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Thirty-two Holstein cows were allocated to receive intranasal vaccination with modified live bovine herpesvirus-1 (BHV-1), bovine respiratory syncytial virus (BRSV) and parainfluenza type 3 virus (PI3V) vaccine either two weeks prior to their projected calving date, or within 24h after calving. Nasal secretions were collected twice at a 12-h interval on the day prior to vaccination (day 0) and at 2, 4, 7, 10 and 14days post vaccination to measure interferon (IFN) alpha, IFN-beta, IFN-gamma, and BHV-1-specific IgA by ELISA. Serum neutralizing antibody titers to BHV-1 and BRSV were measured on days 0, 7, and 14. There was a significant treatment effect (p<0.0004) and interaction (p<0.05) on nasal BHV-1 IgA levels, with higher IgA levels in cows vaccinated within 24h after calving. There was a significant treatment effect on nasal IFN-gamma concentration (p<0.05) and on nasal total IFN concentration (p<0.05), with higher IFN-gamma and total IFN concentrations seen in cows vaccinated within 24h after calving. There was no significant treatment or interaction effect on nasal IFN-alpha or IFN-beta concentrations, or on serum neutralizing titers to BRSV. In spite of prior viral vaccination during the previous lactation, cows vaccinated on the day of calving responded to an intranasal viral vaccination with increased concentrations of IFN-gamma and increased titers of IgA following vaccination which was significantly higher than cows vaccinated precalving. This study is the first to examine respiratory mucosal responses in immunologically mature dairy cattle vaccinated intranasally before and after calving., (Copyright © 2017. Published by Elsevier B.V.)

ABSTRACT The reniform nematode, Rotylenchulus reniformis, is a serious threat to cotton (Gossypium hirsutum) production in the United States, causing an annual loss of about $80 million. The objective of this study was to isolate fungi from eggs of R. reniformis and select potential biocontrol agents for R. reniformis on cotton. We focused on the fungus Pochonia chlamydosporia because it suppresses root-knot and cyst nematodes and because preliminary data indicated that it was present in Arkansas cotton fields. Soil samples were collected from six cotton fields in Jefferson County, Arkansas. A total of 117 isolates of the nematophagous fungus P. chlamydosporia were obtained. In an in vitro test, 105 of the 117 isolates parasitized fewer than 15% of R. reniformis eggs, but 12 isolates parasitized between 16 and 35% of the eggs. These 12 isolates produced from 6.8 x 10(4) to 6.9 x 10(5) chlamydospores per gram of medium in vitro, and chlamydospore production was similar on rice grain and corn grain media. In two greenhouse experiments, a single application of isolate 37 (5,000 chlamydospores per gram of soil) significantly reduced the numbers of R. reniformis on cotton roots and in soil. The three isolates (37, 26, and 14) that parasitized the most eggs in vitro were also the most effective in suppressing numbers of R. reniformis and in increasing cotton growth in the greenhouse.

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 CO collection apparatus, where CO-free air was passed through sterilized cotton to remove the microorganisms in the air and then was passed over the samples, and evolved CO was trapped by KOH. Milligrams of C as CO 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.

The reniform nematode, Rotylenchulus reniformis Linford &Oliveira, has become a serious threat to cotton (Gossypium hirsutum L.) production in the United States during the past decade. The objective of this study is to isolate fungi from eggs of R. reniformis and select potential biological control agents for R. reniformis on cotton. Soil samples were collected from cotton fields located in Jefferson County, Arkansas. Eight genera of fungi were included in the 128 fungal isolates obtained, and among them were five strains of the nematophagous fungus ARF. The mtDNA RFLP pattern, colony growth characteristics, and pathogenicity indicate the five ARF isolates represent one described strain and one new strain. Light and electron microscopic observations suggest ARF is an active parasite of R. reniformis, with parasitism ranging from 48% to 79% in in vitro tests. Three greenhouse experiments demonstrated ARF successfully suppressed the number of reniform nematodes during the first and second generation of the nematode. Reductions in numbers of R. reniformis on the roots for the seven application rates of 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, and 0.5% ARF were 87%, 92%, 94%, 96%, 97%, 98%, and and 98%, respectively.

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.

Five soybean cultivars, one each from Maturity Groups (MG) III, IV, V, VI, and VII, all susceptible to Heterodera glycines, were planted in H. glycines-infested soil in April, May, June, and July at multiple locations in Arkansas and Missouri. The purpose of the study was to determine whether planting early reduces yield losses due to H. glycines. The population levels of cysts, eggs, and secondstage juveniles of H. glycines were determined from soil samples collected at planting and harvest. Yields were measured from the two middle rows of 4.9 or 6.1-m four-row plots. The population dynamics of the nematode varied with planting date, cultivar, MG, and initial nematode population. Generally, the lowest reproductive factor (Rf = final population/initial population) for any year was on the MG III cultivar, regardless of planting date or field location. Also, the Rf was generally lower on each cultivar planted in April than in June or July. The highest Rf values were on cultivars in MG VI or VII planted in June or July. Yields were not consistently correlated with initial nematode population level. Early planting of late-maturing soybean cultivars may be profitable, but early planting of MG III cultivars, though supporting little if any increase in nematode numbers, did not result in high yields.

Rhizobacteria were isolated from the rhizoplane and rhizosphere of soybean plants from fields in Arkansas and tested for their effect on numbers of soybean cyst nematode (Heterodera glycines). In initial greenhouse tests in heat-treated silt loam soil, 138 of the 201 bacterial isolates tested had no influence on numbers of cysts and eggs + second-stage juveniles (J2) of H. glycines, 36 reduced (suppressive isolates) and 27 increased (enhancing isolates) numbers of cysts and (or) eggs + J2 when compared to the controls (P

Sixty-four chitinolytic bacterial isolates from soybean fields in Arkansas were tested for suppression of soybean cyst nematode (Heterodera glycines) in a heat-treated silt loam soil amended with 0.6% (w/w) chitin in a greenhouse. Five isolates consistently reduced numbers of H. glycines compared to controls receiving neither chitin nor bacteria, or only chitin. Four of the five isolates interacted with the chitin substrate to enhance its effectiveness in reducing numbers of the nematode. The size of the clear-zone produced by some of the isolates in colloidal chitin medium, an indication of chitinolytic activity in vitro, was not related to suppression of nematode numbers in soil.

Seven populations of Heterodera trifolii from Arkansas, Kentucky, Pennsylvania, and Australia plus 3 or 4 single-cyst isolates (SCI) from each population were tested for reproduction on seven species of plants to compare the host preferences among and within populations. Common lespedeza, Kummerowia striata cv. Kobe, was a good host for all populations and isolates. Therefore, a plant was considered to be a host if the number of females produced on it was 10% or more of the number on Kobe. All seven populations reproduced on Trifolium repens and T. pratense. None reproduced on Beta vulgaris or Glycine max. One single-cyst isolate from the Australian population produced a few females on T. pratense. The Australian population maintained on carnation, Dianthus caryophyllus, produced females on carnation but not on curly dock, Rumex crispus. However, its subpopulation maintained on T. repens produced females on R. crispus but not on carnation. Four of the other six populations produced females on R. crispus, and four produced females on carnation. Differences in host range were observed among seven of the mother populations and their SCI, and among isolates within each population. Five host range patterns were found in populations and SCI of H. trifolii. Significant quantitative differences occurred among populations in the numbers of females on most hosts, between isolates and their original populations, and among isolates from the same population. SCI selected from white clover produced fewer females on a series of test hosts and had host ranges the same as or narrower than those of the original populations. However, SCI selected from Kobe lespedeza had more females on some hosts and had host ranges the same as or wider than those of the original populations. The host ranges of all populations and SCI of H. trifolii were different from those of populations and SCI of race 3 of H. glycines and H. lespedezae.

ABSTRACT Isolates of a sterile fungus designated ARF (Arkansas fungus) can be separated into two groups, ARF-C and ARF-L, that differ morphologically and in their ability to suppress numbers of Heterodera glycines on soybean. Our objectives were to determine if the two ARF groups differed in their ability to parasitize juveniles, females, and eggs in the rhizo-sphere of soybean and to proliferate in soil. The experiments were conducted in a greenhouse using soil infested with homogenized ARF mycelium. The ARF-L isolates parasitized more juveniles and young females than did the ARF-C isolates. Suppression of these stages was 67% for ARF-L and 12% for ARF-C isolates 14 days after nematode inoculation. When soybean plants containing gravid females were transplanted into fungus-infested soil, ARF-L isolates parasitized 55 to 98% of nematode eggs, whereas ARF-C isolates parasitized 0 to 22%. In both heat-treated and nonheated soil, the biomass of mycelial mats, a measure of relative proliferation, tended to be greater for ARF-L than for ARF-C isolates. The ability of ARF-L isolates to parasitize a large percentage of both pre-reproductive stages and eggs of H. glycines may contribute to its effectiveness as a biological control agent.

An unnamed fungus, designated ARF, that parasitizes eggs and sedentary stages of cyst nematodes is a potential biological control agent of Heterodera glycines. The objectives of this study were to determine whether ARF isolates differ in their ability to suppress nematode numbers in soil and to compare the efficacy of ARF in heat-treated and native soil. The effectiveness of 11 ARF isolates was compared by introducing homogenized mycelium into heat-treated soil. Soybean seedlings were transplanted into pots containing fungus-infested soil and inoculated with H. glycines. After 30 or 60 days, the number of nematodes and the percentage of parasitized eggs were determined. Three isolates (907, 908, and TN14), which were previously reported to be weak egg parasites in vitro, consistently suppressed nematode numbers by 50% to 100%. Of the isolates previously reported to be aggressive egg parasites, four (903, BG2, MS3, and TN12) reduced nematode numbers by 56% to 69% in at least one experimental trial, but the other four had no effect on nematode numbers. When the efficacy of isolate TN14 was tested in heat-treated and native soil, nematode suppression was greater in the heat-treated soil in only one of two trials. In both soil treatments, nematode numbers were reduced by more than 60%. We conclude that virulence toward nematode eggs in vitro is a poor indicator of effectiveness of an ARF isolate in soil, and that the presence of soil microbes may reduce, but does not completely inhibit, activity of isolate TN14.

Eight wild perennial Glycine species (G. argyrea, G. canescens, G. curvata, G. cyrtoloba, G. latifolia, G. microphylla, G. tabacina, and G. tomentella) were evaluated for resistance to isolates of races 1, 3, and 14 of Heterodera glycines. In a second experiment, reproduction of isolates of races 3, 5, and 14 of H. glycines on five of the wild perennial species was determined. Seventy-one derived fertile lines (2n = 40) that were hybrids between G. max cv Clark 63 and G. tomentella also were evaluated for resistance to isolates of races 3, 5, and 14. All of the wild perennial Glycine species were resistant (Female Indices [FI] less than 10) to all of the isolates that were tested on them. In most cases no females matured. The soybean cvs. Clark 63 and Altona, which were tested at the same time as the hybrids, were susceptible to all isolates of H. glycines tested. When the tests were combined and a single FI calculated with the average number of females on Lee 74, one derived fertile line was resistant to race 3, three derived fertile lines were resistant to race 5, and five derived fertile lines were resistant to race 14. Thus, transfer of resistance to H. glycines from G. tomentella to G. max apparently occurred.

Production of females by stock populations of races 1, 2, 3, 4, 5, 6, 9, and 14 of Heterodera glycines on 'Lee 74', 'Pickett', 'Peking,' PI 88788, and PI 90763 soybean cultivars and lines at inoculum densities of 100, 1,000, 4,000, 5,000, and 10,000 eggs and second-stage juveniles/pot (2.2, 21.8, 87.1, 108.9, and 217.9/cm(3) soil) was tested to determine the effects of soil population density on race identification using the 16-race scheme. Tests of all races were repeated 1 to 3 times during the year, except for the race 9 test, which was conducted once. Only races 3 and 9 had the same race designation at all infestation densities in repeated tests. Races 2 and 4 consistently were identified at all except the lowest infestation densities. Race 5 was identified as race 15 at the 100 infestation density in one test, and as race 1 at the 1,000 density in another test. Race 6 had significant numbers of females on Peking and P190763 that resulted in female indices of 34 on Peking and 14 on P190763 when the infestation density was 10,000. Race 14 was consistently identified between infestation densities of 4,000 and 10,000 but was identified as race 6 or 9 at the 100 infestation density and as race 4 or 14 at the 1,000 infestation density. Race 1 was identified as race 5 in a few instances because Pickett was not resistant to this population. The results of this series of experiments suggest that an infestation density of 4,000 eggs and second-stage juveniles/pot is best for race identification. Races were very poorly differentiated at the lowest density, differentiation was inconsistent at the 1,000 infestation density, and densities higher than 4,000 had reduced numbers of females on Lee 74 and relatively high numbers on the differentials which resulted in poor race differentiation with some races.

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.

The effects of 22 pesticides on the mycelial growth and pathogenicity of the biocontrol fungus ARFI8 to Heterodera glycines were tested in vitro. The chemicals were added to agar at 10, 100, and 1,000 ppm a.i.; a block of agar containing the fungus was added to each test concentration; and fungal growth was measured. Subsequently, a block of the fungus on the pesticide-containing agar was used to determine the ability of the fungus to parasitize eggs of H. glycines. Aldicarb, bentazone, and chlorothalonil had little or no effect on fungal growth, whereas benomyl and thiophanate methyl completely inhibited growth of the fungus at 10 ppm. The relative insensitivity of ARF18 to certain pesticides would permit selected use of those pesticides with ARF18 in an integrated control program if the effects on the fungus in the field are similar to results from petri dish studies.

ABSTRACT Seventy-six populations of Heterodera glycines were collected from 33 counties in 10 states of the United States along the Mississippi and Missouri Rivers in 1992 and 1993. A sterile hyphomycete fungus of an unnamed taxon, designated ARF18 and shown to be a parasite of eggs of H. glycines, was isolated from eggs and cysts of 10 of the populations from Kentucky, Louisiana, Mississippi, and Tennessee. Ten isolates of ARF18 obtained in this study and seven isolates obtained in earlier studies were characterized for cultural morphology on several growth media, the ability to produce sclerotium-like structures (SLS) on cornmeal agar, growth rates, pathogenicity to eggs of H. glycines in vitro, and mitochondrial (mt) DNA restriction fragment length polymorphisms (RFLPs). All 17 isolates of ARF18 readily grew on potato dextrose agar, cornmeal agar, and nutrient agar. Based on colony morphology and SLS appearance on cornmeal agar, the isolates could be grouped into two morphological phenotypes. Isolates that produced SLS that were composed of a compact mass of hyphae were designated ARF18-C, whereas isolates that produced SLS composed of a mass of loosely clumped hyphae were designated ARF18-L. Only minor differences in growth rates were detected among the ARF18-C and ARF18-L isolates. All 10 ARF18-C isolates, which were from Arkansas, Louisiana, Mississippi, and Tennessee, belonged to a single mtDNA RFLP haplotype. The seven ARF18-L isolates shared many comigrating mtDNA restriction fragments with one another, but belonged to three distinct mtDNA RFLP haplotypes. Ability to infect eggs of H. glycines in vitro varied considerably among the various isolates of ARF18. In particular, several of the ARF18-C isolates were consistently able to infect over 50% (mean = 70.0%, standard deviation = 16%) of the eggs of H. glycines, whereas ARF18-L infected eggs to a lesser degree (mean = 25%, standard deviation = 27%). ARF18-C was isolated only from H. glycines populations from below 37 degrees N latitude. The presence of ARF18 was associated with soils with Mg levels <314 kg/ha, cyst numbers >4.5 per 100 cm(3), and iron levels >203.5 kg/ha; or with Mg levels >314 kg/ha and Na levels <121 kg/ha. The widespread distribution of ARF18 and the ability of some isolates to aggressively colonize eggs of H. glycines are indications that it has potential as a biological control agent for H. glycines.

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 degrees N latitude, and races 1 and 3 were found north of 37 degrees 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.

Experiments to determine the effects of extraction techniques and the influence of shipping on extraction of Heterodera glycines life stages gave variable results. Shipping did not significantly affect numbers of nematodes extracted. More second-stage juveniles (J2) were extracted with Baermann funnels than with an elutriator, probably because incubation of encysted eggs on the Baermann funnel for 1 week allowed hatching to occur. Sieving was more efficient than elutriation for extracting cysts. Adding air agitation to the water pressure during elutriation increased extraction efficiency of cysts but not J2. Sample sizes of 250 cm(3) and 500 cm(3) did not influence extraction efficiency of cysts; however, sample size did influence extraction of J2.

Many new cultivars of soybean (Glycine max) are released each year. Knowledge of their relative susceptibility to soybean cyst nematode (Heterodera glycines) is of interest to soybean breeders and those making nematode management recommendations. Two-hundred-eighty-one cultivars and breeding lines of soybean were screened for resistance to isolates of H. glycines races 1, 2, 3, 4, 5, 6, 9, and 14. No cultivar or line (except possibly PI 437654 or cultivars developed from this PI line) had resistance to all of the races tested. Twenty-one cultivars and lines were resistant to race 1, 5 to race 2, 146 to race 3, 2 to race 4, 4 to race 5, 24 to race 6, and 24 to race 14; some had resistance to more than one race. In addition, several cultivars and lines had moderate resistance to each of the race isolates tested.

Dry alginate-clay pellets containing mycelium of ARF18 were added to sandy soil in greenhouse tests to determine the formulation's efficacy in the suppression of Heterodera glycines. Pellet formulation variables included quantity of mycelium per pellet (0.0-3.9%), pellet size (2.3 or 8.3 mg), pellet application rate per unit soil (0.4 or 1.0% based on dry soil weight), and pellet storage (0 or 90 days). All of these variables affected efficacy. Nematode suppression was greatest (95%) with 8.3 mg pellets containing 3.9% mycelium that were not stored and applied at the rate of 1.0% of dry soil weight. Storage for 90 days reduced the efficacy of the pellets. The soybean cultivars tested were not equally good hosts of H. glycines, but reproduction of the nematode was reduced equally on all. The average suppression was 96% (range 86-99%). Similar suppression of reproduction occurred in tests with six races of H. glycines. ARF18 appeared to be nonspecific with regard to soybean cultivar and H. glycines race.

For on-site detection of cysts, a portable cyst extraction kit was constructed from nine readily available items. The portable cyst extractor detected cysts in a range of 1-117 cysts/100 g soil from 42 fields. Samples processed by this kit in fields were clean and easy to examine, possibly because the kit is a compact version of the standard wet-sieving technique used in the laboratory. The portable cyst extractor has several advantages over traditional survey methods: i) diagnosis of cyst infestations in the field accurately and rapidly most of the year and ii) reduction in the labor of samplings and transportation of soil samples.

A facultative fungal parasite, Arkansas Fungus 18 (ARF18), was isolated from at least 10 different sources of Heterodera glycines in different areas in the United States. Techniques used for isolation and in vitro evaluation of fungal parasites of H. glycines were described. Successful isolation of probable egg parasites depends on isolation from individual eggs. Selective isolation and a simple, yet definitive, screening system can increase the possibility of identifying effective biocontrol agents.

Large pot (2 years) and field experiments (1 year) were conducted to determine the response of susceptible soybean Glycine max (L.) Merr. cultivars (Essex and Hutcheson) grown in soybean-cyst-nematode (SCN), Heterodera glycines-infested soils at three soil water regimes. The soil water regimes were irrigation whenever soil water potential ([psi]s) 0.30-m deep was i) -30 kPa (I-30) or ii) - 50 kPa (I-50), and iii) no irrigation. Cyst nematode levels in the pot experiment were either 0 or 20,000 second-stage juveniles (J2) per pot. The field experiment was conducted on soil naturally infested with a population of 145 to 475 cysts L(1) of soil. All growth parameters studied were drastically affected in the presence of SCN under nonirrigated conditions for the large pot tests; however, SCN did not influence growth parameters in the field experiment. Seed yield was lowest in the no irrigation treatment when all treatments were compared in both the pot and field experiments. The infested no irrigation treatment in the pot experiment had the lowest yield among soil water treatments.

Locations of syncytia induced by the soybean cyst nematode (SCN), Heterodera glycines race 3, were compared in roots of 'Essex', a susceptible soybean (Glycine max (L.) Merr.) cultivar, at three soil water regimes. The plants were grown in wet (-5 to -20 kPa), moderately wet (-30 to -50 kPa), and moderately dry (-60 to -80kPa) autoclaved Captina silt loam soil (Typic Fragiudult). In the moderately dry soil, syncytia were found only in the stele, but in moderately wet and wet soils, syncytia occurred primarily in the cortex and occasionally in the stele. The location of syncytia in the cortical tissue of roots growing in wet and moderately wet soils may account for the tolerance of susceptible soybean cultivars grown under well-irrigated conditions where there is less interference with water transport through roots. Cell-wall perforations and dense cytoplasm were characteristic of syncytial cells observed in root tissues of all treatments.

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.

Additional tests of 178 cultivars and lines of soybean against soybean cyst nematode are reported. Cultivars are available with some level of resistance to races 1, 2, 3, 4, 5, 6, 9, and 14, but in some cases the resistance level is moderate. No cultivar or line (except possibly PI 437654) has resistance to all of the races tested. Thirteen cultivars and lines, however, were resistant or moderately resistant to race 1, 14 to race 2, 87 to race 3, 2 to race 4, 3 to race 5, 42 to race 6, 18 to race 9, and 36 to race 14. Some have resistance to more than one race.

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.

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.

The influence of selected plant species on egg hatch and subsequent development of Heterodera glycines race 3 was investigated. Plants tested included four soybean cultivars, red clover, alfalfa, hairy vetch, field corn, sweet corn, cabbage, tobacco, cotton, and wheat. Soybean stimulated egg hatching more than any of the other plant species, with H. glycines-resistant cultivars being more stimulating than susceptible ones. Hairy vetch also increased hatch. Roots of cabbage, red clover, alfalfa, and hairy vetch were readily penetrated by juveniles of H. glycines. Maturation to adult occurred only on soybean and hairy vetch.

A tetraploid single-cyst isolate of Heterodera glycines from a field population from Indiana has been propagated in the greenhouse on Lee soybeans since its discovery, in 1973. The tetraploid isolate has n = 18 chromosomes, compared with n = 9 of the diploid H. glycines; it has larger cysts and larvae, but shows the same level of parasitism and host range as the diploid population from which it apparently evolved. Association of chromosomes is irregular at metaphase I, with quadrivalents, trivalents, and univalents often observed in addition to the bivalents. The second maturation division is usually normal. About 80% of the mature oocytes (just before fertilization) have n = 18, and the other 20% have n = 17 or 19. Reproduction of the tetraploid isolate is exclusively by cross-fertilization. The discovery of such a tetraploid provides an experimental tool for the study of polyploidy in nematodes. Many amphimictic plant-parasitic nematodes are suspected of representing polyploids.

Results of surveys from 1978 to 1986 to estimate and identify nematodes in Arkansas soybean fields are presented. The seven most common nematode species in the fields were Heterodera glycines, Quinisulcius acutus, Pratylenchus scribneri, P. alleni, Helicotylenchus pseudorobustus, Tylenchorhynchus ewingi, and Xiphinema americanum. Other nematodes identified from these fields were Pratylenchus brachyurus, P. vulnus, P. zeae, Tylenchorhynchus canalis, T. goffarti, T. martini, Helicotylenchus dihystera, Scutellonema bradys, Xiphinema chambersi, X. rivesi, Hoplolaimus galeatus, H. magnistylus, Paratrichodorus minor (P. christiei), Paratylenchus projectus, P. tenuicaudatus, Criconemella macrodora, C. ornata, and Meiodorus hollisi.

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.

Thirty-five populations of Heterodera glycines and populations of 15 other Heterodera, Globodera, and Punctodera species were studied morphometrically and some were compared serologically. There was a wide range of each measurement within each nematode population. Except for one soybean cyst nematode population from Indiana, which was a tetraploid and considerably larger than the others, morphometric measurements overlapped. In a discriminant function comparison most of the populations were closely grouped but at least three were rather distinctly separated. Morphometrically H. fici, H. cruciferae, H. schachtii, and H. trifolii were closely associated with H. glycines. Serology indicated a close relationship between H. glycines, H. lespedezae, H. trifolii, H. schachtii, and the Heterodera sp. from Rumex, while H. betulae appeared to be more distantly related.

Restriction fragment patterns of mitochondrial DNA from sibling species of cyst nematodes Heterodera glycines and H. schachtii were examined. Fourteen restriction endonucleases recognizing four, five, and six base-pair sequences yielded a total of 90 scorable fragments of which 10% were shared by both species. Mitochondrial genome sizes for H. glycines and H. schachtii were estimated to be 22.5-23.5 kb and 23.0 kb, respectively. A single wild type mitochondrial genome was identified in all populations of H. glycines examined, although other mitochondrial genomes were present in some populations. The H. schachtii genome exhibited 57 scorable fragments, compared with 33 identified in the H. glycines wild type genome. The estimated nucleotide sequence divergence between the two species was p = 0.145. This estimate suggests these species diverged from a common ancestor 7.3-14.8 million years ago.

Progeny from single females of four known races of Heterodera glycines Ichinohe were used to establish relatively uniform populations. Single females from these populations were mated with males of other races in all possible combinations to study compatibility and inheritance patterns. When race 1 or 3 was crossed with either race 2 or 4, there was a significant reduction in number of females and a greater number of eggless females than in crosses of races 1 x 3 and 2 x 4. More females matured and fewer were eggless when matings were of the same race. Parasitic capabilities of races 2 and 4 were dominant or partially dominant over those of races 1 and 3, based on parasitism of F hybrids. Segregation patterns were generally similar for reciprocal crosses between races. There appeared to be either one or two major genes segregating for parasitism of 'Pickett' soybean in the different crosses. A hybrid isolate (race 3 x 4) that differed in parasitic capability from the four known races produced as many females on the resistant soybean genotype, PI 90,763, as on the susceptible Lee cultivar. Those data indicate that isolates of H. glycines with a different parasitic capability may develop from gene recombination.

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.

Soybean cultivars grown in pots in the greenhouse were tested for resistance by inoculation with Meloidogyne incognita or one of six races of Heterodera glycines. Selected cultivars were tested against each nematode isolate. The numbers of cultivars tested against each H. glycines race and the numbers resistant and (or) moderately resistant were as follows: Race 2 - 114 tested, 1 resistant and 9 moderately resistant; race 3 - 170 tested, 56 resistant and 17 moderately resistant; race 4 - 89 tested, 1 resistant and 13 moderately resistant; race 5 - 106 tested, 4 moderately resistant; race 6 - 95 tested, 10 resistant and 25 moderately resistant; race 14 - 81 tested, 2 resistant and 10 moderately resistant. No cultivar was resistant to all races. Meloidogyne incognita was tested on 139 cultivars; 50 were resistant.

A nematicide, dibromochloropropane; the fungicides benomyl and maneb; an insecticide, oxydisulfoton; the herbicides trifluralin, linuron, and dinoseb; and fertilizers were applied to Heterodera glycines-infested soil. A resistant soybean cultivar alone produced the highest yield in one test, and its yield was not affected by application of pesticides or fertilizer. In two tests the cultivar that was supposed to be resistant was not. Application of nematicide alone resulted in higher yields of the susceptible cultivar, compared with the untreated check, in only one of three tests. Various combinations of pesticides also resulted in higher yields, and in all cases the nematicide was included. Pesticides and fertilizer must be used with discretion on soybean.

Twelve populations of Heterodera glycines from the United States (8), China (2), Japan (1), and Colombia (1) were surveyed for phenotypic intraspecific variability in 42 enzyme systems. Activity of 20 enzymes was detected following isoelectric focusing in polyacrylamide gels of extracts from mass homogenates and single females. Five enzymes, aspartate aminotransferase, phosphoglucose isomerase, alpha- and beta-esterases, and hexokinase were the most useful for detecting intraspecific variability. Phenotypic variability between single females was best demonstrated with alpha- and beta-esterases and acid phosphatase enzyme systems. These results suggest that isoelectric focusing in conjunction with sensitive enzyme systems can be used to detect phenotypic variation between individual nematodes from the same population. The unusual phenotypic variability detected in the H. glycines population from Virginia indicates that the genetic diversity of this population is complex.

Surveys from 1986 to 1988 identified 22 phytoparasitic nematode species in Arkansas cotton fields. Meloidogyne spp. was found in ca. 15% of the fields sampled. Of these samples ca. 33% were found to have a population density of 106/100 cm(3) of soil or more. Rotylenchulus reniformis was found in high numbers (5,000 +/100 cm(3) of soil) in 1% of the fields sampled in 1988. Heterodera glycines was found in ca. 22% of the samples, presumably because of past cropping to soybean. Other common species found were Pratylenchus alleni, P. brachyurus, P. scribneri, Tylenchorhynchus ewingi, T. goffarti, Quinisulcius acutus, Helicotylenchus dihystera, H. pseudorobustus, Hoplolaimus magnistylus, Paratrichodorus minor, and Xiphinema americanum. Paratylenchus spp. juveniles were found in ca. 10% of the samples; adults of P. projectus and P. tenuicaudatus were found in ca. 1% of the samples. Other species found only rarely were Pratylenchus zeae, Merlinius brevidens, T. martini, Helicotylenchus multicinctus, Scutellonema brachyurum, and X. chambersi.

Soybean-cyst nematode (Heterodera glycines) was first reported from Japan in 1915. It has since been reported from Korea (1936), Manchuria (1938), the United States (USA) (1954), and Egypt (1968). It is of major concern to soybean producers only in Japan and the USA. Soybean was a major crop in the Orient by 1915 but it was grown very little elsewhere. Since that time its cultivation has spread, and in 1974 there were 37.6 million ha with a production of 51.7 billion kg. As soybean cultivation has spread, the soybean-cyst nematode has been more widely found. Soybean is one of the major food sources for feeding the increasing world population. Soybean-cyst nematodes have been spreading rapidly in recent years and are a major threat to this very important crop.

In Arkansas wheat is commonly grown either in rotation or double cropped with soybean. Surveys from 1986 to 1988 identified 18 phytoparasitic nematode species in Arkansas wheat fields. The frequency of occurrence, as a percentage of the total number of samples (199), for the following nematodes was Heterodera glycines 43%, Quinisulcius acutus 40%, Pratylenchus scribneri 37%, Helicotylenchus pseudorobustus 15 %, Xiphinema americanum 14%, Paratylenchus tenuicaudatus 13 %, Pratylenchus alleni 10%, Merlinius brevidens 8%, Tylenchorhynchus ewingi 7%, Paratrichodorus minor 5%, T. martini 4%, and Hoplolaimus magnistyIus, Paratylenchus projectus, and juveniles of Meloidogyne spp. 2%. Helicotylenchus dihystera, Paratylenchus elachistus, and Tylenchorhynchus claytoni were each found in one wheat field.

The reproductive abilities of four races of Heterodera glycines were compared on soybean cultivars by using single cyst or mass inoculations. Progeny transfers were used to determine changes in reproduction of H. glycines. Reproduction of all races (1, 2, 3, 4) was best on 'Lee' and poorest on P1 88788. The size of females produced and numbers of eggs/female of the different races varied with the cultivar. Races 1 and 3 appeared to contain low populations of Races 2 or 4. Races 2 and 4 were best selected by a series of transfers on 'Pickett' soybean.

Subcellular responses to infection by Race 3 of Heterodera glycines in susceptible ('Lee') and resistant ('Forrest' and 'Bedford') soybean cultivars were compared. Syncytial formation, initiated in susceptible as well as resistant soybean cultivars, was characterized by wall perforations, dense cytoplasm, and increased endoplasmic reticulum, In susceptible plants, syncytia developed continuously until nematode maturity. This included hypertrophy of nuclei, increase of rough endoplasmic reticulum in early stages of infection, and formation of wall ingrowths at a late stage of infection. In the resistant reaction in Forrest, a necrotic layer surrounded syncytium component cells demarcating them from surrounding normal cells and leading to syncytial necrosis. Wall appositions were prominently formed near the necrotic layer, and the cytoplasm of the syncytium component cells was extremely condensed. The whole syncytium became necrotic at a late stage of infection. Bedford had nuclear degeneration prior to cytoplasmic degradation. Chromatin was often scattered throughout the syncytial cytoplasm. Finally the whole syncytium became degenerated with plasmalemma completely detached from the syncytial cell walls. The differences in resistant responses reflect a difference in genetic composition of the soybean cultivars tested.

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.

A total of 66 plants in 50 species were inoculated with eggs and juveniles of soybean cyst nematode, Heterodera glycines. Roots were stained and observed for penetration and development of the nematode. Twenty-six plants were not penetrated; twenty-three were penetrated, but there was no development of the nematode; eight were penetrated with some nematode development; two were penetrated and had considerable nematode development, but few nematodes, if any, matured; and seven were penetrated with many nematodes maturing. The penetration of nonhosts may imply some susceptibility and that populations eventually would build up on the penetrated plants. Plants not penetrated may be useful as rotation plants because no reproduction would occur.

Heterodera betulae, a cyst-forming nematode, originally recovered from roots of river birch in Arkansas, appears to have a limited host range. Of 80 plant species in 20 families tested, only five species of Betula and Cleome spinosa supported reproduction. The minimum time for a complete life cycle was 52 days at 28 C. No reproduction occurred at 31 C or above, and development was very slow below 20 C. Successful population propagations from single larvae demonstrated that males were not necessary for reproduction.

Amino acids emitted and extracted from surface-sterilized larvae and adults of Heterodera glycines were identified by paper chromatography and quantitatively analyzed by column chromatography. Five amino acids (alanine, aspartic acid, glutamic acid, glycine and serine) were emitted by H. glycines larvae and eight others (asparagine, glutamine, leucine/isoleucine, lysine, methionine sulfoxide, threonine, tyrosine, valine/methionine) were found in extracts from crushed larvae.In addition to the amino acids emitted or extracted from larvae, four others were emitted by adults (gamma-aminobutyric acid, histidine, phenylalanine, and proline). Four different amino acids (arginine, cystathionine, hydroxyproline, and ornithine) were found only in the extract from crushed adults. Greater quantities of alanine, aspartic acid and glycine were emitted than could be detected in nematode extracts suggesting selective emission.Subsamples of nematode populations were taken from growing plants 19, 26, 33, and 40 days after inoculation and extracted to determine whether changes in specific amino acid content correlated with aging. Proline content shifted most, increasing from 4.1% to 21.5% of the total amino acid complement from the 19th to the 40th days.