Nonhost Root Penetration by Soybean Cyst Nematode

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.     

A High-Throughput Automated Technique for Counting Females of Heterodera glycines using a Fluorescence-Based Imaging System

The soybean cyst nematode (SCN), Heterodera glycines, is the most damaging pathogen of soybean. Methods to phenotype soybean varieties for resistance to SCN are currently very laborious and time consuming. Streamlining a portion of this phenotyping process could increase productivity and accuracy. Here we report an automated method to count SCN females using a fluorescence-based imaging system that is well suited to high-throughput SCN phenotyping methods used in greenhouse screening. For optimal automated imaging, females were washed from roots at 30 days post-inoculation into small Petri dishes. Using a Kodak Image Station 4000MM Pro, the Petri dishes were scanned using excitation and emission wavelengths of 470 nm and 535 nm, respectively. Fluorescent images were captured and analyzed with Carestream Molecular Imaging Software for automated counting. We demonstrate that the automated fluorescent-based imaging system is just as accurate (r(2) ≥ 0.95) and more efficient (>50% faster) than manual counting under a microscope. This method can greatly improve the consistency and turnaround of data while reducing the time and labor commitment associated with SCN female counting.     

Attempt to Select a Cyst Nematode Population on Soybean Plant Introduction 437654

A method of selecting soybean cyst nematode (Heterodera glycines Ichinohe) on segregating soybean progeny was evaluated for developing a population capable of reproducing on PI 437654. Direct selection on PI 437654 was not possible, since no cysts developed on it. Cysts were selected for 12 nematode generations on F₃ and F₄ plants of Forrest x PI 437654. No cysts of the selected population were produced on PI 437654, but more males were produced on it by the selected population than by the base population. The number of cysts on Forrest and other soybean lines considered to have some of the same genes for resistance increased with selection as expected. The increase in number of males on these other lines with some of the same genes for resistance as Forrest was greater than anticipated, indicating that these lines may have some of the same genes as PI 437654.     

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.     

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

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

Colonization of Soybean Cyst Nematode Females,Cysts, and Gelatinous Matrices by the Fungus Verticillium lecanii

Heterodera glycines was grown in monoxenic culture on soybean roots and then inoculated with the antagonistic fungus Verticillium lecanii. Use of root explant cultures allowed evaluation of the fungus-nematode interaction with the nematode attached to roots or removed from the host, and avoided contamination with other fungi. From 16 hours to 14 days following inoculation, female and cyst samples were examined with the light microscope, or prepared for either conventional or low-temperature scanning electron microscopy. Within 16 hours, hyphae had begun colonizing the gelatinous matrices (GM). The fungus proliferated in the GM of some specimens within a week, but was rarely seen in unhatched eggs. Fungus penetration holes in female and cyst walls were observed 3 days after inoculation; penetration through nematode orifices was not seen at that time. More cysts than females were colonized at the earliest sampling dates. Specimens associated with external hyphae exhibited variable internal colonization, ranging from no fungal penetration to extensive mycelial growth.     

Variation in Efficacy of Isolates of the Fungus ARF Against the Soybean Cyst Nematode Heterodera glycines

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.     

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.     

Microarray Analysis of Gene Expression in Soybean Roots Susceptible to the Soybean Cyst Nematode Two Days Post Invasion

Soybean root cells undergo dramatic morphological and biochemical changes during the establishment of a feeding site in a compatible interaction with the soybean cyst nematode (SCN). We constructed a cDNA microarray with approximately 1,300 cDNA inserts targeted to identify differentially expressed genes during the compatible interaction of SCN with soybean roots 2 days after infection. Three independent biological replicates were grown and inoculated with SCN, and 2 days later RNA was extracted for hybridization to microarrays and compared to noninoculated controls. Statistical analysis indicated that approximately 8% of the genes monitored were induced and more than 50% of these were genes of unknown function. Notable genes that were more highly expressed 2 days after inoculation with SCN as compared to noninoculated roots included the repetitive proline-rich glycoprotein, the stress-induced gene SAM22, ß-1,3-endoglucanase, peroxidase, and those involved in carbohydrate metabolism, plant defense, and signaling.     

Response of Soybean in Cyst Nematode-Infested Soils at Three Soil Water Regimes

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.     

Use of Remote Sensing to Detect Soybean Cyst Nematode-Induced Plant Stress

Integrating remote sensing and geographic information systems (GIS) technologies offers tremendous opportunities for farmers to more cost effectively manage the causes of crop stress. Initial soybean cyst nematode (SCN) population densities from 995 2-×-3-m quadrats were obtained from a soybean field near Ames, Iowa, in 2000. The percentage of sunlight reflected from each quadrat was measured weekly using a multispectral radiometer beginning in mid-May and continuing through mid-September. Aerial images were obtained at heights above the field ranging from 45 to 425 m on 12 dates during the soybean growing season. This was accomplished using color film and infrared film in conjunction with a filter to measure reflectance in the near-infrared region (810 nm). Satellite images (Landsat 7) were obtained for five dates during the 2000 growing season. Maps depicting initial SCN population densities, soybean yield, soy oil, and soy protein were generated using the GIS software program ArcView. Percentage reflectance (810 nm), aerial image intensity, and satellite image intensity data then were regressed against soybean yield, soy oil, and soy protein concentrations obtained from each geospatially referenced soybean quadrat. Percentage reflectance measurements explained up to 60% of the variation in initial SCN population densities within soybean quadrats and up to 91% of the variation in soybean yield. Aerial image and satellite image intensities explained up to 80% and 47% of the variation in soybean yield, respectively. Percentage reflectance data also explained 36% and 54% of the variation in oil and protein concentrations of the harvested soybeans, respectively. These results indicate that remote sensing coupled with GIS technologies may provide new tools to detect and quantify SCN population densities and their impacts on the quantity and quality of soybean yield.     

Genetic Analysis of Esterase Polymorphism in the Soybean Cyst Nematode, Heterodera glycines

The genetic basis of esterase polymorphism in Heterodera glycines was investigated through controlled matings and analysis of F₁ and F₂ progeny. Three nematode lines, each fixed for a different esterase phenotype, were isolated and purified through repeated directional selection and inbreeding. Each phenotype was characterized by its distinct pair of closely spaced bands of esterase activity. Single-female single-male crosses were conducted according to a modified agar-plate mating technique. F₁ progeny were homogeneous, exhibiting both parental esterase phenotypes (codominant heterozygotes) but no hybrid bands. Approximately 1,500 F₂ progeny segregated in a 1:2:1 ratio for expression of the esterase phenotypes of the female parental line, the heterozygote, and the male parental line. Apparently the three esterase phenotypes correspond to three codominant alleles of a single esterase locus. Reciprocal crosses gave similar results, suggesting no maternal inheritance.     

Esterase Allozymes of Soybean Cyst Nematode,Heterodera glycines,from China,Japan, and the United States

Individual females from 19 populations of Heterodera glycines from China, Japan, and the United States were analyzed for esterase allozyme polymorphism. Eight esterase electrophoretic phenotypes were resolved. Four putative loci, est-1, est-2, est-3, and est-4, were identified, having one, one, two, and one allele, respectively. The four loci expressed six genotypes in the four Chinese populations. Loci est-2, est-3, and est-4 were identified in five Japanese populations and expressed five genotypes, whereas only loci est-2 and est-3 were identified in 10 populations from the United States and expressed four genotypes. Putative alleles at each locus were defined as characters for data analysis. Phylogenetic analysis using parsimony (PAUP) was utilized to determine relationships among the 19 populations. More loci and alleles in populations from China and phylogenetic similarities among populations from Japan and the United States are consistent with a founder effect resulting from dissemination of progenitor H. glycines from China to Japan and subsequent introductions of founder populations from Japan to the United States.     

Control of the Soybean Cyst Nematode by Crop Rotation in Combination with a Nematicide

An experiment to evaluate the control of soybean cyst nematodes compared 1-year, 2-year, and 3-year nonhost rotations with continuous soybeans (Glycine max) in 0.2-ha plots. In a second 1-year rotation, the plots were planted to soybean or corn (Zea mays) after fumigation in the spring with a split application of 1,3-dichloropropene (748.2 liters/ha). The effects of the nematicide were apparent the first year. Soybean yield was 1,482 kg/ha compared to 233 kg/ha in the untreated plots. In the second year, the highest yielding plants (2,035 kg/ha) were those following 1 year of corn that had been treated the previous year; plants in untreated plots yielded 288 kg/ha. Average yield of soybean following 1 year of corn was 957 kg/ha compared to 288 kg/ha for continuous soybean. In the third year, the effects of the nematicide were still evident. Soybean plants in plots treated the first year, followed by corn, then soybean, yielded 1,044 kg/ha compared to 761 kg/ ha for soybean following 1 year of corn and 991 kg/ha for soybean following 2 years of corn. Plots planted to soybean for 3 consecutive years yielded 337 kg/ha. Nematicidal effects were no longer evident during the fourth year. Yields were most improved by the greatest number of years in the nonhost crop; highest yields in descending order were from plants following 3 years of corn, 2 years of corn, and 1 year of corn. Plots planted to soybean for 4 consecutive years yielded 130 kg/ha. Highly significant negative correlations occurred each year between initial nematode population densities and seed yield.     

Coating Soybean Seed with Oxamyl for Control of Heterodera glycines

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

DNA Restriction Fragment Length Polymorphism in Races of the Soybean Cyst Nematode,Heterodera glycines

Restriction endonuclease digests of total DNA from races 3, 4, and 5 of the soybean cyst nematode, Heterodera glycines, have been analyzed on agarose gels. DNA fragment patterns of race 4 were completely different from those patterns obtained for races 3 and 5 by all eight restriction enzymes tested. Differences in long and short restriction DNA fragments generated by the enzyme Msp I or its isoschizomer, Hpa II, were detected between race 3 and 5 digestion profiles. Rapid DNA isolation followed by its digestion with either Msp I or Hpa II enzymes and visualization of repetitive DNA fragments in agarose gels provided a diagnostic assay for the populations of the three races examined in this study.     

Identification of Cyst Nematodes of Agronomic and Regulatory Concern with PCR-RFLP of ITS1

The first internally transcribed spacer region (ITS1) from cyst nematode species (Heteroderidae) was compared by nucleotide sequencing and PCR-RFLP. European, Asian, and North American isolates of five heterodefid species were examined to assess intraspecific variation. PCR-RFLP patterns of amplified ITS1 DNA from pea cyst nematode, Heterodera goettingiana, from Northern Ireland were identical with patterns from Washington State. Sequencing demonstrated that ITS1 heterogeneity existed within individuals and between isolates, but did not result in different restriction patterns. Three Indian and two U.S. isolates of the corn cyst nematode, Heterodera zeae, were compared. Sequencing detected variation among ITS1 clones from the same individual, between individuals, and between isolates. PCR-RFLP detected several restriction site differences between Indian and U.S. isolates. The basis for the restriction site differences between isolates from India and the U.S. appeared to be the result of additional, variant ITS1 regions amplified from the U.S. isolates, which were not found in the three India isolates. PCR-RFLP from individuals of the U.S. isolates created a composite pattern derived from several ITS1 types. A second primer set was specifically designed to permit discrimination between soybean (H. glycines) and sugar beet (H. schachtii) cyst nematodes. Fok I digestion of amplified product from soybean cyst nematode isolates displayed a uniform pattern, readily discernible from the pattern of sugar beet and clover cyst nematode (H. trifolii).     

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.     

Fungi Associated with Females and Cysts of Heterodera glycines in a Florida Soybean Field

Fungal colonization was determined for females and cysts of Heterodera glycines on soybean roots or in rhizosphere soil from a Florida soybean field. A total of 1,620 females and cysts were examined in 1991, and 1,303 were examined in 1992. More than 35 species of fungi were isolated from females and cysts. The frequency of fungi colonizing white and yellow females was low, but a high frequency of fungi was encountered in brown cysts, which increased with time of exposure of the cysts to the soil. No single fungal species predominated in the nematode females or cysts in this field. Rarely was a female or cyst colonized by more than one fungus. The common fungi isolated from the females and cysts were Neocosmospora vasinfecta, Fusarium solani, Fusarium oxysporum, Dictyochaeta coffeae, Dictyochaeta heteroderae, Pyrenochaeta terrestris, Exophiala pisciphila, Gliocladium catenulatum, Stagonospora heteroderae, and a black yeast-like fungus. The communities of common fungal species isolated from cysts in several regions in the southeastern United States appear to be similar.