Impact of Pratylenchus penetrans on Soybean Grown in Wisconsin, U.S.A.

Models were developed to quantify the impact of Pratylenchus penetrans to the early season growth and yield of soybean in field and greenhouse environments and to estimate yield loss because of P. penetrans in Wisconsin. There was a negative linear relationship between initial nematode population densities (Pi) and shoot and total plant weight at V2 and yield, pod number, seed number, and seed mass at harvest in the field. Relative yield loss, modeled for the second year of the field experiment, suggested a loss of 4.5% for yield and between 2.4 and 2.8% for yield components at the mean field Pi value. Negative linear relationships were demonstrated for the relative loss in those variables as well as for harvest index and shoot, root, and total plant weight at harvest in the greenhouse. Stress imposed by P. penetrans began within 2 weeks after planting and continued through harvest. Estimates of the percent loss attributed to each nematode Pi value were 0.020% for yield, 0.015% for pod number, and 0.017% for seed number. Pratylenchus spp. was the most widely prevalent pest nematode among samples submitted to a statewide nematode testing program. Molecular identification of a subset of 63 samples suggested 15% were infested with P. penetrans at a mean Pi value of 197 P. penetrans per 100 cm3 soil. Yield loss because of P. penetrans, estimated from prevalence data and our empirical greenhouse model, ranged from 0.23 to 2.76% among Wisconsin's agricultural districts. The cumulative impact for all Pratylenchus spp. is likely much greater, given this loss estimate does not account for the monoecious species present in 79% of the samples.

Intraspecific variation in phenotypic and phylogenetic features among Pratylenchus penetrans isolates from Wisconsin, USA.

Pratylenchus penetrans is a common and important agricultural pest in Wisconsin, a USA state with a diverse agriculture. We compared populations from around the state to each other and to data published for populations around the world to gain insight on the variability of features important for identification of this cosmopolitan species. Thirteen isolates from samples collected in soybean fields in ten Wisconsin counties were established in monoxenic cultures. Analysis of morphological features revealed the least variable feature for all isolates collectively was vulva percentage. Features less variable within than among isolates were body width, lip region height, and stylet length. Some isolates showed only the smooth tail tip phenotype and others had a mix of smooth and annulated tail phenotypes. A suite of features provided sufficient pattern to group isolates into four clusters according to hierarchical agglomerative clustering and canonical discriminative analyses, but not with enough distinction to be useful for classification. Haplotype analysis based on the COI mitochondrial gene of the 13 cultured isolates, 39 Wisconsin field populations, and published sequences representing five additional USA states and six countries revealed 21 haplotypes, 15 of which occurred in Wisconsin. Ten haplotypes represented in Wisconsin were shared with populations from Europe, South America, Africa, or Asia. Five haplotypes were unique to Wisconsin, six were unique to The Netherlands, and one was unique to Japan suggesting that even more COI diversity will be revealed when more COI sequences for P. penetrans become available. The maximum pairwise sequence variation was 6% and the SNPs did not alter amino acids, indicating cryptic biodiversity within the species worldwide. The cosmopolitan to localized scale of distribution of COI haplotypes could be due to frequent and ongoing dispersal events, facilitated by life history traits and the broad host range of P. penetrans. Regions of diverse agriculture, like Wisconsin, show promise for studying this important pest and our study confirms the utility of the COI mtDNA gene for studying variation within a species.

Difference in lesion formation by male and female Pratylenchus penetrans.

Pratylenchus penetrans induce necrotic lesions, the hallmark symptom for the genus, soon after infection. The objective of our study was to characterize and quantify gender differences in lesion development. Independent experiments were conducted in vitro for three hosts; pea (Pisum sativum L. cv. Early Alaskan), dill (Anethum graveolens cv. Long Island Mammoth), and alfalfa (Medicago sativa cv. Vernal). Each experimental unit was an excised radical placed on water agar in a Petri dish and inoculated with either 40 adult males or 40 fourth-stage juvenile females. Length, size, and number of lesions were recorded during the experiment and the radicals were harvested 14 days after introducing nematodes. Lesions were first observed on pea after two days for female-inoculated roots, and 24 hr after introducing both genders to dill and alfalfa. Lesions expanded either by multiple lesions coalescing or individual lesions expanding over time. Males made fewer, smaller lesions with less discoloration for all three hosts. There was no difference among genders for the total number of nematodes recovered per Petri dish or the number of endoparasitic nematodes after 14 days. The survival rate of males and females at harvest was not different, indicating that the difference in lesion formation was not related to nematode population densities. This study verified and quantified the observation that lesions induced by males are less extensive and in smaller numbers than lesions by females.

Incidence of Oscheius onirici (Nematoda: Rhabditidae), a potentially entomopathogenic nematode from the marshlands of Wisconsin, USA.

In a search for an entomopathogenic nematode to control cranberry insect pests, three Oscheius populations (Rhabditidae) were recovered through the Galleria-bait method from one sample taken in a wild cranberry marsh in Jackson County, Wisconsin, USA. Morphological studies with light microscopy and scanning electron microscopy, as well as molecular analyses of the near-full-length small subunit rDNA gene, D2/D3 expansion segments of the large subunit rDNA gene, internal transcribed spacer, and mitochondrial cytochrome oxidase subunit 1 (CoxI) genes revealed this as Oscheius onirici, a species recently described from a karst cave soil of central Italy. The species belongs to the dolichura-group and is characterized by its DNA sequences; hermaphroditic reproduction; and males not found. A Bacillus-like bacterium appears to be associated with this nematode based on our microscopic and SEM observations; however its identity and persistent association with the nematode has not been confirmed. Nonetheless, this nematode is capable of infecting and killing the sparganothis fruitworm Sparganothis sulfureana Clemens (Lepidoptera: Tortricidae), the brown-banded cockroach Supella longipalpa Fabricius (Blattodea: Ectobiidae), and the cranberry fruitworm Acrobasis vaccinii Riley (Lepidoptera: Pyralidae), under laboratory conditions, and each in less than 72 hr. The mealworm Tenebrio molitor Linnaeus (Coleoptera: Tenebrionidae) and the greater wax moth Galleria mellonella Linnaeus (Lepidoptera: Pyralidae), are also susceptible, but take 3.5 and 5.2 days to die, respectively. This species is a new potential bio-control agent on insects.

Development of a Damage Function Model for Pratylenchus penetrans on Corn.

The lesion nematode Pratylenchus penetrans is a common pest of corn in the north-central United States. There are relatively few studies documenting the impact of Pratylenchus spp. on grain yield even though they are recognized as pests of corn and the target of commercial seed treatments. We adapted a component error modeling approach to develop a damage function for P. penetrans that included the influence of year and site in the yield loss relationship. Field data from six site-years was used to derive panel data consisting of all pairwise comparisons of the difference in nematode population densities and the associated proportional yield difference. Fourteen regression models of the relationship between proportional yield loss and the difference in nematode density were developed from soil and root assays at different corn growth stages. Seven models were significant: four models based on nematode population densities in soil (initial and final samples) and three based on nematode densities in seminal roots (corn growth stages V1 to V2 and V6) and adventitious roots (corn growth stage R1 to R2). The model we consider to be the most important, that based on the initial soil assay, estimated the yield loss caused by each nematode to be 0.0142%. The grand mean of the 118 plots we sampled implied a yield loss of 3.79%. The random effects of year and field did not contribute significantly to any of the models but were close to significance for some, suggesting a benefit from larger data sets. Experimental error was the largest component of the variance for all of the models; therefore, the damage function is more useful for demonstrating impact of P. penetrans rather than for accurately predicting yield loss at the field level. All of the fields in our study were an irrigated loamy sand soil, with grain yields above the county average; therefore, it is possible that our damage function is conservative. The value of soil sampling has been questioned for P. penetrans and this study shows it to be equal to if not better than root assays for predicting yield.

A nematode demographics assay in transgenic roots reveals no significant impacts of the Rhg1 locus LRR-Kinase on soybean cyst nematode resistance.

BACKGROUND: Soybean cyst nematode (Heterodera glycines, SCN) is the most economically damaging pathogen of soybean (Glycine max) in the U.S. The Rhg1 locus is repeatedly observed as the quantitative trait locus with the greatest impact on SCN resistance. The Glyma18g02680.1 gene at the Rhg1 locus that encodes an apparent leucine-rich repeat transmembrane receptor-kinase (LRR-kinase) has been proposed to be the SCN resistance gene, but its function has not been confirmed. Generation of fertile transgenic soybean lines is difficult but methods have been published that test SCN resistance in transgenic roots generated with Agrobacterium rhizogenes. RESULTS: We report use of artificial microRNA (amiRNA) for gene silencing in soybean, refinements to transgenic root SCN resistance assays, and functional tests of the Rhg1 locus LRR-kinase gene. A nematode demographics assay monitored infecting nematode populations for their progress through developmental stages two weeks after inoculation, as a metric for SCN resistance. Significant differences were observed between resistant and susceptible control genotypes. Introduction of the Rhg1 locus LRR-kinase gene (genomic promoter/coding region/terminator; Peking/PI 437654-derived SCN-resistant source), into rhg1- SCN-susceptible plant lines carrying the resistant-source Rhg4+ locus, provided no significant increases in SCN resistance. Use of amiRNA to reduce expression of the LRR-kinase gene from the Rhg1 locus of Fayette (PI 88788 source of Rhg1) also did not detectably alter resistance to SCN. However, silencing of the LRR-kinase gene did have impacts on root development. CONCLUSION: The nematode demographics assay can expedite testing of transgenic roots for SCN resistance. amiRNAs and the pSM103 vector that drives interchangeable amiRNA constructs through a soybean polyubiqutin promoter (Gmubi), with an intron-GFP marker for detection of transgenic roots, may have widespread use in legume biology. Studies in which expression of the Rhg1 locus LRR-kinase gene from different resistance sources was either reduced or complemented did not reveal significant impacts on SCN resistance.

Mortality and behavior in Heterodera glycines juveniles following exposure to isothiocyanate compounds.

For this report, we examined the toxic effects of three plant-derived isothiocyanate compounds on second-stage juveniles (J2) of Heterodera glycines. We found significant differences among compounds in the concentration required to affect nematodes, according to mortality and behavioral measurements. The concentrations required to affect behavior were significantly lower than those required for mortality. Both mortality and behavioral measurements were used to investigate whether nematodes in a quiescent state display decreased sensitivity to isothiocyanates compared with actively moving nematodes. Mortality measurements revealed that quiescent nematodes were significantly less sensitive to isothiocyanates than active nematodes. All behavioral measurements following exposure to benzyl- and phenyl isothiocyanate showed significant differences in sensitivity between quiescent and active nematodes. However, significant differences between quiescent and active nematodes were observed in only one of the five behavioral measurements following exposure to allyl isothiocyanate. These results expand the list of plant-derived compounds toxic to H. glycines and illustrate the impact of behavioral quiescence on nematode sensitivity to exogenous toxins.

A Field Study on the Influence of Verticillium dahliae and Pratylenchus penetrans on Gas Exchange of Potato.

Field experiments were conducted for three consecutive years to study the effects of low populations of Verticillium dahliae and Pratylenchus penetrans on leaf gas exchange of Russet Burbank potato. Treatments were P. penetrans, V. dahliae, the combination of the nematode with the fungus, and a no-pathogen control. Gas exchange was measured nondestructively on young, fully expanded, asymptomatic leaves one to three times per week starting the ninth week after planting. Infection with either pathogen alone had little or no effect on leaf gas exchange parameters. However, co-infection by both pathogens resulted in reduced leaf light use efficiency (mole of CO2 fixed per mole of photon), lower leaf stomatal conductance, lower leaf water use efficiency (mole of CO2 fixed per mole of water used), and increased intercellular CO2 compared with the no-pathogen control. These effects, additive relative to the impact of each pathogen alone, were first observed 9 weeks after inoculation in the first 2 years of the study and 15 weeks after inoculation in the third year.