Association of RFLP markers with loci conferring broad-based resistance to the soybean cyst nematode (Heterodera glycines)

Soybean cyst nematode (SCN) is a major soybean yield-limiting pest. The present study was conducted to map broad-based SCN resistance loci from the cultivar Hartwig. Two-hundred F₂₃ lines derived from the cross Williams 82 x Hartwig were screened with a fourth-generation SCN inbred and 56 polymorphic molecular markers. Allele states and phenotypes were analyzed using stepwise regression and the model selection was made at P 0.01. Four unlinked RFLP markers (A006, A567, A487, A112) were associated with SCN resistance and the partial coefficient of determinations (R²) were 91%, 1%, 1%, and 1%. We have mapped a new, major SCN resistance locus (A006) and three minor loci (A567, A487, A112). This complete mapping will accelerate the transfer of broad-based resistance without linkage drag and aid in the determination of relationships among various SCN-resistant germplasm sources.     

Variability in Race Tests with Heterodera glycines

Tests of Heterodera glycines on differential host plants to determine races were run in Arkansas, Illinois, and North Carolina to check the uniformity of results of the test. Methods used at the three locations varied somewhat. Results indicate that the race test is highly variable. Isolates previously identified as race 1 were identified as race 1 or race 3; those identified as race 2 were identified in these tests as race 2, 4, 9, or 14; those previously identified as race 3 were identified as race 1 or race 3; those identified as race 4 were identified in these tests as race 4 or race 14; those previously identified as race 5 were identified as race 2; and those previously identified as race 6 were identified as race 1, 2, 4, 5, or 6. Part of the variability resulted from the use of differential host plants from different sources and part from nonstandard differential host plants. Other variations may be due to inability to obtain completely uniform inoculum or to recover all nematodes that penetrated.     

Tolerance of Soybean to Heterodera glycines

Seven soybeans were selected from 200 entries evaluated for tolerance to soybean cyst nematode (SCN), Heterodera glycines. Tolerance to SCN was measured by comparing the seed yield from aldicarb-treated vs. nontreated plots. A yield response index (YRI) was calculated for each entry: YRI = (seed yield from nontreated plot/seed yield from treated plot) × 100. The soybean entries Coker 156, PI 97100, and S79-8059 exhibited high tolerance (YRI) to SCN when compared to Essex even though they became heavily infected with SCN. Tolerance in soybeans to SCN may be useful in pest management programs designed to stabilize soybean yield.     

Identification of a new major QTL associated with resistance to soybean cyst nematode (Heterodera glycines)

Resistance of soybean [Glycine max (L.) Merr.] to cyst nematode (SCN) (Heterodera glycines Ichinohe), one of the most destructive pathogens affecting soybean, involves a complex genetic system. The identification of QTLs associated with SCN resistance may contribute to the understanding of such system. The objective of this work was to identify and map QTLs for resistance to SCN Race 14 with the aid of molecular markers. BC₃F_2:3 and F_2:3 populations, both derived from an original cross between resistant cv. Hartwig and the susceptible line BR-92–31983 were screened for resistance to SCN Race 14. Four microsatellite (Satt082, Sat_001, Satt574 and Satt301) and four RAPD markers (OPAA-11₇₉₅, OPAE-08₈₃₇, OPR-07₅₄₈ and OPY-07₂₀₃₀) were identified in the BC₃F_2:3 population using the bulked segregant analysis (BSA) technique. These markers were amplified in 183 F_2:3 families and mapped to a locus that accounts for more than 40% of the resistance to SCN Race 14. Selection efficiency based on these markers was similar to that obtained with the conventional method. In the case of the microsalellite markers, which identify homozygous resistant genotypes, the efficiency was even higher. This new QTL has been mapped to the soybean linkage group D2 and, in conjunction with other QTLs already identified for SCN resistance, will certainly contribute to our understanding of the genetic basis of resistance of this important disease in soybean. Received: 12 October 1999 / Accepted: 14 April 2000     

Impact of Herbicides on Heterodera glycines Susceptible and Resistant Soybean Cultivars

Several abiotic and biotic stresses can affect soybean in a growing season. Heterodera glycines, soybean cyst nematode, reduces yield of soybean more than any other pathogen in the United States. Field and greenhouse studies were conducted to determine whether preemergence and postemergence herbicides modified the reproduction of H. glycines, and to determine the effects of possible interactive stresses caused by herbicides and H. glycines on soybean growth and yield. Heterodera glycines reproduction factor (Rf) generally was less on resistant than susceptible cultivars, resulting in a yield advantage for resistant cultivars. The yield advantage of resistant cultivars was due to more pods per plant on resistant than susceptible cultivars. Pendimethalin reduced H. glycines Rf on the susceptible cultivars in 1998 at Champaign, Illinois, and in greenhouse studies reduced dry root weight of H. glycines-resistant and susceptible cultivars, therefore reducing Rf on the susceptible cultivars. The interactive stresses from acifluorfen or imazethapyr and H. glycines reduced the dry shoot weight of the resistant cultivar Jack in a greenhouse study. Herbicides did not affect resistant cultivars'' ability to suppress H. glycines Rf; therefore, growers planting resistant cultivars should make herbicide decisions based on weeds present and cultivar tolerance to the herbicide.     

Spatial Analysis of Heterodera glycines Populations in Field Plots

Spatial heterogeneity in nematode population densities presents an obstacle to the precise determination of infestation levels. Three field plots were intensively sampled for soybean cyst nematode (Heterodera glycines Ich.) cysts before and after spring cultivation to quantify the spatial attributes of the population. Population density strata were detected running parallel to plant rows. Highest population densities before cultivation were found in the plant row and the middle furrow, Population density in the plant row averaged 26% higher and 4% lower than the whole-plot mean before and after cultivation, respectively. Cysts containing fewer than 25 eggs were not stratified, indicating that most were produced before the previous season. Sample population counts were fit to the negative binomial distribution model before cultivation, but distributions differed among plots. The Neyman type A and negative binomial distributions both fit the data after cultivation disturbed the soil. Population clusters 1-3 m long were detected in plant beds before cultivation. Heterogeneity in population density increased with plant row length after cultivation. Optimum plot length for minimal spatial heterogeneity in four-row mechanically tended field plots was estimated at 6 m after trimming plot ends.     

Molecular Characterization of a Soybean Cyst Nematode (Heterodera glycines) Homolog of unc-87

In Caenorhabditis elegans the unc-87 gene encodes a protein that binds to actin at the I band and is important in nematodes for maintenance of the body-wall muscle. Caenorhabditis elegans mutant phenotypes of unc-87 exhibit severe paralysis in larvae and limp paralysis in the adult. We cloned and characterized a full-length cDNA representing a Heterodera glycines homolog of the unc-87 gene from C. elegans that encodes a protein that contains a region of seven repeats similar to CLIK-23 from C-elegans and has 81% amino acid identity with that of C. elegans unc-87 variant A. In the EST database clones labeled "unc-87'''' encode mainly the 3'' portion of unc-87, while clones labeled "calponin homolog OV9M'''' contain mainly DNA sequence representing the 5'' and middle transcribed regions of unc-87. A 1770 nucleotide cDNA encoding H. glycines unc-87 was cloned and encodes a predicted UNC-87 protein product of 375 amino acids. The expression of unc-87 was determined using RT-PCR and, in comparison to its expression in eggs, unc-87 was expressed 6-fold higher in J2 juveniles and 20-fold and 13-fold (P = 0.05) higher in nematodes 15 and 30 days after inoculation, respectively. In situ hybridization patterns confirmed the expression patterns observed with RT-PCR.     

Flow Cytometric Analysis and Sorting of Heterodera glycines Eggs

A nondestructive technique was developed to characterize and separate eggs of soybean cyst nematode, Heterodera glycines, by developmental stage using flow cytometry. Eggs from cysts cultured on susceptible soybean roots were suspended in 0.1% xanthan gum or 59% sucrose and loaded into either a Coulter EPICS 752 or EPICS 753 flow cytometer. Eggs were analyzed and sorted according to forward angle and 90° light scatter, flow cytometric parameters that are relative measures of object size and granularity, respectively. Mature eggs containing vermiform juveniles were less granular and slightly larger than eggs in earlier stages of embryogeny, allowing for separation of mature eggs from immature eggs. The effectiveness of flow cytometric sorting was evaluated by comparing the developmental stages of subpopulations of unsorted and sorted eggs. Of a subpopulation of unsorted eggs, 62% contained vermiform juveniles, whereas 85 to 95% of sorted subpopulations of larger, less granular eggs contained vermiform juveniles. Suspending H. glycines eggs in 0.1% xanthan gum or 59% sucrose for flow cytometric analysis had no effect on subsequent egg hatch in vitro. This technique is an efficient and effective means to collect large, relatively homogeneous quantities of H. glycines eggs in early or late embryogeny, and would likely be useful for analyzing and sorting eggs of other nematode species for use in developmental, genetic, or physiological research, or for identification and collection of parasitized eggs.     

Effect of Soybean Tip Removal on Penetration and Development of Heterodera glycines

On a few occasions, soybeans with broken root tips were included in tests to evaluate resistance to Heterodera glycines. Although females developed on these plants, the numbers tended to be lower than on similarly treated intact roots. To test the possibility that removal of the root meristem affected nematode development, a culture system using pruned soybeans was devised that permitted access to the roots without disturbing the plants. Treatments included removal of 2 mm of root tip at various times ranging from 24 hours before to 10 days after inoculation, or roots left intact. In each experiment, all roots were inoculated at the same time with equal numbers of freshly hatched second-stage juveniles of Heterodera glycines. No differences in nematode development were detected in plants with root tips removed after inoculation compared to the control. When tips were removed at or before inoculation, fewer juveniles entered roots and relatively fewer nematodes developed. Penetration levels and development correlated with root tip removal such that progressively fewer nematodes entered roots and relatively greater numbers of nematodes remained undeveloped as the time interval between root tip removal and inoculation was increased.     

FMRFamide-like Immunoactivity in Heterodera glycines (Nemata: Tylenchida)

Material antigenically related to the neuromodulatory peptide FMRFamide was detected and examined in preparations of the soybean cyst nematode, Heterodera glycines, and in the free-living nematodes Caenorhabditis elegans and Panagrellus redivivus. FMRFamide-related peptides were quantified by an enzyme-linked immunosorbent assay. Specific activities were remarkably similar among all of the vermiform members of the three species. FMRFamide-related peptide immunoactivity was present in both sexes and all stages of H. glycines examined. The highest specific activity was present in second-stage juveniles and in males, and the lowest in white and yellow females. Total FMRFamide-related peptide level per individual was highest in brown females, with 90% of the activity associated with the eggs. Peptide levels in these eggs and in second-stage juveniles were comparable and increased in adults, especially in females. Chromatographic analysis of FMRFamide-related peptide preparations from H. glycines juveniles, C. elegans, and P. redivivus revealed distinct qualitative differences between the infective plant parasite and the free-living nematodes.     

Coating Soybean Seed with Oxamyl for Control of Heterodera glycines

Oxamyl coated on soybean (Glycine max (L.) Merr. cv. Elgin) seeds in solutions of 20, 40, 80, and 160 mg/ml had no serious deleterious effects on seedling emergence and growth when planted in sterile soil. Seedling emergence on day 3 was less than that of the uncoated control, but by day 7 emergence was equal to, or greater than, the control. Shoot and root growth from seed coated with oxamyl in 40 and 80 mg/ml solutions was greater than that of the control. In soil infested with soybean cyst nematode, Heterodera glycines, shoot weight of soybean plants from seeds coated with oxamyl in 80 mg/ml solution was 11 and 9% greater at weeks 3 and 7, respectively, than from uncoated seeds. Numbers of juveniles (J3 and J4) and adults of H. glycines observed on the roots of plants from oxamyl-coated seeds were 83, 42, and 49% less at weeks 3, 5, and 7, respectively, than numbers on the roots of the untreated control. Numbers of J2 extracted from the roots of plants from oxamyl-coated seeds were 75% less at weeks 5 and 7 than those extracted from roots of uncoated seeds. The numbers of J2 extracted from the soil planted to oxamyl-coated seeds were 51 and 33% less at weeks 5 and 7, respectively, than from soil planted to uncoated seed.     

Effects of Zinc Fertilization of Corn on Hatching of Heterodera glycines in Soil

Experiments were conducted to determine the effects of zinc fertilizers on hatching and soil population densities of Heterodera glycines. In vitro egg hatching in solutions of reagent-grade zinc sulfate and zinc chloride and fertilizer-grade zinc sulfate was significantly greater than hatching in deionized water, whereas zinc chelate fertilizer significantly inhibited egg hatching relative to deionized water. In greenhouse experiments, no differences in cumulative percentage egg hatch were detected in soil naturally infested with H. glycines amended with fertilizer-grade zinc sulfate and zinc chelate at rates equivalent to 0, 1.12, 11.2, and 112 kg Zn/ha and subsequently planted with corn (Zea mays L.). In a field experiment, no significant differences in H. glycines egg population densities and corn yields were detected among plots fertilized with 0, 11.2, and 22.4 kg Zn/ha rates of zinc chelate. Yields of H. glycines-susceptible soybean planted in plots 1 year after zinc fertilization of corn plots also were not significantly affected. Zinc compounds significandy affected H. glycines egg hatching in vitro, but had no effect on hatching in natural soils.     

Characterization of the Virulence Phenotypes of Heterodera glycines in Minnesota

Knowledge of the virulence phenotypes of soybean cyst nematode, Heterodera glycines populations is important in choosing appropriate sources for breeding resistant cultivars and managing the nematode. We investigated races of 59 H. glycines populations collected from 1997 to 1998 and races and HG Types of 94 populations collected in 2002 from soybean fields across southern and central Minnesota. In the 1997 to 1998 samples, race 3 was predominant and represented 78% of the populations. The remaining populations were 11.9% race 1, 1.7% race 4, 6.8% race 6, and 1.7% race 14. In the 2002 samples, the populations were classified as 15.3% race 1, 77.6% race 3, 2.4% race 5, 3.5% race 6 and 1.2% race 9. Percentage of 1997 to 1998 populations with female indices (FI) higher than 10 were 10.2% on Pickett 71, 3.4% on Peking, 13.6% on PI 88788, 3.4% on PI 90763, 1.7% on PI 209332, and 1.7% on PI 437654. Percentage of 2002 populations with FI >10 was 1.1% on Peking, 17.0% on PI88788, 14.9% on PI 209332, 33.0% on PI 548316, 11.7% on Pickett 71, and 0% on the other three indicators, PI 90763, PI 437654, and PI 89772. The line PI 548316 was relatively susceptible to the Minnesota H. glycines populations and may not be recommended for breeding resistant cultivars in the state. There was no noticeable change of frequencies of virulence phenotypes in response to the use of resistant cultivars during 1997 to 2002 in Minnesota except that FI increased on the PI 209332.     

Effect of Soybean Monoculture on the Bacterial Communities Associated with Cysts of Heterodera glycines

The soybean cyst nematode (SCN), Heterodera glycines, can cause significant reductions in soybean yield and quality in many parts of the world. Natural biological control may play an important role in regulating SCN population. In this study the bacterial communities associated with SCN cysts obtained from fields under different lengths of soybean monoculture were explored. Soil samples were collected in 2010 and 2011 from six fields that had been used for soybean monoculture for 2 to 41 yr. SCN population densities were determined and bacterial communities from SCN cysts were investigated by Biolog and PCR-DGGE methods. SCN population densities initially increased in the first 5 yr of soybean monoculture but then declined steeply as years of soybean monoculture increased. Catabolic diversity of bacterial communities associated with cysts tended to decline as number of years of monoculture increased. Some specific PCR-DGGE bands, mainly representing Streptomyces and Rhizobium, were obtained from the cysts collected from the long-term monoculture fields. Principal component analysis of Biolog and PCR-DGGE data revealed that bacterial communities associated with cysts could be divided into two groups: those from cysts obtained from shorter (< 8 yr) vs. longer (> 8 yr) monoculture. This research demonstrates that the composition of the bacterial communities obtained from SCN cysts changes with length of soybean monoculture; the suppressive impact of these bacterial communities to SCN is yet to be determined.     

Variation in Resistance of Soybean Lines to Races of Heterodera glycines

The objective of this study was to determine the interrelationships of Heterodera glycines races based on their resistance to soybean (Glycine max) cultivars and lines against which they were tested. Greenhouse tests determined the numbers of females of each of eight races of H. glycines that developed on 277 to 522 soybean cultivars and lines. A Female Index (number of females on a test cultivar as a percentage of the number on ''Lee 74'') was calculated and used in frequency distributions, correlations, and duster analyses of the resistance reactions to the different races in an attempt to determine relationships among cultivars. Frequency distribution patterns of all cultivars and lines tested against each race were skewed in favor of resistance, and in some cases bimodality was observed. The majority of correlations between pairs of races were highly significant. Cluster analyses based on the correlations divided eight races into four clusters that explained 73% of the variation in resistance. Cluster 1 was comprised of races 2, 4, and 14; Cluster 2 was comprised of races 6 and 9; Cluster 3 was comprised of races 1 and 3; and Cluster 4 was comprised of race 5. The information obtained in this study could increase the efficiency of testing resistant soybean breeding lines for resistance to H. glycines.