Reduction of Pythium Damping-Off in Soybean by Biocontrol Seed Treatment.

Pythium spp. is one of the major groups of pathogens that cause seedling diseases on soybean, leading to both preemergence and postemergence damping-off and root rot. More than 100 species have been identified within this genus, with Pythium irregulare, P. sylvaticum, P. ultimum var ultimum, and P. torulosum being particularly important for soybean production given their aggressiveness, prevalence, and abundance in production fields. This study investigated the antagonistic activity of potential biological control agents (BCAs) native to the U.S. Midwest against Pythium spp. First, in vitro screening identified BCAs that inhibit P. ultimum var. ultimum growth. Scanning electron microscopy demonstrated evidence of mycoparasitism of all potential biocontrol isolates against P. ultimum var. ultimum and P. torulosum, with the formation of appressorium-like structures, short hyphal branches around host hyphae, hook-shaped structures, coiling, and parallel growth of the mycoparasite along the host hyphae. Based on these promising results, selected BCAs were tested under field conditions against six different Pythium spp. Trichoderma afroharzianum 26 used alone and a mix of T. hamatum 16 + T. afroharzianum 19 used as seed treatments protected soybean seedlings from Pythium spp. infection, as BCA-treated plots had on average 15 to 20% greater plant stand and vigor than control plots. Our results also indicate that some of these potential BCAs could be added with a fungicide seed treatment with minimum inhibition occurring, depending on the fungicide active ingredient. This research highlights the need to develop tools incorporating biological control as a facet of soybean seedling disease management programs. The harnessing of native BCAs could be integrated with other management strategies to provide efficient control of seedling diseases.

Convergent evolution of saccate body shapes in nematodes through distinct developmental mechanisms.

BACKGROUND: The vast majority of nematode species have vermiform (worm-shaped) body plans throughout post-embryonic development. However, atypical body shapes have evolved multiple times. The plant-parasitic Tylenchomorpha nematode Heterodera glycines hatches as a vermiform infective juvenile. Following infection and the establishment of a feeding site, H. glycines grows disproportionately greater in width than length, developing into a saccate adult. Body size in Caenorhabditis elegans was previously shown to correlate with post-embryonic divisions of laterally positioned stem cell-like 'seam' cells and endoreduplication of seam cell epidermal daughters. To test if a similar mechanism produces the unusual body shape of saccate parasitic nematodes, we compared seam cell development and epidermal ploidy levels of H. glycines to C. elegans. To study the evolution of body shape development, we examined seam cell development of four additional Tylenchomorpha species with vermiform or saccate body shapes. RESULTS: We confirmed the presence of seam cell homologs and their proliferation in H. glycines. This results in the adult female epidermis having approximately 1800 nuclei compared with the 139 nuclei in the primary epidermal syncytium of C. elegans. Similar to C. elegans, we found a significant correlation between H. glycines body volume and the number and ploidy level of epidermal nuclei. While we found that the seam cells also proliferate in the independently evolved saccate nematode Meloidogyne incognita following infection, the division pattern differed substantially from that seen in H. glycines. Interestingly, the close relative of H. glycines, Rotylenchulus reniformis does not undergo extensive seam cell proliferation during its development into a saccate form. CONCLUSIONS: Our data reveal that seam cell proliferation and epidermal nuclear ploidy correlate with growth in H. glycines. Our finding of distinct seam cell division patterns in the independently evolved saccate species M. incognita and H. glycines is suggestive of parallel evolution of saccate forms. The lack of seam cell proliferation in R. reniformis demonstrates that seam cell proliferation and endoreduplication are not strictly required for increased body volume and atypical body shape. We speculate that R. reniformis may serve as an extant transitional model for the evolution of saccate body shape.

Immobility in the sedentary plant-parasitic nematode H. glycines is associated with remodeling of neuromuscular tissue.

The sedentary plant-parasitic nematodes are considered among the most economically damaging pathogens of plants. Following infection and the establishment of a feeding site, sedentary nematodes become immobile. Loss of mobility is reversed in adult males while females never regain mobility. The structural basis for this change in mobility is unknown. We used a combination of light and transmission electron microscopy to demonstrate cell-specific muscle atrophy and sex-specific renewal of neuromuscular tissue in the sedentary nematode Heterodera glycines. We found that both females and males undergo body wall muscle atrophy and loss of attachment to the underlying cuticle during immobile developmental stages. Male H. glycines undergo somatic muscle renewal prior to molting into a mobile adult. In addition, we found developmental changes to the organization and number of motor neurons in the ventral nerve cord correlated with changes in mobility. To further examine neuronal changes associated with immobility, we used a combination of immunohistochemistry and molecular biology to characterize the GABAergic nervous system of H. glycines during mobile and immobile stages. We cloned and confirmed the function of the putative H. glycines GABA synthesis-encoding gene hg-unc-25 using heterologous rescue in C. elegans. We found a reduction in gene expression of hg-unc-25 as well as a reduction in the number of GABA-immunoreactive neurons during immobile developmental stages. Finally, we found evidence of similar muscle atrophy in the phylogenetically diverged plant-parasitic nematode Meloidogyne incognita. Together, our data demonstrate remodeling of neuromuscular structure and function during sedentary plant-parasitic nematode development.

Embryogenesis in the parasitic nematode Heterodera glycines is independent of host-derived hatching stimulation.

BACKGROUND: Many parasites regulate their development to synchronize their life cycle with a compatible host. The parasitic nematode Heterodera glycines displays incomplete host-mediated hatching behavior wherein some H. glycines individuals hatch only in the presence of a host-derived cue while others hatch in water alone. Furthermore, H. glycines shows variable hatching behavior based on oviposition location. The mechanisms regulating this hatching variability are unknown. In this study, we established a detailed timeline of the H. glycines pre-hatch development from early embryogenesis to the pre-hatched J2. These descriptive data were then used to test hypotheses regarding the effect of host stimulus and oviposition location on pre-hatch development. RESULTS: We found that H. glycines develops from a single-cell egg to a fully formed J2 in approximately 172 hours. The stylet-based mouthpart, which is used to pierce the eggshell during hatching, is not completely formed until late in pre-hatch J2 development and is preceded by the formation of stylet protractor muscles. We also found that the primary motor nervous system of H. glycines did not complete development until late in pre-hatch J2 development. These data suggest possible structural requirements for H. glycines hatching. As expected, exposure of H. glycines eggs to host-derived cues increased the percentage of nematodes that hatched. However, exposure to hatching cues did not affect pre-hatch development. Similarly, we found no obvious differences in the pre-hatch developmental timeline between eggs laid in an egg sac or retained within the mother. CONCLUSIONS: The pattern of early embryonic development in H. glycines was very similar to that recently described in the related parasitic nematode Meloidogyne incognita. However, the speed of H. glycines pre-hatch development was approximately three times faster than reported for M. incognita. Our results suggest that hatching stimulants do not affect embryogenesis itself but only influence the hatching decision once J2 development is complete. Similarly, the oviposition location does not alter the rate of embryogenesis. These results provide insight into the primary survival mechanism for this important parasite.

Baseline Sensitivity of Fusarium virguliforme to Fluopyram Fungicide.

Fluopyram, a succinate dehydrogenase inhibitor (SDHI) fungicide, was recently registered for use as a soybean seed treatment for management of sudden death syndrome (SDS) caused by Fusarium virguliforme. Although registered and now used commercially, in vitro baseline fungicide sensitivity of F. virguliforme to fluopyram has not yet been established. In this study, the baseline sensitivity of F. virguliforme to fluopyram was determined using in vitro growth of mycelium and germination of conidia assays with two collections of F. virguliforme isolates. A total of 130 and 75 F. virguliforme isolates were tested using the mycelial growth and conidia germination assays, respectively, including a core set of isolates that were tested with both assays. In the mycelial growth inhibition assay, 113 out of 130 isolates (86.9%) were inhibited 50% by effective concentrations (EC50) less than 5 µg/ml with a mean EC50 of 3.35 µg/ml. For the conidia germination assay, 73 out of 75 isolates (97%) were determined to have an estimated EC50 of less than 5 µg/ml with a mean EC50 value of 2.28 µg/ml. In a subset of 20 common isolates that were phenotyped with both assays, conidia germination of F. virguliforme was determined to be more sensitive to fluopyram (mean EC50 = 2.28 µg/ml) than mycelial growth (mean EC50 = 3.35 µg/ml). Hormetic effects were observed in the mycelial growth inhibition assay as 22% of the isolates demonstrated more growth on medium amended with the lowest fluopyram concentration (1 µg/ml), as compared with the nonfluopyram amended control. It was not possible to determine EC50 values for nine out of 185 isolates (4.8%), as those isolates were not inhibited by 50% even at the highest fluopyram concentrations of 100 µg/ml for mycelial growth and 20 µg/ml for conidia germination inhibition assays. On the whole, the F. virguliforme population appears to be sensitive to fluopyram, and this study enables future monitoring of fungicide sensitivity.