Variability in Host Preference Among Field Populations of Heterodera glycines

Eighteen soybean fields, six each with race 3, race 4, or Bedford population of Heterodera glycines, were selected for testing of host variability. Each field was divided into three sections, and a bulk soil sample was taken from each section. The 54 bulk soil populations (BSP) and 270 single cyst populations (SCP) were subjected to race determination tests. Tests of the 18 BSP and 90 SCP from the race 3 fields revealed that race 3 was the predominant race; however, 68 of the populations tested were other races. Tests of the 18-BSP and 90 SCP from race 4 fields demonstrated that races 2 and 4 were predominant, with 38 and 39 populations; respectively. Tests of the 18 BSP and 90 SCP from the Bedford population fields revealed tremendous variability. Races 2, 4, and 6 were the predominant races, with 32, 31, and 28 populations, respectively. These results indicate that of the three races studied, the Bedford population is the most variable, race 3 shows considerable variability, and race 4 shows very little.     

A Revised Classification Scheme for Genetically Diverse Populations of Heterodera glycines

Heterodera glycines, the soybean cyst nematode, is a major yield-limiting pathogen in most soybean production areas worldwide. Field populations of H. glycines exhibit diversity in their ability to develop on resistant soybean cultivars. Since 1970, this diversity has been characterized by a bioassay used to assign a race classification to a population. The value of the race scheme is reflected in the number and quality of resistant soybean cultivars that have been developed and released by soybean breeders and nematologists working in concert. However, the race scheme also has been misapplied as a means of studying H. glycines genotypes, in part due to the use of the term "race." For fungal and bacterial pathogen species, "race" can theoretically be applied to individuals of a population, thus allowing inference of individual genotypes. Application of a race designation to an individual egg or second-stage juvenile (J2) of H. glycines is not possible because a single J2 cannot be tested on multiple hosts. For other nematode species, "race" is defined by host ranges involving different plant species, whereas the H. glycines race test involves a set of lines of the same plant species. Nonetheless, because H. glycines populations vary in genetic diversity, and this variation has implications for management strategies, a mechanism is needed for documenting and discussing population differences. The HG Type scheme described herein avoids the implication of genetic uniformity or predictability in contrast to the way the race scheme has been used.     

Overwinter Population Dynamics of Heterodera glycines

The purpose of this research was to compare the overwinter survival of populations of Heterodera glycines at different latitudes in the United States and the effect of changing latitudes before and after the initiation of dormancy. Soil samples infested with H. glycines were collected in August or October in 1992 to 1994 from soybean fields in two to four states (combinations of Arkansas, Florida, Minnesota, Missouri, and Wisconsin). The samples were mixed thoroughly, divided into subsamples, shipped to an overwinter location, and buried until time for processing. To determine survival, cysts, eggs, and second-stage juveniles were extracted from replicated subsamples and counted each month from December to May. Survival generally was between 50% and 100%, and often was best in the state of origin. In Florida, survival was at the 50 to 100% level in soil from most locations, and in Wisconsin was near 100%. Survival of H. glycines in Arkansas and Missouri varied more than at the other locations. In a separate test, survival in microplots in Arkansas, in a more natural environment than that of buried samples, was 70 to 94% for field isolates from Arkansas, Minnesota, and Missouri and 100% for isolates of races 1, 3, and 14 that had been maintained in a greenhouse for several years. Survival appears to be better than previous tests had indicated. High survival rates require cultivars with high levels of resistance and long-term rotations for management.     

Optimization of the Heterodera glycines Race Test Procedure

Effects of pot size, length of seedling radicle at the time of inoculation with Heterodera glycines, transplanting after inoculation, type and amount of inoculum, and temperature were tested to determine the optimum procedure for the H. glycines race test. Numbers of H. glycines females extracted from plants in 7.5-cm-d pots tended to be higher than numbers from 10-cm-d pots, but not significantly so. Radicle lengths from 2.5 cm to 7.5 cm had no effect. Transplanting after inoculation reduced the variation in the number of females extracted, but the numbers of females produced were very low. Plump females (40 per pot) or eggs (4,000 per pot) were the best inocula. A constant temperature of 28 C appeared to be optimum. More H. glycines females were extracted from plants 28 days after inoculation than at 35 days. Race tests in which all of these factors were included were still highly variable in the number of H. glycines females extracted from different replications of the same test host. Tests of several susceptible cultivars revealed differences in their capabilities as hosts of H. glycines races.     

Effects of Biosolid Soil Amendment on Heterodera glycines Populations

The high degree of parasitic variability in Heterodera glycines and its distribution in a wide range of soybean production systems present multiple challenges for management, which necessitate increased understanding of the biology of H. glycines. Soil amendments are being considered either as stand-alone and/or as part of integrated management approaches. A recycled municipal biosolid with nutrition supplement and liming qualities, N-Viro Soil (NVS) has potential as a multi-purpose soil amendment. In three greenhouse experiments, the effects of 0, 1.0 or 4.0 g NVS/100 cm³ of sandy loam soil on three H. glycines populations (GN1, GN2 and GN3) were investigated on soybean grown for 557 ± 68 degree-days (base 10°C). The response of the three H. glycines populations to NVS treatment varied by experiment. The overall numbers of preadult stages and cysts generally decreased with increasing levels of NVS in all experiments, and the high rate was more effective than the low rate in suppressing H. glycines numbers. This suggests that the high NVS treatment can affect the three populations in the experimental soil type under controlled conditions. Field studies to determine efficacy of the soil amendment in a wide range of environments should be initiated.     

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.     

Complete Characterization of the Race Scheme for Heterodera glycines

One hundred thirty-eight isolates of Heterodera glycines from nine states in the United States, People''s Republic of China, and Indonesia were tested on the four standard soybean race differentials. A total of 12 variants were found, including the five races described previously. The seven variants that did not correspond to one of the described races and reports from other areas of populations that could not be classified are evidence that the present race classification system needs to be fully characterized. Eleven additional races are described; this expands the total to 16 races, the maximum possible using the four prescribed differentials and a + or - rating for each. The seven new races are designated as 6, 9, 10, 13, 14, 15, and 16. This complete characterization of the race scheme will allow for immediate communication of the discovery of the remaining four races plus the identification of previous undescribed races.     

An Alternative Method for Evaluating Soybean Tolerance to Heterodera glycines in Field Plots

Alternate planting dates and periodic destruction of the previous year''s soybean crop as well as 1-year bare fallow were used to establish a range of population densities ofHeterodera glycines for the subsequent year. Soybean cultivar Coker 156 (susceptible, moderately tolerant) was compared to cultivars Essex (susceptible, intolerant) and Bedford (resistant) to evaluate tolerance at different H. glycines population densities established through the previous year''s treatments. Yield of Coker 156 was consistently intermediate between yields of Bedford and Essex in 1986 and 1987. Yield of Essex was negatively correlated (P = 0.05) with preplant egg numbers of H. glycines in 1987, whereas yield of Bedford and Coker 156 were not related to nematode density. Reproduction of H. glycines was greater (P = 0.05) on the moderately tolerant Coker 156 than on either of the other cultivars.     

Population Density and Spatial Pattern of Heterodera glycines in Relation to Soybean Phenology

Population dynamics of Heterodera glycines (SCN) were influenced by initial nematode population density in soil, soybean root growth pattern, soil type, and environmental conditions in two field experiments. Low initial populations (Pi) of SCN increased more rapidly during the growing season than high Pi and resulted in greater numbers of nematodes at harvest. Egg and juvenile (J2) populations increased within 2-6 weeks after planting when early-season soil temperatures were 20 C and above and were delayed by soil temperatures of 17 C or below in May and early June. Frequencies of occurrence and number of nematodes decreased with increasing depth and distance from center of the soybean row. Spatial pattern of SCN paralleled that of soybean roots. Higher clay content in the subsoil 30-45 cm deep in one field restricted soil penetration by roots, indirectly influencing vertical distribution of SCN. Shoot dry weight was a good indicator of the effect of SCN on seed yield. Root dry weight was poorly correlated with soybean growth and yield. The relationship of yield (seed weight) to Pi was best described by a quadratic equation at one site, but did not fit any regression model tested at the second site.     

Improving the Accuracy of Sampling Field Plots for Plant-Parasitic Nematodes

The validity of nematode data from field experiments depends largely on how well samples represent the nematode population. Data from an intensive sampling of three field plots before and after spring cultivation were used to compare eight simulated sampling schemes. Average deviation from the plot mean ranged from 10% to 34% before cultivation and from 7% to 16% after cultivation. Samples taken from only the plant row erred most before cultivation but were comparable to other schemes after cultivation. Several schemes achieved a 25% deviation or less in 90% of the sample simulations. Sampling a nematode population usually involves subsampling a composite bulk sample, however, and this increases error by an estimable amount. A random sample with 35 cores and four random subsamples estimated mean plot densities within 25% with probabilities ranging from 0.77 to 0.85. The probability of a sample-subsample combination coming within a specified percent error of the true mean can be extended cautiously to any field mean and variance more-or-less independent of species and area using formulae presented herein. The most economical method of increasing sample accuracy was to increase the number of soil cores.     

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.     

Fitness Components and Selection of Biotypes of Heterodera glycines

Survival of biotypes of Heterodera glycines was studied in microplots and in the field. The field population was subjected to various cropping sequences. Viability of eggs overwintered in microplots was determined each spring by percentage hatch, percentage of hatched eggs penetrating roots, and numbers of females developing on Peking and PI 88788 soybeans. Eggs from the field were collected in the spring and fall and assayed for ability to develop on Peking and PI 88788. Hatch of isolates overwintered in the microplots averaged 13% in May 1989 and 19% in 1990. No differences in hatch were detected among the isolates in 1989. Numbers of juveniles penetrating susceptible roots averaged less than 20% of the hatched eggs each year. An isolate of a biotype parasitic on susceptible soybeans and the resistant soybean PI 88788 penetrated roots more successfully than other biotypes. A second isolate from North Carolina, parasitic on susceptible soybeans, PI 88788, and the resistant soybean Peking experienced selection against development on Peking during two winters. Only 17 % of the expected numbers of females developed on Peking from this isolate. In the microplot experiment, parasitism of PI 88788 and Peking had a selective disadvantage (selection coefficient) of s = 0.29 and 0.62 over all isolates, respectively. In the field experiment, the relative numbers of cysts on Peking and PI 88788 increased between the spring and fall on soybean, then decreased over the winter and under corn. Selection coefficients against parasitism of PI 88788 and Peking averaged 0,19 and 0.3 in the field population. In neither experiment did juveniles lose their ability to parasitize susceptible soybeans.     

Influence of Temperature and Soybean Phenology on Dormancy Induction of Heterodera glycines

Growth room and field experiments were conducted to determine the influence of soil temperature and soybean phenology on dormancy induction of a North Carolina population of Heterodera glycines race 1. Three temperature regimes and two photoperiods to regulate plant phenology were investigated in growth rooms. H. glycines hatch was greatest from the 26 and 22 C (day and night) temperature treatment, intermediate at 22 and 18 C, and least from the decreasing regime (26 and 22 C, 22 and 18 C, and 18 and 14 C). More eggs hatched and greater nematode reproduction occurred on pod-producing soybeans than on those that remained vegetative. In the field study, hatching patterns were not different between depodded and naturally senescing soybeans nor between the different maturity groups of soybean cultivars (groups V through VIII). Egg hatch (9-16%) was greatest in August and September when mean soil temperatures were between 25 and 29 C. Hatch declined to 1% in vitro and was not detectable in the bioassay in November. Greatest nematode numbers were observed on the latest maturing cultivar (group VIII) and fewest on the cultivar which matured earliest (group V). Decreasing temperature appears to be more important than soybean phenology in dormancy induction of H. glycines.     

Dormancy of Heterodera glycines in Missouri

A 2-year study was conducted in field microplots to determine the relative importance of soybean phenology and soil temperature on induction of dormancy in Heterodera glycines in Missouri. Four near-isogenic soybean lines differing for maturity date were planted in microplots infested with a race 5 isolate of H. glycines. Soil temperature was monitored at a depth of 15 cm. Eggs of H. glycines, extracted from cysts collected monthly from each microplot, were used in hatching tests and bioassays to determine dormancy. Egg hatching and second-stage juvenile (J2) infectivity rates decreased sharply from their highest levels in midsummer (July-August) to a low level by October of each year and remained low (< 10% hatching and < 0.2 J2/cm root) until May or June of the following year. The patterns of numbers of females and eggs in the bioassays were similar. The decreases were not related to soil temperature and did not differ consistently among soybean isolines. The monophasic changes in all nematode responses with peak midsummer rates suggest that H. glycines produces one primary generation per year in central Missouri. Changes in hatching rates and the timing of minimum and maximum rates suggested that H. glycines eggs exhibit more than one type of dormancy.     

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.     

Scanning Electron Microscopy of Second-Stage Juvenile Cephalic Morphology in Heterodera glycines Races

Scanning electron microscopy (SEM) was used to compare juvenile cephalic morphology of the five described races of Heterodera glycines. Races 1, 2, 3, and 4 were obtained in the United States and race 5 was obtained from Japan. Differences in the gross morphology o f labial discs; ventral, dorsal and lateral lips; amphidial apertures; and fissures on the labial disc o f some specimens were observed. There was considerable interracial and intraracial variation which precluded separation o f juveniles of H. glycines races by SEM.     

Host Suitability of Diverse Lines of Phaseolus vulgaris to Multiple Populations of Heterodera glycines

The host suitability of diverse races and gene pools of common bean (Phaseolus vulgaris) for multiple isolates of Heterodera glycines was studied. Twenty P. vulgaris genotypes, representing three of the six races within the two major germplasm pools, were tested in greenhouse experiments to determine their host suitability to five H. glycines isolates. Phaseolus vulgaris genotypes differed in their host suitability to different H. glycines isolates. While some common bean lines were excellent hosts for some H. glycines isolates, no common bean line was a good host for all isolates. Some bean lines from races Durango and Mesoamerica, representing the Middle America gene pool, were resistant to all five nematode isolates. Other lines, from both the Andean and Middle America gene pools, had differential responses for host suitability to the different isolates of H. glycines.     

Separation and Characterization of Heterodera glycines Acetylcholinesterase Molecular Forms

The composition and biochemical properties of acetylcholinesterases isolated from Heterodera glycines were determined. Heterodera glycines contains three separable AChE molecular forms that can be grouped into two classes corresponding to classes A and C found in some other nematode species. The apparent lack of class B AChE is unusual and may have significant behavioral ramifications. The class C enzyme isolated from H. glycines is similar to that from Meloidogyne arenaria and M. incognita but is somewhat more sensitive to AChE inhibitors such as eserine. Heterodera glycines possesses a larger percentage of its total acetylcholinesterase as class C than other nematodes thus far examined.     

Diversity Among a Heterodera glycines Field Isolate and Derived Inbreds Based on RAPD Analysis and Reproduction on Soybean Genotypes

A field population of Heterodera glycines was inbred by a combination of controlled male-female matings and inoculation of soybean with second-stage juveniles (J2) from single cysts. The initial and four F₆ inbred populations were subjected to random amplified polymorphic DNA analysis and were also tested for their ability to reproduce on race differentials. The RAPD patterns of the inbred populations had a lower number of total bands and a lower percentage of polymorphic bands among individual cysts than the initial population. The estimated number of polymorphic loci detected by RAPD analysis was about 25% for the initial population and 4% to 7% for the inbred lines. Reproduction of H. glycines decreased for 6 of 24 inbred-soybean combinations. In particular, reproduction of three inbred populations on PI 90763 was greatly reduced. Inbreeding did not decrease variance of cyst number on soybean genotypes. The inbreeding coefficient calculated from RAPD data was greater than that derived from the known inbreeding pedigree.