Density-Dependent Multiplication and Survival Rates in Heterodera glycines

Seasonal multiplication and overwinter survival are density-dependent in Heterodera glycines. At low to moderate population densities, the nematode is capable of large population increases on susceptible soybean cultivars and high rates of oversummer or overwinter survival in the absence of a host. To improve estimates of H. glycines multiplication and survival rates, egg densities were monitored for 12 cropping sequences across 10 years. Log-linear regression analysis was used to describe and compare density-dependent relationships. Growing-season change in H. glycines egg densities was density-dependent for all crops (susceptible soybean, resistant soybean, and nonhost), with slope estimates for the density-dependent relationship greater for susceptible soybean compared with a non-host crop. Overwinter population change also was density-dependent, with similar declines in survival rates observed for all crops as population densities increased. Survival was greater following susceptible soybean compared with resistant soybean, with an intermediate rate of survival associated with non-host crops. Survival estimates greater than 100% frequently were obtained at low population densities, despite attempts to account for sampling error. Rates of growing-season multiplication and survival, when standardized for population density, declined with year of the study. Standardized overwinter survival rates were inversely related to average daily minimum temperature and monthly snow cover.     

Distribution of Races of Heterodera glycines in the Central United States

A total of 62 populations of Heterodera glycines were collected in 10 states along the Mississippi and Missouri rivers, and 206 populations were collected in Arkansas. Among the 62 populations, races 2, 3, 4, 5, 6, 9, and 14 were found south of 37°N latitude, and races 1 and 3 were found north of 37°N latitude. In Arkansas samples, races 2, 4, 5, 6, 9, and 14 comprised 87% of the populations. In both groups of samples, H. glycines populations with genes that enabled the population to parasitize cv. Pickett occurred the most frequently, followed by those with genes for parasitism of cv. Peking, then PI88.788, and the fewest with genes for parasitism of PI90.763. The diversity of races in this study raises questions about the effectiveness of race-specific cultivars for the management of soybean cyst nematodes. The greater diversity of races of H. glycines in the southern United States may be because of a longer history of planting resistant cultivars.     

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.     

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.     

Decomposition of Plant Debris by the Nematophagous Fungus ARF

In the study of the biological control of plant-parasitic nematodes, knowledge of the saprophytic ability of a nematophagous fungus is necessary to understand its establishment and survival in the soil. The objectives of this study were (i) to determine if the nematophagous fungus ARF (Arkansas Fungus) shows differential use of plant residues; and (ii) to determine if ARF still existed in the soil of a field in which ARF was found originally and in which the population level of Heterodera glycines had remained very low, despite 15 years of continuous, susceptible soybean. Laboratory studies of the decomposition of wheat straw or soybean root by ARF were conducted in two separate experiments, using a CO₂ collection apparatus, where CO₂-free air was passed through sterilized cotton to remove the microorganisms in the air and then was passed over the samples, and evolved CO₂ was trapped by KOH. Milligrams of C as CO₂ was used to calculate the percentage decomposition of the plant debris by ARF. Data indicated ARF decomposed 11.7% of total organic carbon of the wheat straw and 20.1% of the soybean roots in 6 weeks. In the field soil study, 21 soil samples were taken randomly from the field. Only 3 months after the infestation of the soil with H. glycines, the percentage of parasitized eggs of H. glycines reached 64 ± 19%, and ARF was isolated from most parasitized eggs of H. glycines. Research results indicated ARF could use plant residues to survive.     

Characteristics and Efficacy of a Sterile Hyphomycete (ARF18), a New Biocontrol Agent for Heterodera glycines and Other Nematodes

A filamentous, nonsporulating fungus, designated Arkansas Fungus 18 (ARF18), was isolated from 9 of 95 populations of Heterodera glycines, the soybean cyst nematode, in Arkansas. In petri dishes, ARF18 parasitized 89% of H. glycines eggs in cysts. The fungus also infected eggs of Meloidogyne incognita and eggs in cysts of Cactodera betulae, H. graminophila, H. lespedezae, H. leuceilyma, H. schachtii, and H. trifolii. In pot tests, reproduction of SCN was 70% less in untreated field soil that was naturally infested by ARF18 than in autoclaved field soil. Although ARF18 grew well at 25 C on cornmeal agar over a wide pH range, it did not sporulate on 28 media and thus could not be identified to genus or species.     

Glomus fasciculatum,a Weak Pathogen of Heterodera glycines

The occurrence ofchlamydospores of Glomus fasciculatum (Gf) within cysts of the soybean cyst nematode, Heterodera glycines, and the effects of vesicular-arbuscular mycorrhizae on nematode population dynamics and soybean (Glycine max) plant growth were investigated. Chlamydospores occupied 1-24% of cysts recovered from field soil samples. Hyphae of Missouri isolate Gfl penetrated the female nematode cuticle shortly after she ruptured the root epidermis. Convoluted hyphae filled infected eggs, and sporogenesis occurred within infected eggs. G. microcarpum, G. mosseae, and two isolates of Gf were inoculated with H. glycines on plants of ''Essex'' soybeans. Each of the two Gf isolates infected about 1% of the nematode eggs in experimental pot cuhures. The Gfl isolate decreased the number of first-generation adult females 26%, compared with the nonmycorrhizal control. The total numbers of first-generation plus second-generation adult females were similar for both Gf isolates and 29-41% greater than the nonmycorrhizal control. Soybean plants with Gf and H. glycines produced more biomass than did nonmycorrhizal plants with nematodes, but only Gfl delayed leaf senescence.     

Changes in Heterodera glycines Egg Population Density in Continuous Glycine max over Four Years

Soybean cyst nematode, Heterodera glycines, is found throughout soybean production areas of the United States, but the nematode''s distribution is not uniform within states, counties, and individual fields. The goal of this research was to determine the spatial pattern of H. glycines population density in a field in southeastern Missouri and whether it changed over time in the absence of management practices. Geostatistical methods were used to describe and map the distribution of H. glycines over 4 years in a soybean (Glycine max) field in southeastern Missouri. Semivariograms and kriging, an interpolation method, were used to prepare isoarithmic contour maps and associated error maps. In the field studied, fall H. glycines population density (Pf) was poorly related to density the following spring (Pi). The distribution of peak H. glycines population density within the field changed from year to year, although high densities were often detected in the same general region of the field. The patchiness of H. glycines distribution within a field was verified. Yield was not related to H. glycines egg population density at planting, indicating that unmeasured variables were also reducing yield.     

Seasonal Biochemical Changes in Eggs of Heterodera glycines in Missouri

Changes in the carbohydrate (glucose, trehalose, and glycogen) and total protein contents of eggs retained within Heterodera glycines cysts were monitored monthly in a field microplot experiment conducted from March 1993 to March 1995. Treatments included two near-isogenic lines of soybean cv. Clark differing for date of maturity, and one corn hybrid. The soybean lines were planted in microplots infested with H. glycines at a high average initial population density (Pi) (23,810 eggs/100 cm³ soil), and the corn was planted in microplots infested at high (24,640) and low (5,485) Pi. Soil temperatures at 15 cm depth and rainfall were monitored. Carbohydrate contents varied in the same pattern, with the highest levels measured before planting (May) and after harvest (October) in both years. Neither Pi nor soybean isoline had an effect on any measured response, but the carbohydrate contents of eggs from corn and soybean microplots differed during the overwinter (October-May) periods (P < 0.0001). Trehalose accumulation was negatively correlated with soil temperature (r = -0.78 and r = -0.84, P = 0.0001, July through November 1993 and 1994, respectively), which reflects its role as a cryoprotectant. In contrast to the pattern for carbohydrates, total protein was lowest before planting and after harvest, and highest (>20 μg/1,000 eggs) June through October. Protein content was unaffected by plant cultivar or species. Protein and carbohydrate levels in H. glycines eggs showed seasonal changes that appeared to be primarily temperature-dependent.     

Development of Heterodera glycines as Affected by Fusarium solani,the Causal Agent of Sudden Death Syndrome of Soybean

The effects of the blue form of Fusarium solani, the causal agent of sudden death syndrome (SDS), on Heterodera glycines were examined in the greenhouse. Roots of soybean cv. Coker 156 were inoculated with either H. glycines alone or F. solani + H. glycines in combination. Population levels of H. glycines were reduced 47% in the presence of F. solani. Life-stage development of H. glycines increased 3% in 30 days in the presence of F. solani. Fusarium solani colonized epidermal and cortical cells adjacent to developing juveniles of H. glycines and the nematode-induced syncytia within the soybean root tissue. At 40 days after inoculation, F. solani was isolated from 37% of the cysts in soil recovered from the F. solani + H. glycines combination treatment. Fusarium solani significantly affected H. glycines population density, life-stage development, and succeeding populations.     

Competition between Heterodera glycines and Meloidogyne incognita or Pratylenchus penetrans: Independent Infection Rate Measurements

Competition on soybean between Heterodera glycines (race 3) and Meloidogyne incognita or H. glycines and Pratylenchus penetrans were investigated in greenhouse experiments. Each pair of nematode species was mixed in 3-ml suspensions at ratios of 1,000:0, 750:250, 500:500, 250:750, and 0:1,000 second-stage juveniles or mixed stages for P. penetrans. Nematodes from a whole root system were counted and infection rates standardized per 1,000 nematodes (per replication) prior to testing the null hypothesis through a lack-of-fit F-test. Although the effect of increasing H. glycines proportions on the infection rate of M. incognita was generally adverse, the rate deviated significantly from a trend of linear decline at the 75% H. glycines level in one of two experiments. All lack-of-fit F-tests for the H. glycines and P. penetrans mix were significant, indicating that infection rates for both nematodes varied considerably across inocula. The infection rate of H. glycines decreased with increasing P. penetrans proportions. The rate of P. penetrans infection increased with increasing H. glycines proportions up to the 50% level, but declined at the 75% level. Competition had no effect on nematode development. The general adverse relationships between M. incognita and H. glycines and those between P. penetrans and H. glycines showed a linear trend. The relationship between H. glycines and P. penetrans indicates that the former may be competitive when present at higher proportions than the latter. In this study we have evaluated nematode competition under controlled conditions and provide results that can form a basis for understanding the physical and physiological trends of multiple nematode interactions. Methods critical to data analyses also are outlined.     

Distribution and Morphology of Longidorus breviannulatus Norton &Hoffman, 1975 and Longidorus fragilis Thorne, 1974 (Nematoda: Longidoridae) from North America

In a survey of ecotypes for longidorids, primarily from the rhizosphere hardwood trees growing in sandy soil along stream banks, 828 soil samples were collected from 37 Arkansas counties in 1999-2001. Eight populations of Longidorus breviannulatus were identified from the Arkansas survey samples. A total of 19 populations from California, Illinois, Iowa, Kansas, Michigan, Nebraska, New Jersey, New York, and Wisconsin were identified from the collection of the second author. A few males were found in New York and Nebraska populations and are described herein. Seven populations of L. fragilis were identified in the Arkansas survey samples, and one population was found from Indiana. Four juvenile stages of L. fragilis are present, and data are given for them herein.     

Molecular Characterization of Aldolase from Heterodera glycines and Globodera rostochiensis

Fructose-bisphosphate aldolase (EC 4.1.2.13) is a key enzyme in glycolysis. We have characterized full-length coding sequences for aldolase genes from the cyst nematodes Heterodera glycines and Globodera rostochiensis, the first for any plant-parasitic nematode. Nucleotide homology is high (83% identity), and the respective sequences encode 40 kDa proteins with 89% amino acid identity. Genomic sequences contain six introns located at identical positions in both genes. Intron 4 in the H. glycines gene is >500 bp. Partial genomic sequences determined for seven other cyst nematode species reveal that the large fourth intron is characteristic of Heterodera but not Globodera aldolase genes. Total aldolase-like specific activity in homogenates from H. glycines was 2-fold lower than in either Caenorhabditis elegans or Panagrellus redivivus (P = 0.001). Activity in H. glycines samples was higher in juvenile stages than in adults (P = 0.003). Heterodera glycines aldolase has Km = 41 µM and is inhibited by treatment with carboxypeptidase A or sodium borohydride.     

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.     

Influence of Herbicide Application to Soybeans on Soybean Cyst Nematode Egg Hatching

The hatching of Heterodera glycines eggs in soybean root exudates collected after postemergence application of three herbicides, and the hatching potential of H. glycines eggs from females feeding on herbicide-treated plants, were measured in vitro. Hatching in all root exudate solutions (RES) was greater than in deionized water but less than in 0.003 M ZnSO₄ solution. Filtering RES with a 0.22-μm-filter increased H. glycines hatching in RES. Application of acifluorfen, bentazon, and lactofen to foliage of soybean plants inhibited hatching of H. glycines eggs from the same plants. Hatching in RES from the different herbicide-treated soybeans was similar. Application of crop oil concentrate and non-ionic surfactant adjuvant to foliage did not affect hatching of H. glycines eggs from soybean plants.     

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.     

Susceptibility of Soybean Introductions to Races 1, 2, 3, and 4 of Heterodera glycines

Thirteen soybean plant introduction (PI) lines, selected for their apparent susceptibility to Heterodera glycines, were compared with cultivar Lee 74 as hosts of H. glycines races 1, 2, 3, and 4. Race 3 produced the highest average number of females of the four races. Compared to Lee 74, more (P = 0.05) females of H. glycines race 1 were extracted from eI 274420, PI 274423, and PI 317333; PI 86457 had more females of H. glycines race 2; and PI 86443, PI 86457, PI 261467, PI 274420, PI 274421, and PI 274423 had more females of H. glycines race 3. Similar numbers of females of H. glycines race 4 developed on all of the soybean lines and Lee 74. PI 274421, PI 274420, or PI 196159 could provide a more or equally susceptible host for H. glycines races 1, 2, 3, and 4 than Lee 74. One of these three lines could be substituted for Lee as the standard susceptible cultivar in the race determination test.     

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.     

Effects of Continuous Cropping of Resistant and Susceptible Cultivars on Reproduction Potentials of Heterodera glycines and Globodera tabacum solanacearum

The reproductive potentials of Heterodera glycines (mixture of races 3 and 4 and unidentified races) and a tobacco cyst nematode Globodera tabacum solanacearum were studied in the field. The experiments involved four cultivars of soybean Glycine max and four cultivars of Nicotiana tabacum. The reproductive potential of the H. glycines population was high on Essex and Lee 74 soybean but low on Forrest and Bedford over the 3 years (1982-84) of continuous cropping. The reproductive potential of H. glycines was 12% on Forrest and 6% on Bedford in 1982 but increased to 37 and 35% in 1983 and to 71 and 41% in 1984, respectively, on these two cultivars. The reproductive potential of G. tabacum solanacearum was high on McNair 944 and Coker 319 tobacco cultivars and low on VA 81 and PD 4 over the 3 years of cropping. The reproductive potential of G. tabacum solanacearum on VA 81 and PD 4 was 18 and 17% in 1982, 7 and 16% in 1983, and 5 and 5% in 1984, respectively. The changes in reproductive potentials of H. glycines and G. tabacum solanacearum may be related to inherent genetic variability in the systems that control reproduction of the two cyst nematodes and nature of resistance incorporated in the soybean and tobacco cultivars.     

Hematozoa of wild turkeys from the midwestern United States: translocation of wild turkeys and its potential role in the introduction of Plasmodium kempi

Twenty-three of 310 blood samples taken from live-trapped eastern wild turkeys (Meleagris gallopavo silvestris) from Missouri (USA), and hunter-killed birds from Wisconsin, North Dakota and Minnesota (USA), and inoculated into domestic broad-breasted-white turkey poults were positive for two species of Plasmodium. Twenty-one of the positive samples were infected with P. (Novyella) kempi, and two samples from Wisconsin were infected with P. (Giovannolaia) lophurae. Twenty percent of 310 blood smears were positive for Haemoproteus melagridis, while only 3% were infected with Leucocytozoon smithi. A statistically higher prevalence of Plasmodium spp. from 1983 to 1984 was observed in Wisconsin, and in the samples from Minnesota when compared with both Missouri and Wisconsin. Turkeys from Wisconsin and Minnesota had both a statistically higher prevalence and mean intensity of H. meleagridis than birds from Missouri. Evidence indicates that P. kempi has been introduced into other states along with the vertebrate hosts. It is suggested that greater care should be exercised when translocated wild turkeys are introduced into areas where there are other endangered or threatened avian species.