Interactions Among Heterodera glycines, Macrophomina phaseolina, and Soybean Genotype.

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.

Sampling Depth and Crop Growth Stage Affect Estimates of the Population Density of Lesion Nematodes (Pratylenchus spp.) in Corn Fields in Ohio.

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.

Giant Starship Elements Mobilize Accessory Genes in Fungal Genomes.

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.

Incidence, Population Density, and Spatial Heterogeneity of Plant-Parasitic Nematodes in Corn Fields in Ohio.

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.

Cropping Practices and Soil Properties Associated with Plant-Parasitic Nematodes in Corn Fields in Ohio.

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.

Evaluation of Cultivar Resistance to Soybean Cyst Nematode with a Quantitative Polymerase Chain Reaction Assay.

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.

Discovery and initial analysis of novel viral genomes in the soybean cyst nematode.

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.

Plant-Parasitic Nematodes Are Potential Pathogens of Miscanthus × giganteus and Panicum virgatum Used for Biofuels.

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 × âˆšfrequency 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.

Selection of Heterodera glycines chorismate mutase-1 alleles on nematode-resistant soybean.

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.

A chorismate mutase from the soybean cyst nematode Heterodera glycines shows polymorphisms that correlate with virulence.

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.