, eds. Cyst nematodes. New York: Plenum Press. 4. Powers, T. O., and D. S. Wysong. 1987. First report of soybean cyst nematode (Heterodera glycines) in Nebraska. Plant Disease 71:1146. 5. Riggs, R. D., and D. P. Schmitt. 1988. Com- plete characterization f the race scheme for Heter- odera glycines. Journal of Nematology 20:392-395. 

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Distribution of Soybean Cyst Nematode in Nebraska

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