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Charcoal rot, caused by the soilborne fungus Macrophomina phaseolina (Mp) poses a serious threat to soybean health and harvests at a global scale. Mp exhibits varying distribution patterns across fields, which complicates our ability to predict disease occurrences and outbreaks. Therefore, determining the spatial distribution of Mp abundance and its relation with soil physicochemical properties would help to inform precision management decisions for mitigating charcoal rot. To achieve this, Mp colony forming units (CFU) and edaphic properties were evaluated in 297 soybean fields located in the main soybean growing regions across 7 Departments of Paraguay. A pattern of decreasing CFU density was observed from the south-eastern to the western part of the country. While several edaphic factors are positively correlated with Mp CFU, pH showed a significant negative correlation with CFU. Both spatial and non-spatial model suggest that cation exchange capacity, percentage of clay, and pH could be potential predictors of Mp CFU abundance. Including spatial dependence of edaphic factors improved the prediction of Mp CFU more effectively than classical statistical models. We demonstrated that the occurrence of Mp shows a significant spatial clustering pattern as indicated by Moran's I. Our findings will help growers and policy-makers make informed decisions for managing Mp by improving our ability to predict which agricultural fields and soils are at greatest risk for charcoal rot.

Heterodera glycines , the soybean cyst nematode (SCN), and fungal pathogen Macrophomina phaseolina are economically important soybean pathogens that may coinfest fields. Resistance remains the most effective management tactic for SCN, and the rhg1-b resistance allele derived from plant introduction 88788 is most commonly deployed in the northern United States. The concomitant effects of SCN and M. phaseolina on soybean performance, as well as the effect of the rhg1-b allele in two different genetic backgrounds, were evaluated in three environments (during 2013 to 2015) and a greenhouse bioassay. Within two soybean populations, half of the lines had the rhg1-b allele, and the other half had the susceptible allele in the backgrounds of the cultivars IA3023 and LD00-3309. Significant interactions between soybean rhg1-b allele and M. phaseolina -infested plots were observed in 2014. In all experiments, initial SCN populations (Pi) and M. phaseolina in roots were associated with reduced soybean yield. SCN reproduction factor (RF = final population/Pi) was affected by SCN Pi, rhg1-b , and genetic background. A background-by-genotype interaction on yield was observed only in 2015, with a stronger rhg1-b effect in the LD00-3309 background, which suggested that the susceptible parent 'IA3023' is tolerant to SCN. SCN female index from greenhouse experiments was compared with field RF, and Lin's concordance and Pearson's correlation coefficients decreased with increasing field SCN Pi in soil. In this study, both SCN and M. phaseolina reduced soybean yield asymptomatically, and the impact of SCN rhg1-b resistance was dependent on SCN virulence but also population density.

The effects of sampling depth and crop growth stage on the population density of lesion nematodes were investigated in three commercial fields in Wayne and Fulton Counties, Ohio, during the 2016 and 2017 growing seasons. Soil samples were collected at five growth stages by removing 15 soil cores to a depth of 70 cm from each of 25 plots per field-year. Cores were divided into seven 10-cm sections, and nematodes were extracted from the soil and root fractions of each of them. Pratylenchus crenatus and P. thornei were detected in approximately 84 and 78% of the samples collected in Wayne and Fulton Counties, respectively. Depth significantly affected total population density of both species as well as densities in the soil and root factions in all field-years, but the effects of growth stage and its interaction with depth varied with field-year. In most cases, mean population densities were higher from 10 to 40 cm soil depth than at the reference 40 to 50 cm depth and lower from 50 to 70 cm. There were quadratic relationships between population density (on the log link scale) and depth, with the highest peaks in estimated predicted densities generally occurring between 20 and 40 cm, depending on crop growth stage and growing conditions. These findings suggest that the standard practice of sampling between growth stages V3 and V6 to a depth of 45 to 50 cm and using the entire core for extraction and enumeration could lead to underestimation of population densities of P. crenatus and P. thornei .

Accessory genes are variably present among members of a species and are a reservoir of adaptive functions. In bacteria, differences in gene distributions among individuals largely result from mobile elements that acquire and disperse accessory genes as cargo. In contrast, the impact of cargo-carrying elements on eukaryotic evolution remains largely unknown. Here, we show that variation in genome content within multiple fungal species is facilitated by Starships, a newly discovered group of massive mobile elements that are 110 kb long on average, share conserved components, and carry diverse arrays of accessory genes. We identified hundreds of Starship-like regions across every major class of filamentous Ascomycetes, including 28 distinct Starships that range from 27 to 393 kb and last shared a common ancestor ca. 400 Ma. Using new long-read assemblies of the plant pathogen Macrophomina phaseolina, we characterize four additional Starships whose activities contribute to standing variation in genome structure and content. One of these elements, Voyager, inserts into 5S rDNA and contains a candidate virulence factor whose increasing copy number has contrasting associations with pathogenic and saprophytic growth, suggesting Voyager's activity underlies an ecological trade-off. We propose that Starships are eukaryotic analogs of bacterial integrative and conjugative elements based on parallels between their conserved components and may therefore represent the first dedicated agents of active gene transfer in eukaryotes. Our results suggest that Starships have shaped the content and structure of fungal genomes for millions of years and reveal a new concerted route for evolution throughout an entire eukaryotic phylum., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Heterodera glycines , the soybean cyst nematode, and Macrophomina phaseolina , causal agent of charcoal rot, are economically important soybean pathogens. The impact and effect of these pathogens on soybean yield in coinfested fields in the Midwest production region is not known. Both pathogens are soilborne, with spatially aggregated distribution and effects. Spatial regression analysis, therefore, is an appropriate method to account for the spatial dependency in either the dependent variable or regression error term from data produced in fields naturally infested with H. glycines and M. phaseolina . The objectives of this study were twofold: to evaluate the combined effect of H. glycines and M. phaseolina on soybean yield in naturally infested commercial fields with ordinary least squares and spatial regression models; and to evaluate, under environmentally controlled conditions, the combined effect of H. glycines and M. phaseolina through nematode reproduction and plant tissue fungal colonization. Six trials were conducted in fields naturally infested with H. glycines and M. phaseolina in Ohio. Systematic-grid sampling was used to determine the population densities of H. glycines and M. phaseolina , and soybean yield estimates. Though not used in any statistical analysis, M. phaseolina colony forming units from plant tissue, charcoal rot severity, and H. glycines type were also recorded and summarized. In two greenhouse experiments, treatments consisted of H. glycines alone, M. phaseolina alone, and coinfestation of soybean with both pathogens. Moran's I test indicated that the yield from five fields was spatially correlated ( P < 0.05) and aggregated. In these fields, to account for spatial dependence, spatial regression models were fitted to the data. Spatial regression analyses revealed a significant interaction effect between H. glycines and M. phaseolina on soybean yield for fields with high initial population densities of both pathogens. In the greenhouse experiments, H. glycines reproduction was significantly ( P < 0.05) reduced in the presence of M. phaseolina ; however, soybean tissue fungal colonization was not affected by the presence of H. glycines . The direct mechanisms by which H. glycines and M. phaseolina interact were not demonstrated in this study. Future studies must be conducted in the field and greenhouse to better understand this interaction effect.

Soil samples were collected from 425 corn fields in 28 Ohio counties between growth stages V3 and V6 during the 2013 and 2014 growing seasons. Ten morphological groups of plant-parasitic nematodes, namely spiral, lesion, lance, dagger, stunt, pin, ring, stubby-root, cyst, and "tylenchids" (several genera morphologically similar to members of the subfamily Tylenchinae [NCBI Taxonomy] including Cephelenchus, Filenchus, Malenchus, and Tylenchus) were identified. Eight species belonging to six of these groups were characterized. Spiral, tylenchids, lesion, pin, lance, stunt, and dagger nematodes were detected in 94, 96, 80, 57, 48, 48, and 37% of the fields, respectively, whereas the stubby-root, cyst, and ring nematodes were present in fewer than 14% of the samples. Averaged across fields, the spiral, tylenchids, and pin nematodes had the highest mean population densities. For all groups, incidence and population density varied among counties, and in some cases, among soil regions and cropping practices. Both population parameters were heterogeneous at multiple spatial scales, with the lowest heterogeneity among soil regions and the highest among fields within county and soil region. Estimated variances at the soil region level were not significantly different from zero for most of the nematodes evaluated. Stunt and lance were two of the most variable groups at all tested spatial scales. In general, the population densities were significantly more heterogeneous at the field level than at the county level. Findings from this study will be useful for developing sampling protocols and establishing on-farm trials to estimate losses and evaluate nematode management strategies.

Ten morphological groups of plant-parasitic nematodes (spiral, lesion, lance, dagger, stunt, pin, ring, stubby-root, cyst, and miscellaneous tylenchids) were detected in corn fields in Ohio, but the presence and population density of these groups varied among fields. Binary and ordinal logistic regression models were fitted to the data to estimate the odds of each group being present, and the lesion, lance, spiral, and pin nematode population densities being at moderate-high risk levels based on soil region, cropping sequence, tillage, and soil pH, silt content, and electrical conductivity. All covariates were associated with at least one nematode group, but soil region had the greatest and most consistent effect. Dagger and ring nematodes were more likely to be present in region 6 than in any of the other regions, whereas lance, stunt, pin, stubby-root, and spiral nematodes were more likely to be present in regions 1 to 5 than 6. Spiral, lance, and pin nematode population densities were more likely to be at moderate-high risk levels in regions 3 and 4 than in region 6. Fields under conservation tillage were two times more likely to have moderate-high risk lance nematode population densities than fields under conventional tillage. Similarly, pin nematode population densities were two times more likely to be at moderate-high risk levels in fields under rotation than in continuous corn. For every unit increase in soil pH, the odds of the spiral nematode population density being at moderate-high risk levels increased by 43%, but the odds of the lesion and pin nematode population densities being at the same risk level decreased by 63 and 29%, respectively. The predicted probability of lesion and lance population densities being at moderate-high risk levels decreased as the silt content of the soil increased. These finding will be useful for developing future nematode sampling protocols and for assessing the risk associated with nematodes in corn fields in Ohio.

Stubby-root nematodes (family Trichoridae) are an economically important group of ectoparasites that feed on roots, vector tobraviruses, and cause substantial crop loss (1,2,3). In June 2013, 48 soil samples were submitted to the Nematology Laboratory at Ohio State University for nematode analysis from a field planted to corn in Wood County, Ohio. The soil texture was sandy and the field was previously planted to wheat and soybean in 2011 and 2012, respectively. Nematodes were extracted from 100 cm 3 soil by decanting and sieving followed by sucrose centrifugal flotation. Phytoparasitic nematodes were identified and counted based on morphological traits to genus at 40× to 63× magnification. Nematode genera parasitic to corn recovered from these samples included Helicotylenchus, Hoplolaimus, Paratrichodorus, Pratylenchus, and Tylenchorhynchus. Stubby-root nematodes (Paratrichodorus sp.) were detected in more than 60% of the samples with a maximum count of 52 per 100 cm 3 soil. Individual stubby-root nematodes were hand-picked and identified to species under a compound light microscope as Paratrichodorus allius (Jensen, 1963) Siddiqi, 1974 according to morphological and morphometric characteristics (1). Females (n = 14) were observed with the intestine not anteriorly overlapping the esophagus, posterior subventral esophageal glands overlapping the intestine, caudal pores, absence of spermatheca, and vaginal sclerotization reduced in lateral view. Body length ranged from 475.8 to 840.5 μm (mean = 652.2 μm), and onchiostylet length ranged from 37.7 to 47.4 μm (mean = 42.9 μm). DNA was extracted from single adult females (n = 4) and the 18S rRNA region was amplified with 18S (TTGATTACGTCCCTGCCCTTT) and 26S (TTTCACTCGCCGTTACTAAGG) primers (4). PCR products were purified and sequenced. The sequence was deposited in GenBank (Accession No. KF887974) and was compared with previously deposited sequences by means of BLAST search. The comparison revealed a sequence similarity of 98 to 99% with both P. allius and P. teres (AM269895, AM087124, AJ439572, FJ040484, AJ439575, and AM087125). P. allius and P. teres can be difficult to discriminate using both morphological characteristics and molecular sequencing (3). Therefore, a universal primer (BL18: 5' CCCGTCGMTACTACCGATT 3') and species-specific primers designed to produce PCR products of 432 bp (PAR2: 5'-CCGTTCAAACGCGTATATGATC-3') and 677 bp (PTR4: 5'-CCTGACAAGC'IWGCACTAGC-3') were used for P. allius and P. teres, respectively (3). DNA from individuals used for sequencing was used in PCR reactions with each species-specific primer. DNA samples yielded PCR products of 432 bp with the P. allius-specific primer set and had no reaction with the P. teres-specific primer set. Molecular results and morphological observations confirmed the presence of P. allius in the samples. P. allius is a polyphagous migratory ectoparasite and a vector for Tobacco rattle virus (TRV). The known distribution of P. allius has previously been limited to the Pacific Northwest, where it was originally described as an important pathogen in potato production (2,3). Corn and wheat have been reported as suitable hosts; although they are not susceptible to TRV, crop loss may result from direct damage to roots (2,3). Nematode management recommendations for corn and wheat will depend on the distribution of this nematode. To our knowledge, this is the first report of P. allius in Ohio. References: (1) W. Decreamer. Rev. Nematol. 3:81, 1980. (2) H. Mojtahedi and G. S. Santo. Am. J. Potato Res. 76:273, 1999. (3) E. Riga et al. Am. J. Potato Res. 84:2, 2007. (4) T. C. Vrain et al. Fundam. Appl. Nematol. 15:563, 1992.

Heterodera glycines, the soybean cyst nematode, is a major pathogen of soybean. Effective management of this pathogen is contingent on the use of resistant cultivars; thus, screening for resistant cultivars is essential. The purpose of this research was to develop a method to assess infection of soybean roots by H. glycines with real-time quantitative polymerase chain reaction (qPCR). This method will serve as a prelude to differentiation of resistance levels in soybean cultivars. A reproducible inoculation method was developed by means of a sand column to provide active second-stage juveniles (J2). Two-day-old soybean roots were infested with 0 or 1,000 J2/ml distilled water per seedling. Twenty-four hours after infestation, the roots were surface-sterilized and genomic DNA (gDNA) was extracted. For the qPCR assay, a primer pair for the single copy gene HgSNO, which codes for a protein involved in the production of vitamin B6, was selected for H. glycines gDNA amplification within soybean roots. Compatible 'Lee 74', incompatible 'Peking', and cultivars with different levels of resistance to H. glycines were infested with 0 or 1,000 J2/ml distilled water per seedling. Twenty-four hours postinfestation, infected seedlings were transplanted into pasteurized soil. Subsequently, they were harvested at 1, 7, 10, 14, and 21 days postinfestation for gDNA extraction. With the qPCR assay, the time needed to differentiate highly resistant cultivars from the rest was reduced. Quantification of H. glycines infection by traditional means (numbers of females produced in 30 days) is a time-consuming practice. This qPCR assay has the potential to replace the traditional Female Index-based screening and improve precision in determining infection levels.

Boxwood (Buxus sempervirens L. and other species) is a popular evergreen shrub used in landscaping. In January 2012, three nursery-grown plants of cv. Green Gem boxwood were submitted from Warren County, Ohio to the C. Wayne Ellet Plant and Pest Diagnostic Clinic at The Ohio State University, an Ohio Plant Diagnostic Network laboratory. The plants, established for 4 years, exhibited orange to bronze discoloration of the foliage; foliage was not desiccated and dieback was not evident although stunting was present. Plant root symptoms ranged from nearly complete necrosis to distinct black lesions on living roots. A root scraping showed nematodes present in the lesions. Nematodes were extracted from root and soil subsamples with a Baermann funnel apparatus for 48 h (3). A high number of lesion nematodes (Pratylenchus sp.) were observed from both soil and root samples. Individual nematodes were handpicked and identified under a compound light microscope as Pratylenchus vulnus Allen & Jensen, 1951 according to morphologic and morphometric characteristics (2). Males and females were observed with stylets having rounded knobs, labial regions continuous with the body contour, and three to four lip annuli. The lateral field contained four incisures, with the two inner incisures closer to each other than to the outer ones. The esophagus overlapped the intestine ventrally. Female (n = 12) body length ranged from 410.3 to 654.5 μm (mean 583.0 μm), stylet length from 15.0 to 17.8 μm (mean 16.8 μm), tail length from 23.2 to 37.5 μm (mean 29.2 μm), vulva position from 78.9 to 85.6% (mean 81.7%), dorsal esophageal outlet (DGO) from 2.6 to 3.5 μm (mean 3.1 μm), and with functional oblong spermathecae. De Man ratios were as follows: a = 25.3 to 33.3 (mean 28.4), b = 4.1 to 7.6 (mean 6.0), c = 16.1 to 23.5 (mean 20.1), and c' = 1.8 to 2.6 (mean 2.1). Male (n = 16) body length ranged from 478.0 to 589.0 μm (mean 537.9 μm), stylet length from 15.0 to 17.2 μm (mean 16.2 μm), tail length from 22.7 to 28.1 μm (mean 25.5 μm), spicule from 15.0 to 17.5 μm (mean 16.4 μm), gubernaculum from 3.5 to 4.7 μm (mean 4.0 μm), and DGO from 2.6 to 3.7 μm (mean 3.1 μm). De Man ratios were as follows: a = 26.4 to 36.3 (mean 30.5), b = 5.0 to 7.9 (mean 5.8), c = 19.1 to 23.0 (mean 21.1), and c' = 1.6 to 2.4 (mean 2.0). DNA was extracted from single adult females and the D2-D3 expansion region of the 28S rRNA gene was amplified using forward primer ACAAGTACCGTGAGGGAAAGTTG and reverse primer TCGGAAGGAACCAGCTACTA (4). The PCR product was purified and sequenced. The sequence was deposited in GenBank (Accession No. JQ692308) and was compared with sequences previously deposited in GenBank by means of BLAST search. The comparison revealed a sequence similarity of 98 to 99% with P. vulnus (e.g., GenBank Accession Nos. HM469437.1, EU130886.1, and JQ003994.1). P. vulnus is a known pathogen of boxwood (1). To our knowledge, this is the first report of P. vulnus in Ohio. References: (1) K. R. Barker. Plant Dis. Rep. 58:991, 1974. (2) P. Castillo and N. Vovlas. Pratylenchus (Nematoda: Pratylenchidae): Diagnosis, Biology, Pathogenicity and Management. Koninklijke Brill NV, Leiden, the Netherlands, 2007. (3) D. J. Hooper. In: Laboratory Methods for Work with Plant and Soil Nematodes. J. F. Southey, ed. Reference book 402. Ministry of Agriculture, Fisheries and Food, London, 1986. (4) G. C. Tenente et al. Nematropica 34:1, 2004.

Sugar beet (Beta vulgaris L.) and canola (Brassica napus L.) are major crops in North Dakota, with sugar beet production primarily in the eastern part of the state in the Red River Valley and canola production across the northern half of the state. Both crops are hosts of sugar beet cyst nematode (SBCN), Heterodera schachtii Schmidt. In April 2011, soil samples were collected from four sugar beet fields belonging to three growers who believed the fields were infested with SBCN. The fields were located in a 65-km 2 area in the Yellowstone Valley of western North Dakota. Cysts were extracted by sieving and Heterodera-like cysts with eggs were observed in all four soil samples. Population densities in the four fields ranged from 100 to 1,750 eggs/100 cm 3 soil. Sugar beet seedlings (cv. M832224) were grown in a potting mix for 6 weeks in the greenhouse and then transferred to conetainers (type D40; volume 656 ml) containing autoclaved river sand. Conetainers were placed in sand in plastic pots immersed in a water bath at 27°C. Three plants were each infested with 800 eggs from field No. 2. After 55 days of incubation, the average number of females was 115 per plant. A similar experiment was conducted with canola cvs. Hyclass 940, Caliber 30, and Westar, which were inoculated with 500 eggs each from field No. 2. After 53 days of incubation, there was an average of 39, 20, and 30 females for each respective cultivar. Flask-shaped cysts (n = 26) from canola roots were light to dark brown; the vulval cone was ambifinestrate with dark brown, molar-shaped bullae positioned underneath the vulval bridge. Body length (excluding neck) ranged from 600 to 850 μm (mean 701.2 μm); body width, 350 to 580 μm (mean 469.2 μm); and length/width ratio, 1.2 to 1.8 (mean 1.5). Second-stage juvenile (J2) (n = 21) body length ranged from 400 to 485 μm (mean 437.1 μm); stylet length was 25 μm (no variation) with forwardly directed knobs; conical tail with rounded tip ranged from 37.5 to 55.0 μm long (mean 46.6 μm) with hyaline region from 20.0 to 32.5 μm (mean 27.3 μm); and lateral field presented four incisures. These morphometrics were used to identify H. schachtii according to Subbotin et al. (4). Confirmation of identification was by amplification and sequencing of a 28S rDNA gene fragment (1) from individual females (GenBank Accession No. JQ040526), which was 100% identical to H. schachtii 28S rDNA sequence (GenBank Accession No. GU475088). To our knowledge, this is the first confirmed report of H. schachtii in North Dakota. A 1958 report of SBCN in North Dakota (2) was not subsequently confirmed (3). Because there is extensive canola production across the northern part of the state bordering western and eastern sugar beet- production areas, canola may serve as a bridge for movement of SBCN from west to east. SBCN is a potential threat to these two important crops. References: (1) A. Amiri et al. Eur. J. Plant Pathol. 108:497, 2002. (2) F. Caveness. J. Sugar Beet Res. 10:544, 1958. (3) P. Donald and R. Hosford. Plant Dis. 64:45, 1980. (4) S. A. Subbotin et al. Systematics of Cyst Nematodes (Nematoda: Heteroderinae). Nematology Monographs and Perspectives. Vol. 8B. Brill, The Netherlands. 2010.

Nematodes are the most abundant multicellular animals on earth, yet little is known about their natural viral pathogens. To date, only two nematode virus genomes have been reported. Consequently, nematode viruses have been overlooked as important biotic factors in the study of nematode ecology. Here, we show that one plant parasitic nematode species, Heterodera glycines, the soybean cyst nematode (SCN), harbours four different RNA viruses. The nematode virus genomes were discovered in the SCN transcriptome after high-throughput sequencing and assembly. All four viruses have negative-sense RNA genomes, and are distantly related to nyaviruses and bornaviruses, rhabdoviruses, bunyaviruses and tenuiviruses. Some members of these families replicate in and are vectored by insects, and can cause significant diseases in animals and plants. The novel viral sequences were detected in both eggs and the second juvenile stage of SCN, suggesting that these viruses are transmitted vertically. While there was no evidence of integration of viral sequences into the nematode genome, we indeed detected transcripts from these viruses by using quantitative PCR. These data are the first finding of virus genomes in parasitic nematodes. This discovery highlights the need for further exploration for nematode viruses in all tropic groups of these diverse and abundant animals, to determine how the presence of these viruses affects the fitness of the nematode, strategies of viral transmission and mechanisms of viral pathogenesis.

A survey of Miscanthus × giganteus and switchgrass plots throughout the midwestern and southeastern United States was conducted to determine the occurrence and distribution of plant-parasitic nematodes associated with these biofuel crops. During 2008, rhizosphere soil samples were collected from 24 Miscanthus × giganteus and 38 switchgrass plots in South Dakota, Iowa, and Illinois. Additional samples were collected from 11 Miscanthus × giganteus and 10 switchgrass plots in Illinois, Kentucky, Georgia, and Tennessee the following year. The 11 dominant genera recovered from the samples were Pratylenchus, Helicotylenchus, Xiphinema, Longidorus, Heterodera, Hoplolaimus, Tylenchorhynchus, Criconemella, Paratrichodorus, Hemicriconemoides, and Paratylenchus. Populations of Helicotylenchus, Xiphinema, and Pratylenchus were common and recorded in 90.5, 83.8, and 91.9% of the soil samples from Miscanthus × giganteus, respectively, and in 91.6, 75, and 83.3% of the soil samples from switchgrass, respectively. Prominence value (PV) (PV = population density × √frequency of occurrence/10) was calculated for the nematodes identified. Helicotylenchus had the highest PV (PV = 384) and was followed by Xiphinema (PV = 152) and Pratylenchus (PV = 72). Several of the nematode species associated with the two biofuels crops were plant parasites. Of these, Pratylenchus penetrans, P. scribneri, P. crenatus, Helicotylenchus pseudorobustus, Hoplolaimus galeatus, X. americanum, and X. rivesi are potentially the most damaging pests to Miscanthus × giganteus and switchgrass. Due to a lack of information, the damaging population thresholds of plant-parasitic nematodes to Miscanthus × giganteus and switchgrass are currently unknown. However, damage threshold value ranges have been reported for other monocotyledon hosts. If these damage threshold value ranges are any indication of the population densities required to impact Miscanthus × giganteus and switchgrass, then every state surveyed has potential for yield losses due to plant-parasitic nematodes. Specifically, Helicotylenchus, Xiphinema, Pratylenchus, Hoplolaimus, Tylenchorhynchus, Criconemella, and Longidorus spp. were all found to have population densities within or above the threshold value ranges reported for other monocotyledon hosts.

Heterodera glycines, the soybean cyst nematode (SCN), is a damaging agricultural pest that could be effectively managed if critical phenotypes, such as virulence and host range could be understood. While SCN is amenable to genetic analysis, lack of DNA sequence data prevents the use of such methods to study this pathogen. Fortunately, new methods of DNA sequencing that produced large amounts of data and permit whole genome comparative analyses have become available. In this study, 400 million bases of genomic DNA sequence were collected from two inbred biotypes of SCN using 454 micro-bead DNA sequencing. Comparisons to a BAC, sequenced by Sanger sequencing, showed that the micro-bead sequences could identify low and high copy number regions within the BAC. Potential single nucleotide polymorphisms (SNPs) between the two SCN biotypes were identified by comparing the two sets of sequences. Selected resequencing revealed that up to 84% of the SNPs were correct. We conclude that the quality of the micro-bead sequence data was sufficient for de novo SNP identification and should be applicable to organisms with similar genome sizes and complexities. The SNPs identified will be an important starting point in associating phenotypes with specific regions of the SCN genome.

A lack of diversity and durability of resistant soybean varieties complicates management of the soybean cyst nematode (SCN), Heterodera glycines, exemplified by the current overdependence on the PI 88788 source of resistance. Of interest is the effect of adaptation of a SCN population to a source of resistance on its subsequent ability to develop on others. Female indices (FI) from virulence assays (race, HG Type and SCN Type tests) for SCN field populations and inbred lines were analyzed. Female indices on PI 88788, PI 209332 and PI 548316 were highly correlated, as were those of PI 548402, PI 90763, PI 89772 and PI 438489B. Previous studies on resistant SCN-infected soybean roots indicated that the cellular resistance response was similar within these two groups of soybean genotypes. In field populations, highly significant correlations were also found between FI on PI 88788 and PI 548402 and those on PI 89772 and PI 437654. In inbred lines, FI on PI 437654 were correlated with PI 90763 and PI 438489B. To avoid further adaptation, rotation of cultivars with resistance from these groups should be carefully monitored, including those from the most promising source of resistance, PI 437654, such as CystX. In a separate test, 10 soybean varieties developed from CystX were tested against HG Type 0, HG Type 2.5.7 and HG Type 1-7. Female development occurred in all tests but one. Although identification and deployment of unique resistance is needed, management strategies to prevent and detect adaptation should be emphasized.

ABSTRACT The soybean cyst nematode, Heterodera glycines, and the fungus that causes sudden death syndrome (SDS) of soybean, Fusarium solani f. sp. glycines, frequently co-infest soybean (Glycine max) fields. The interactions between H. glycines and F. solani f. sp. glycines were investigated in factorial greenhouse experiments with different inoculum levels of both organisms on a soybean cultivar susceptible to both pathogens. Measured responses included root and shoot dry weights, H. glycines reproduction, area under the SDS disease progress curve, and fungal colonization of roots. Both H. glycines and F. solani f. sp. glycines reduced the growth of soybeans. Reproduction of H. glycines was suppressed by high inoculum levels but not by low levels of F. solani f. sp. glycines. The infection of soybean roots by H. glycines did not affect root colonization by the fungus, as determined by real-time polymerase chain reaction. Although both pathogens reduced the growth of soybeans, H. glycines did not increase SDS foliar symptoms, and statistical interactions between the two pathogens were seldom significant.

The soybean cyst nematode Heterodera glycines (SCN) is of major economic importance and widely distributed throughout soybean production regions of the United States where different maturity groups with the same sources of SCN resistance are grown. The objective of this study was to assess SCN-resistant and -susceptible soybean yield responses in infested soils across the north-central region. In 1994 and 1995, eight SCN-resistant and eight SCN-susceptible public soybean cultivars representing maturity groups (MG) I to IV were planted in 63 fields, either infested or noninfested, in 10 states in the north-central United States. Soil samples were taken to determine initial SCN population density and race, and soil classification. Data were grouped for analysis by adaptation based on MG zones. Soybean yields were 658 to 3,840 kg/ha across the sites. Soybean cyst nematode-resistant cultivars yielded better at SCN-infested sites but lost this superiority to susceptible soybean cultivars at noninfested sites. Interactions were observed among initial SCN population density, cultivar, and location. This study showed that no region-wide predictive equations could be developed for yield loss based on initial nematode populations in the soil and that yield loss due to SCN in our region was greatly confounded by other stress factors, which included temperature and moisture extremes.

The soybean cyst nematode, Heterodera glycines, adversely affects the production of soybean, Glycine max, in many areas of the world, particularly in the United States, where it is the most economically important soybean pathogen. Despite the availability of hundreds of H. glycines-resistant soybean cultivars, the nematode continues to be a major limiting factor in soybean production. The use of nonhost rotation and resistance are the primary means of reducing losses caused by the nematode, but each of these options has disadvantages. As a subject for study of nematode parasitism and virulence, H. glycines provides a useful model despite its obligately parasitic nature. Its obligately sexual reproduction and ready adaptation to resistant cultivars, formerly referred to as "race shift," presents an excellent opportunity for the study of virulence in nematodes. Recent advances in H. glycines genomics have helped identify putative nematode parasitism genes, which, in turn, will aid in the understanding of nematode pathogenicity and virulence and may provide new targets for engineering nematode resistance.

The most effective management program for soybean cyst nematode, Heterodera glycines, is a crop rotation that uses nonhost crops and resistant soybean cultivars. However, little is known about the effects of rotation crops and overwintering on H. glycines biology. These experiments were initiated to determine the effects of seven alternative crops on H. glycines' ability to infect and mature on subsequent soybean crops, and to assess the viability of eggs during the overwintering months. Rotation studies were conducted for 2 years in each of two naturally infested fields, and overwintering tests were conducted in three consecutive growing seasons in one naturally infested field. Rotation crop and fallow treatments did not have a consistent effect on the ability of H. glycines to infect soybean or mature. Soybean yields were often higher following fallow or a nonhost crop than following soybean, although not usually significantly so. Heterodera glycines egg viability did not differ (P < 0.05) between overwintering months at 0-to-10 or 10-to-20-cm soil depths. These results suggest that H. glycines' ability to infect a subsequent soybean crop and develop to maturity is not diminished by nonhost crops or during the winter months.

The soybean cyst nematode Heterodera glycines is the most destructive pathogen of soybean in the Unites States. Diversity in the parasitic ability of the nematode allows it to reproduce on nematode-resistant soybean. H. glycines chorismate mutase-1 (Hg-CM-1) is a nematode enzyme with the potential to suppress host plant defense compounds; therefore, it has the potential to enhance the parasitic ability of nematodes expressing the gene. Hg-cm-1 is a member of a gene family where two alleles, Hg-cm-1A and Hg-cm-1B, have been identified. Analysis of the Hg-cm-1 gene copy number revealed that there are multiple copies of Hg-cm-1 alleles in the H. glycines genome. H. glycines inbred lines were crossed to ultimately generate three F2 populations of second-stage juveniles (J2s) segregating for Hg-cm-1A and Hg-cm-1B. Segregation of Hg-cm-1A and 1B approximated a 1:2:1 ratio, which suggested that Hg-cm-1 is organized in a cluster of genes that segregate roughly as a single locus. The F2 H. glycines J2 populations were used to infect nematode-resistant (Hartwig, PI88788, and PI90763) and susceptible (Lee 74) soybean plants. H. glycines grown on Hartwig, Lee 74, and PI90763 showed allelic frequencies similar to Hg-cm-1A/B, but nematodes grown on PI88788 contained predominately Hg-cm-1A allele as a result of a statistically significant drop of Hg-cm-1B in the population. This result suggests that specific Hg-cm-1 alleles, or a closely linked gene, may aid H. glycines in adapting to particular soybean hosts.

To determine whether currently used sources of resistance (soybean Plant Introductions [PI] 548402, 88788, 90763, 437654, 209332, 89772, and 548316) influence sex ratios in H. glycines, four inbred lines of the nematode characterized by zero or high numbers of females on resistant soybean were used to observe the number of adult males produced. Nematodes were allowed to infect soybean roots for 5 days in pasteurized sand. Infected plants were washed and transferred to hydroponic culture tubes. Males were collected every 2 to 3 days up to 30 days after infestation (DAI), and females were collected at 30 DAI. Resistance that suppressed adult females also altered adult male numbers. On PI 548402, 90763, and 437654, male numbers were low and close to zero, whereas on PI 88788, male numbers were higher (alpha = 0.05). In a separate experiment, the same PIs were infected by an inbred line that tested as an HG Type 0 (i.e., the numbers of females that developed on each PI were less than 10% of the number that developed on the standard susceptible soybean cultivar Lee). In this experiment, male numbers were similar to female numbers on PI 548402, 90763, 437654, and 89772, whereas male numbers on PI 88788, 209332, and 548316 were higher than those of females (alpha = 0.05). In all experiments, the total number of adults that developed to maturity relative to the number of second-stage juveniles that initially penetrated the root was less on resistant than on susceptible soybean (P

A genetic linkage map of the soybean cyst nematode (SCN) Heterodera glycines was constructed using a population of F2 individuals obtained from matings between two highly inbred SCN lines, TN16 and TN20. The AFLP fingerprinting technique was used to genotype 63 F2 progeny with two restriction enzyme combinations (EcoRI/MseI and PstI/TaqI) and 38 primer combinations. The same F2 population was also genotyped for Hg-cm-1 (H. glycines chorismate mutase-1), a putative virulence gene, using real-time quantitative PCR. Some of the markers were found to be distributed non-randomly. Even so, of the 230 markers analyzed, 131 could be mapped onto ten linkage groups at a minimum LOD of 3.0, for a total map distance of 539 cM. The Hg-cm-1 locus mapped to linkage group III together with 16 other markers. The size of the H. glycines genome was estimated to be in the range of 630-743 cM, indicating that the current map represents 73-86% of the genome, with a marker density of one per 4.5 cM, and a physical/genetic distance ratio of between 124 kb/cM and 147 kb/cM. This genetic map will be of great assistance in mapping H. glycines markers to genes of interest, such as nematode virulence genes and genes that control aspects of nematode parasitism.

Fusarium solani f. sp. glycines is the causal organism of soybean sudden death syndrome (SDS). This organism is difficult to detect and quantify because it is a slow-growing fungus with variable phenotypic characteristics. Reliable and fast procedures are important for detection of this soybean pathogen. Protocols were optimized for extraction of DNA from pure fungal cultures and fresh or dry roots. A new procedure to test polymerase chain reaction (PCR) inhibitors in DNA extracts was developed. Novel real-time quantitative PCR (QPCR) assays were developed for both absolute and relative quantification of F. solani f. sp. glycines. The fungus was quantified based on detection of the mitochondrial small-subunit rRNA gene, and the host plant based on detection of the cyclophilin gene of the host plant. DNA of F. solani f. sp. glycines was detected in soybean plants both with and without SDS foliar symptoms to contents as low as 9.0 × 10 -5 ng in the absolute QPCR assays. This is the first report of relative QPCR using the comparative threshold cycle (Ct) method to quantify the DNA of a plant pathogen relative to its host DNA. The relative QPCR assay is reliable if care is taken to avoid reaction inhibition and it may be used to further elucidate the fungus-host interaction in the development of SDS or screen for resistance to the fungus.

The soybean cyst nematode, Heterodera glycines, is the most economically important pathogen of soybean in Missouri. Knowledge of the nematode's distribution and ability to adapt to resistant varieties is important for determining crop losses and establishing research priorities. No previous surveys of Missouri have provided reliable population density and phenotypic diversity data; therefore, we conducted a random survey to obtain both. Two samples from each of 200 fields were collected; 392 samples were processed for extractions of cysts and eggs. Two hundred and forty seven (63%) of the samples had detectable cyst nematode populations, which ranged from 15 to 149,700 eggs per 250 cm 3 of soil. The lowest average population densities were observed in the east-central region of Missouri (2,260 eggs per 250 cm 3 of soil), and the highest were observed in the northeast (9,238 eggs per 250 cm 3 of soil), but among the eight regions sampled, mean population densities did not differ significantly. These population densities were potentially responsible for losses worth over $58 million in 1999 in Missouri. Race tests were conducted on populations from 183 samples. In order of frequency, races 3, 1, and 2 accounted for 86% of H. glycines populations. Nearly 60% of the populations were virulent (able to produce females) on plant introduction (PI) 88788, which is the source of resistance for most H. glycines-resistant cultivars. More than a third of the populations were virulent on cv. Peking, another common resistance source. Very few populations were virulent on PI 90763 or PI 437654, suggesting that these sources of resistance should be exploited more frequently.

Field experiments were conducted at locations in northern and southern Illinois, central Iowa, and central Missouri from 1997 to 1999 to investigate the effects of Heterodera glycines on soybean growth, development, and yield. A wide range of infestation levels was present at all locations. Two locally adapted cultivars, one resistant to H. glycines, were grown at each location. Cultivars were planted in alternating four-row strips with 76 cm between rows. For each cultivar, 20 1-m-long single-row plots were sampled every 2 weeks starting 4 weeks after planting. Infection by H. glycines reduced plant height and leaf and stem weight on the resistant cultivars in the first 12 weeks after planting, and delayed pod and seed development 12 to 14 weeks after planting. Biomass accumulation was not reduced on the susceptible cultivars until 10 weeks after planting; reduction in pod and seed development occurred throughout the reproductive stages. Susceptible cultivars produced significantly lower yields than resistant cultivars, but the yield reductions were not accompanied by visually detectable symptoms.

Parasitism genes from phytoparasitic nematodes are thought to be essential for nematode invasion of the host plant, to help the nematode establish feeding sites, and to aid nematodes in the suppression of host plant defenses. One gene that may play several roles in nematode parasitism is chorismate mutase (CM). This secreted enzyme is produced in the nematode's esophageal glands and appears to function within the plant cell to manipulate the plant's shikimate pathway, which controls plant cell growth, development, structure, and pathogen defense. Using degenerate polymerase chain reaction primers, we amplified and cloned a chorismate mutase (Hg-cm-1) from Heterodera glycines, the soybean cyst nematode (SCN), and showed it had CM activity. RNA in situ hybridization of Hg-cm-1 cDNA to SCN sections confirms that it is specifically expressed in the nematodes' esophageal glands. DNA gel blots of genomic DNA isolated from SCN inbred lines that have differing virulence on SCN resistant soybean show Hg-cm-1 is a member of a polymorphic gene family. Some Hg-cm family members predominate in SCN inbred lines that are virulent on certain SCN resistant soybean cultivars. The same polymorphisms and correlation with virulence are seen in the Hg-cm-1 expressed in the SCN second-stage juveniles. Based on the enzymatic activity of Hg-cm-1 and the observation that different forms of the mutase are expressed in virulent nematodes, we hypothesize that the Hg-cm-1 is a virulence gene, some forms of which allow SCN to parasitize certain resistant soybean plants.

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

Soybean cultivars with different sources of resistance to Heterodera glycines were grown at three locations initially infested with races 2, 3, and 6 in order to investigate H. glycines race shift in field populations. Each spring and fall, soil samples were taken from each plot and race tests were conducted to evaluate effects of cultivar and time of sampling. Field experiments were paired field plots rotated annually with corn since 1991. Cultivars included at the northern and central Missouri sites were Williams 82 (susceptible to H. glycines), Linford (PI 88788 source of resistance), MFA 9043 (Peking) replaced by Morsoy 9345 (Peking and PI 88788) from 1995 to 1997, and Jackson II (Peking + PI 88788) replaced by Asgrow 3431 (Peking and PI 88788) in 1996-97. Cultivars at the southern Missouri site were Essex or Hutcheson (susceptible to H. glycines), Forrest (Peking), Hartwig (PI 437654), and Rhodes (PI 88788 + Peking). In 1995, race tests were performed at four temperature regimes to determine temperature effects on race designations. Race shifts were not predictable based on the source of resistance of the soybean cultivar planted. Variability in female numbers on Lee 74 among tests caused changes in female indices (FI). Furthermore, race designations were influenced by the time of sampling and temperature at which the race tests were conducted. The variability of H. glycines populations in both field and greenhouse situations diminishes the value of race test results when making cultivar recommendations.

ABSTRACT This study explored the possibilities that changes in the egg shell/lipid layer electrical potential or pH communicate external hatching conditions to the Heterodera glycines second-stage juvenile (J2) within the mature egg and that electrophysiology could measure effects of chemicals on emergence. Potentials were measured following application of the emergence inducers (ZnSO(4) and ZnCl(2)), ions that do not affect emergence, or synthetic emergence inhibitors. Results were compared with pH measurements and emergence bioassays. Healthy appearing eggs had negative resting potentials. Application of ZnSO(4) caused a smooth depolarization. However, eggs containing J2 and immature eggs depolarized to a similar degree when ZnSO(4) was added. In addition, ZnSO(4), synthetic emergence inhibitors, and CaCl(2) caused similar depolarization, and some depolarization was measured in dye-permeable eggs and empty shells. Results suggest that change in cation surface charge contributed to depolarization and that Cl penetrated the egg shell/lipid layer without causing potential changes. In bioassays, zinc consistently stimulated emergence to a greater degree than H(2)O, other cations, or buffers, and counteracted emergence inhibitors. Zinc-caused emergence stimulation was independent of pH. In summary, it is concluded that depolarization and pH are not emergence signals and electrophysiology is unlikely to measure effectiveness of emergence stimulators or inhibitors.

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.

An experiment was conducted in Heterodera glycines-infested fields in 40 north central U.S. environments (21 sites in 1994 and 19 sites in 1995) to assess reproduction of this nematode. Two resistant and two susceptible soybean cultivars from each of the maturity groups (MG) I through IV were grown at each site in 6.1 m by 4 row plots. Soil samples were collected from each plot at planting and harvest and processed at Iowa State University to determine H. glycines initial (Pi) and final (Pf) population densities as eggs per 100 cm 3 of soil. Overall, reproduction (Pf/Pi) of H. glycines on susceptible cultivars in all MG was similar. Reproduction was higher on MG III and IV susceptible cultivars than on those in MG I and II. Resistant MG I and II cultivars reduced nematode population densities more consistently than those in MG III and IV. Reproduction of the nematode was similar among sites within the same maturity zone (MZ), defined as the areas of best adaptation of the corresponding MG. Nonetheless, careful monitoring of nematode population densities is necessary to assess changes that occur over time in individual fields.

High-performance liquid chromatography and Sinorhizobium fredii USDA191 nodC-lacZ gene fusion were used to monitor changes in the isoflavonoid content of soybean roots infected with Heterodera glycines isolate TN1. Isoflavonoid concentrations in infected roots of both H. glycines-resistant Hartwig and susceptible Essex soybean were two to four-fold higher than those of uninfected roots 2 and 3 days after inoculation. The isoflavonoids produced activated the transcription of nodC-lacZ fusion. The most abundant isoflavonoids were identified as daidzein and genistein by HPLC and GC/MS. Heterodera glycines increased the number of nodules formed on Essex roots inoculated with B. japonicum (USDA110) but reduced shoot weight and decreased the net nitrogenase activity of the nodules. Heterodera glycines infection of resistant Hartwig did not affect the total number of nodules or their nitrogen- fixing capacity.

Polyclonal antibodies were raised against Heterodera glycines eggshells to determine the feasibility of developing an immunoassay for H. glycines eggs. An indirect enzyme-linked immunosorbent assay (ELISA) was developed from anfisera collected 10 weeks after the initial injection. From serial dilutions of sonicated eggshells or whole eggs, a sensitivity of detection to 5 ng/ml sonicated eggshells or 1 egg of H. glycines was determined. The method of eggshell preparation had no effect on the antibodies produced; however, the antibodies cross-reacted with sonicated J2 of H. glycines and eggs of Meloidogyne incognita and H. schachtii. Most of the proteins in both life stages of H. glycines and eggs of M. incognita and H. schachtii had similar migration properties when separated on SDS-PAGE gels and stained with Coomassie blue. Western blot analysis, with antisera adsorbed with homogenized J2 of H. glycines, showed proteins that were specifically localized to eggshells of H. glycines. Monoclonal antibodies might provide a useful immunoassay where polyclonal antibodies lack sufficient specificity.

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 cm(3) 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 mug/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.

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.

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

Greenhouse tests were conducted to determine the persistence of nonviable Heterodera glycines eggs in soil. Laboratory and greenhouse tests were conducted to determine the usefulness of the vital stains acridine orange and tetrazolium red for differentiating viable and nonviable eggs of H. glycines. Nonviable (boiled) egg preparations were compared with preparations that were not boiled for their persistence in methyl bromide-fumigated soil. Boiled eggs persisted longer (>200 days) than untreated eggs, perhaps due to disinfestation of the egg suspensions by boiling. Neither stain was a good indicator of egg viability as inferred from infectivity of juveniles in a bioassay of the same egg preparations exposed to the stains.

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 degrees 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.

Field plots of tall rescue (Festuca arundinacea) at two locations on the same experimental farm in southwestern Missouri were sampled (one in 1987-88, the other in 1988-89) to inventory root-parasitic nematodes and to determine whether cultivars or endophyte (Acremonium coenophialum) infection frequencies (EIF) affected nematode population densities within single growing seasons. Plots were planted with seven tall rescue cultivars: Kentucky-31, Kenhy, Johnstone, Martin, Mozark, Missouri-96, and Forager. Kentucky-31 seed with high and low EIF were planted in separate plots. Plant-parasitic nematodes were extracted from soil samples, identified to genus, and enumerated four and three times per year for the 1987-1988 and 1988-1989 studies, respectively. Several plant-parasitic genera were identified from both fields, including Helicotylenchus, Heterodera, Hoplolaimus, Paratylenchus, Pratylenchus, Tylenchorhynchus, and members of genera grouped in the family Tylenchidae. Densities of five of these seven groups of nematodes differed among tall fescue cultivars in the 1987-88 study, but only two out of eight groups did so in the 1988-89 study. Irrespective of tall rescue cultivar, EIF had no consistent impact on nematode densities. The putative suppressive effect of endophyte infection on infection by plant-parasitic nematodes is not detectable within single growing seasons and deserves long-term study in field situations.

Between May, 1988, and July, 1992, the University of Missouri Plant Nematode Diagnostic Laboratory collected data on Heterodera glycines populations in Missouri by offering diagnostic services to soybean farmers. A greenhouse bioassay (free), egg count ($10/sample), and race determination test ($25) were conducted by request on soil samples submitted to the lab. Each test was offered for a specific purpose: the bioassay for fields not known to be infested; the egg count for population monitoring in fields with known infestations; and the race test for fields with a history of resistant cultivars. Of 818 samples submitted for bioassay on a H. glycines-susceptible soybean cultivar, 13 (1.6%) contained brown cysts but no white females, and 364 (45%) contained white females after 35 days in the greenhouse. Of 6,193 egg counts, 39% were either free of H. glycines or contained fewer than 500 eggs/250 cm(3) soil, the action threshold for Missouri. The remaining 61% ranged from 500-400,000 eggs/250 cm(3) soil (mean = 10,617). Eleven races were detected, with races 3 (45%) and 1 (23%) the most common. The data show that H. glycines is widespread in Missouri (with confirmed infestations in 80 of 114 counties), that most infested fields have population densities above the action threshold, and that there is considerable genetic diversity among H. glycines field populations.

Eighteen species in eight genera of plant-parasitic nematodes were identified from soil samples collected from soybean fields throughout Missouri. The genera represented were Helicotylenchus, Heterodera, Hoplolaimus, Meloidogyne, Paratylenchus, Pratylenchus, Tylenchorhynchus, and Xiphinema. Three fields, each with high densities of Helicotylenchus pseudorobustus, Pratylenchus hexincisus, or Paratylenchus projectus, were planted in 1989 with six soybean cultivars, with plots of each cultivar either not treated or treated with 5.43 kg/ha aldicarb, to determine whether economically important relationships existed. In none of the sites were nematode densities affected by either aldicarb treatment or cultivar, nor were seed yields related to nematode densities; however, mean seed yield was significantly lower in the P. projectus site. In 1990, seed yield was negatively correlated (r = -0.34, P < 0.05) with P. projectus density at planting. Based on the present and previous studies, H. pseudorobustus and P. hexincisus do not appear to be of economic interest on soybean, but P. projectus probably deserves more study.

During September 1990, 30 cotton fields in each of three Missouri counties were surveyed for plant-parasitic nematodes. Soil samples for nematode analysis consisted of a composite of 20 cores collected in a zig-zag pattern within a 1-ha block in each field. Cores were taken from within weed-free cotton rows. Nine genera of plant-parasitic nematodes were found (Rotylenchulus, Helicotylenchus, Hoplolaimus, Meloidogyne, Paratylenchus, Pratylenchus, Tylenchorhynchus, Heterodera, and Trichodorus), and five species were identified: Meloidogyne incognita, Rotylenchulus reniformis, Hoplolaimus galeatus, Pratylenchus vulnus, and P. scribneri. This is the first report of R. reniformis, H. galeatus, P. vulnus, and P. scribneri in Missouri cotton fields and the first report of R. reniformis and P. vulnus in Missouri. The known cotton pathogens M. incognita, R. reniformis, and H. galeatus were found in 30%, 3%, and 2% of the fields sampled, respectively. The correlation between sand content of the soil sample and the number of vermiform M. incognita in the sample was not significant, with r(2) = 0.13. Select fields where H. galeatus and R. reniformis were found in 1990 were sampled more intensely in 1991. The 1-ha block sampled in 1990 was sampled in 1991, along with three other 1-ha blocks uniformly distributed within the field. In addition, a 1-ha block was sampled in each of eight nearby fields, within 2 km of the first field. The nine plant-parasitic nematode genera identified in the 1990 survey were observed again in 1991. Within-field distribution of M. incognita, R. reniformis, and H. galeatus was not uniform. When M. incognita, R. reniformis, or H. galeatus were present in a field, the same species was found in 38%, 25%, or 50% of nearby fields, respectively.

Tall fescue (Festuca arundinacea Schreb.) is a C-3 perennial grass noted for its persistence in harsh environments. Tall fescue persistence is enhanced byAcremonium coenophialum, a mutualistic fungal endophyte that increases resistance to drought, pathogens, and insects. This research was conducted to identify and elicit biochemical mechanism(s) that could account for tall fescue persistence. In initial studies, two cultivars known to differ in persistence were analyzed for chitinase, an antifungal hydrolase associated with disease resistance in other plants.Acremonium-infected Kentucky 31 (KY31), a persistent cultivar, and Johnstone, a nonpersistent cultivar, were inoculated with the parasitic nematode,Meloidogyne marylandi, grown for 50 days, and analyzed at 10-day intervals. Chitinase fluctuated throughout the 50-day period of seedling development, and activity was highest in the persistentAcremonium-infected KY31. In addition, chitinase was elicited by parasiticM. marylandi and expressed systemically. Subsequent studies were conducted to determine whether or not mutualisticAcremonium could increase chitinase activity. Genetically identical KY31, with and withoutAcremonium, were grown for 25 days and analyzed for chitinase at 5-day intervals. After 20 days,Acremonium-infected KY31 expressed more chitinase thanAcremonium-free KY31. We concluded that chitinase is related to tall fescue persistence; it was highest in the most persistent cultivar, increased under pathogen attack, and increased in the presence ofAcremonium, a symbiont known to enhance disease resistance.

Soybean cultivars in maturity groups III, IV, and V that were resistant or susceptible to Heterodera glycines were evaluated for their response to in-furrow applications (5.43 kg a.i./ha) of aldicarb. Tests were conducted over three years (1988-1990) in various H. glycines-infested locations in Missouri for a total of 16 environments. In 7 environments, overall yields were higher (P

Five soybean cultivars (Braxton, Gordon, Jeff, Bragg, and Wright) resistant to Meloidogyne incognita (Mi) and three susceptible cultivars (Coker 156, GaSoy 17, and Coker 237) were grown at two locations for four seasons in microplots with increasing initial soil population densities (Pi) of Mi. The resistant cultivars and Coker 156 yielded better than GaSoy 17 and Coker 237 at all Pi. Yield response was dependent on environmental conditions and at one location was stimulated on Braxton, Gordon, Jeff, and Bragg by low Pi. Although Mi reproduced well on all cultivars, the pattern of reproduction differed. Population densities of Mi leveled off after 90 days on GaSoy 17 and Coker 237 but were still increasing after 120 days on the resistant cultivars; population densities were lower on resistant than on the susceptible cultivars. The population density of Mi on Coker 156 after 120 days was intermediate between those on the other susceptible and on the resistant cultivars. Mi population densities followed the same pattern under varying environmental conditions.

Greenhouse and field microplot studies were conducted to compare soybean shoot and root growth responses to root penetration by Heterodera glycines (Hg) and Meloidogyne incognita (Mi) individually and in combination. Soybean cultivars Centennial (resistant to Hg and Mi), Braxton (resistant to Mi, susceptible to Hg), and Coker 237 (susceptible to Hg and Mi) were selected for study. In the greenhouse, pot size and number of plants per pot had no effect on Hg or Mi penetration of Coker 237 roots; root weight was higher in the presence of either nematode species compared with the noninoculated controls. In greenhouse studies using a sand or soil medium, and in field microplot studies, each cultivar was grown with increasing initial population densities (Pi) of Hg or Mi. Interactions between Hg and Mi did not affect early plant growth or number of nematodes penetrating roots. Root penetration was the only response related to Pi. Mi penetration was higher in sand than in soil, and higher in the greenhouse than in the field, whereas Hg penetration was similar under all conditions. At 14 days after planting, more second-stage juveniles were present in roots of susceptible than in roots of resistant plants. Roots continued to lengthen in the greenhouse in the presence of either Mi or Hg regardless of host genotype, but only in the presence of Mi in microplots; otherwise, responses in field and greenhouse studies were similar and differed only in magnitude and variability.

Nursery blocks (48 dogwood, 27 red maple, and 17 peach) distributed among 20 Tennessee nurseries were sampled for nematodes in March, July, and October 1981. Plant-parasitic nematodes were extracted from soil, counted by genera, and identified to species after fixation. A total of 57 species in 24 genera were found, with 1-16 species occurring in a site. The species most commonly detected were Paratylenchus projectus and Xiphinema americanum, which were found in 88% and 78% of the sites, respectively. Relationships existed between distribution and densities of some species present in more than 10% of the sites and certain soil factors (pH, bulk density, texture, and organic matter content). Plant-parasitic nematode community diversity was related to tree age, percentage of weed ground cover, and number of weed species. Site similarities in community ordinations were dependent on the individual nurseries sampled, tree age, and soil type, but clusters of sites of similar tree ages and soil types were not exclusive.