Escape of Steinernema feltiae from Alginate Capsules Containing Tomato Seeds

The entomogenous nematode Steinernema feltiae was encapsulated in an alginate matrix containing a tomato seed. When these capsules were placed on 0.8% agar for 7 days, the seed germinated and ca. 20% of the nematodes escaped from the capsules, whereas only 0.1% escaped from capsules without seeds. When capsules containing nematodes and a seed were planted into sterilized or nonsterilized soil, nematodes escaped to infect Galleria mellonella larvae. When seed in capsules containing ca. 274 nematodes per capsule were planted in nonsterilized soil, Galleria mortality was 90% 1 week later. Galleria mortality declined to 27%, 23%, and 0% in weeks 2, 4, and 8 postplant, respectively. In sterilized soil, Galleria mortality was 96% and did not differ significantly from the nonsterilized soil in week 1, but was significantly higher in sterilized soil over nonsterilized soil for week 2 (81%) and week 4 (51%). When capsules containing nematodes only were used, Galleria mortality was 71% in sterilized soil 1 week after planting and 58%, 33%, and 12% in weeks 2, 4, and 8 postplant, respectively. In nonsterilized soil, Galleria mortality was 8%, 30%, 21%, and 28% after 1, 2, 4, and 8 weeks, respectively, using only encapsulated nematodes. When the number of nematodes per capsule was increased to ca. 817, Galleria mortality was 92 % or higher in sterilized soil from week 1 to week 4.     

Penetration of Celery and Alfalfa Roots by Pratylenchus penetrans as Affected by Temperature

A greater percentage of females than juveniles or males of P. penetrans penetrated celery roots grown in infested soil at 5, 18, or 30 C; the difference was greatest at 5 C. The time of initial penetration of alfalfa seedlings inoculated with single nematodes on water agar varied with temperature. Females penetrated the seedlings earlier and over a wider range of temperatures than did males or juveniles. The rate of penetration was highest for females. After initial penetration, the penetration rate decreased with time. At 13-28 C, approximately 80% of roots were penetrated by females and only 25-30% by males and juveniles by the end of the experiment.     

Occurrence of Pasteuria-like Organisms on Selected Plant-Pamsitic Nematodes of Pineapple in the Hawaiian Islands

Soils from 320 sites representing diverse undisturbed habitats from five Hawaiian Islands were assessed for occurrence of Pasteuria-like organisms. Mean annual rainfall at sites ranged from 125-350 cm, elevation from 69-2,286 m, and annual mean temperature from 12-24 C. Seven different natural communities were represented: wet lowland, mesic lowland, wet montane, mesk montane, dry montane, mesic subalpine, and dry alpine. Pasteuria spp. in a soil sample was detected by baiting with infective stages of Helicotylenchus dihystera, Meloidogyne javanica, Pratylenchus brachyurus, and Rotylenchulus reniformis, followed by cultivation of the nematodes on pineapple plants for 10-11 months. All nematode baits except R. reniformis were readily recovered from the soil samples. A sample was considered Pasteuria-positive if at least 5 % of the nematode specimens showed endospore attachment. Thirteen percent of all samples were positive for Pasteuria-like organisms. The frequencies of association between Pasteuria spp. and Meloidogyne, Helicotylenchus, or Pratylenchus species were 52%, 24%, and 24%, respectively. Positive samples were more prevalent on the older islands of Kauai and Oahu (75%), in lowland communities (61%), and in areas with introduced vegetation (60%). More than 27% of the positive samples were associated with plant species in a few selected families that included Meliaceae and Myrtaceae. Occurrence of Pasteuria spp. seemed to be positively associated with mean annual rainfall or temperature, but negatively associated with elevation.     

Parasitism of Xiphinema rivesi and X. americanum by Zoosporic Fungi

Living Xiphinema americanum (Xa) and X. rivesi (Xr) extracted from soil samples and stored for 1-5 days at 4 or 20 C contained aseptate fungal hyphae. The fungi directly penetrated the nematode''s cuticle from spores encysted near the head. Penetration through the stoma, vulva, or anus was rare. Catenaria anguillulae (Cat), Lagenidium caudatura (Lag), Aphanomyces sp. (Aph), and Leptolegnia sp. (Lep) were isolated into pure culture from infected nematodes. The pathogenicity of these zoosporic fungi was determined by incubating mixed freshly extracted Xa and Xr in 2% soil extract (pH = 6.7, conductivity = 48 μmhos, 20 ± 2 C) containing zoospores obtained from single-spore isolates. After 4 days, Cat, Lag, Aph, and Lep had infected 78, 18, 13, and 22%, respectively, of the nematodes. Both Xa and Xr were infected by every fungus; however, the relative susceptibility of Xa and Xr to these fungi was not determined. All noninoculated control nematodes remained uninfected and alive. In a second experiment, parasitism of Xa and Xr by Aph and Lep was increased when nematodes were incubated in 2% soil extract for 4 days before exposure to zoospores. In a third experiment, parasitism of Xa and Xr by Cat was greater in diluted saturation soil extract (conductivity = 100-400 μmhos) than in undiluted saturation extract (conductivity = 780 μmhos). Cat produced small zoospores (4-μm-d), bulbous infection hyphae, and assimilative hyphae of varying diameters in nematodes, whereas Lag, Aph, and Lep produced large zoospores (8-μm-d) and tubular, uniform infection and assimilative hyphae in nematodes.     

Accelerated Movement of Nematodes from Soil in Baermann Funnels with Temperature Gradients

Baermann funnels were modified to eliminate or reverse the small temperature gradient (1-2 C/cm) across the soil layer that normally results from water evaporation. Effects of modifications on extraction efficiency were examined at various ambient temperatures and after overnight adaptation of three nematode species at 20 and 30 C. Extraction of Meloidogyne incognita from sandy loam, Tylenchulus semipenetrans from sandy clay loam, and Rotylenchulus reniformis from silt was greatly accelerated simply by covering funnels to prevent evaporation. In most cases, covering increased the nematodes extracted by 10-100 times after 5.5-48 hours. Faster and more efficient extraction of R. reniformis occurred over a wide range of ambient temperature (18-29 C). Effects of ambient temperature and temperature gradient direction on Baermann funnel extraction of R. reniformis were partly inconsistent with the behavior of R. reniformis in agar. Nematodes in agar moved toward cold at some ambient temperatures and toward heat at other temperatures. They always appeared to move toward cold on Baermann funnels. Differences were not attributable to blockage of gas exchange by covers. In agar and in funnels, the patterns of response to ambient temperature were shifted in the direction of the storage temperature.     

An Assay for Hirsutella rhossiliensis Spores and the Importance of Phialides for Nematode Inoculation

A spore assay was developed to measure the relative density of spores of the nematophagous fungus Hirsutella rhossiliensis in soil. Orchard soil containing H. rhossiliensis-parasitized Criconemella xenoplax was placed in vials and incubated for 0-120 days before the addition of probe nematodes, Heterorhabditis heliothidis juveniles. After 18 hours, H. heliothidis were extracted from the soil and examined for adhering spores of H. rhossiliensis. No spores were detected when H. heliothidis were added to freshly mixed soil, but the percentage of H. heliothidis with spores increased rapidly if soil was incubated undisturbed. Because mixing soil detaches spores from phialides, the results indicate that spores must be attached to phialides to adhere to nematodes. The spore assay was compared with a plate assay that measures the population density of H. rhossiliensis-parasitized C. xenoplax. Results from the two assays were highly correlated, suggesting that spores occur in three phases: reserves in nematodes that may be converted into spores; spores on phialides and therefore capable of adhering to nematodes; and spores detached from phialides and thus incapable of adhering to nematodes.     

Biocontrol: Bacillus penetrans and Related Parasites of Nematodes

Bacillus penetrans Mankau, 1975, previously described as Duboscqia penetrans Thorne 1940, is a candidate agent for biocontrol of nematodes. This review considers the life stages of this bacterium: vegetative growth phase, colony fragmentation, sporogenesis, soil phase, spore attachment, and penetration into larvae of root-knot nematodes. The morphology of the microthallus colonies and the unusual external features of the spore are discussed. Taxonomic affinities with the actinomycetes, particularly with the genus Pasteuria, are considered. Also discussed are other soil bacterial species that are potential biocontrol agents. Products of their bacterial fermentation in soil are toxic to nematodes, making them effective biocontrol agents.     

Quantitative Locomotion Study of Freely Swimming Micro-organisms Using Laser Diffraction

Soil and aquatic microscopic organisms live and behave in a complex three-dimensional environment. Most studies of microscopic organism behavior, in contrast, have been conducted using microscope-based approaches, which limit the movement and behavior to a narrow, nearly two-dimensional focal field.¹ We present a novel analytical approach that provides real-time analysis of freely swimming C. elegans in a cuvette without dependence on microscope-based equipment. This approach consists of tracking the temporal periodicity of diffraction patterns generated by directing laser light through the cuvette. We measure oscillation frequencies for freely swimming nematodes.Analysis of the far-field diffraction patterns reveals clues about the waveforms of the nematodes. Diffraction is the process of light bending around an object. In this case light is diffracted by the organisms. The light waves interfere and can form a diffraction pattern. A far-field, or Fraunhofer, diffraction pattern is formed if the screen-to-object distance is much larger than the diffracting object. In this case, the diffraction pattern can be calculated (modeled) using a Fourier transform.²C. elegans are free-living soil-dwelling nematodes that navigate in three dimensions. They move both on a solid matrix like soil or agar in a sinusoidal locomotory pattern called crawling and in liquid in a different pattern called swimming.³ The roles played by sensory information provided by mechanosensory, chemosensory, and thermosensory cells that govern plastic changes in locomotory patterns and switches in patterns are only beginning to be elucidated.⁴ We describe an optical approach to measuring nematode locomotion in three dimensions that does not require a microscope and will enable us to begin to explore the complexities of nematode locomotion under different conditions.     

Soil Sampling and Isolation of Entomopathogenic Nematodes (Steinernematidae,Heterorhabditidae)

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to two families: Steinernematidae and Heterorhabditidae. Until now, more than 70 species have been described in the Steinernematidae and there are about 20 species in the Heterorhabditidae. The nematodes have a mutualistic partnership with Enterobacteriaceae bacteria and together they act as a potent insecticidal complex that kills a wide range of insect species.Herein, we focus on the most common techniques considered for collecting EPN from soil. The second part of this presentation focuses on the insect-baiting technique, a widely used approach for the isolation of EPN from soil samples, and the modified White trap technique which is used for the recovery of these nematodes from infected insects. These methods and techniques are key steps for the successful establishment of EPN cultures in the laboratory and also form the basis for other bioassays that consider these nematodes as model organisms for research in other biological disciplines. The techniques shown in this presentation correspond to those performed and/or designed by members of S. P. Stock laboratory as well as those described by various authors.     

Detection and Description of Soils with Specific Nematode Suppressiveness

Soils with specific suppressiveness to plant-parasitic nematodes are of interest to define the mechanisms that regulate population density. Suppressive soils prevent nematodes from establishing and from causing disease, and they diminish disease severity after initial nematode damage in continuous culturing of a host. A range of non-specific and specific soil treatments, followed by infestation with a target nematode, have been employed to identify nematode-suppressive soils. Biocidal treatments, soil transfer tests, and baiting approaches together with observations of the plant-parasitic nematode in the root zone of susceptible host plants have improved the understanding of nematode-suppressive soils. Techniques to demonstrate specific soil suppressiveness against plant-parasitic nematodes are compared in this review. The overlap of studies on soil suppressiveness with recent advances in soil health and quality is briefly discussed. The emphasis is on methods (or criteria) used to detect and identify soils that maintain specific soil suppressiveness to plant-parasitic nematodes. While biocidal treatments can detect general and specific soil suppressiveness, soil transfer studies, by definition, apply only to specific soil suppressiveness. Finally, potential strategies to exploit suppressive soils are presented.     

Parasitic and Saprophytic Abilities of the Nematode-Attacking Fungus Hirsutella rhossiliensis

The ability of Hirsutella rhossiliensis to colonize various substrates in sterile and nonsterile soil was measured. Hirsutella rhossiliensis was recovered from 67% and 77% of living, inoculated Criconemella xenoplax incubated in sterile and nonsterile soil, respectively. In contrast, the fungus was recovered from 100% and 18% of heat-killed, inoculated nematodes incubated on sterile and nonsterile soil, respectively. Hirsutella rhossiliensis was readily recovered from inoculated, autoclaved wheat seeds incubated in sterile soil but not from seeds incubated in nonsterile soil. Autoclaved peach roots were a poor substrate for the fungus. Germination of H. rhossiliensis spores incubated on agar disks above soil was about 90% regardless of soil treatment. However, germ tube length was greatly suppressed by nonsterile soil. Our results suggest that H. rhossiliensis is a better parasite than saprophyte and that the fungus may be specialized for attacking nematodes.     

Infection of Pratylenchus penetrans by Nematode-pathogenic fungi

Eleven fungal isolates were tested in agar dishes for pathogenicity to Pratylenchus penetrans. Of the fungi that produce adhesive conidia, Hirsutella rhossiliensis was a virulent pathogen; Verticillium balanoides, Drechmeria coniospora, and Nematoctonus sp. were weak or nonpathogens. The trapping fungi, Arthrobotrys dactyloides, A. oligospora, Monacrosporium dlipsosporum, and M. cionopagum, killed most of the P. penetrans adults and juveniles added to the fungus cultures. An isolate of Nematoctonus that forms adhesive knobs trapped only a small proportion of the nematodes. In 17-cm³ vials, soil moisture influenced survival of P. penetrans in the presence of H. rhossiliensis; nematode survival decreased with diminishing soil moisture. Hirsutella rhossiliensis and M. ellipsosporum were equally effective in reducing numbers of P. penetrans by 24-25% after 4 days in sand. After 25 days in soil artificially infested with H. rhossiliensis, numbers of P. penetrans were reduced by 28-53%.     

Soil Applications of Steinernematid and Heterorhabditid Nematodes for Control of Colorado Potato Beetles,Leptinotarsa decemlineata (Say)

Three strains of Steinernema feltiae Filipjev (All, Mexican, and Breton strains) and one of Heterorhabditis heliothidis (Khan, Brooks, and Hirschmann) were evaluated for their potential to control Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say), larvae and pupae in the soil. In laboratory studies, H. heliothidis and S. feltiae (Mexican strain) produced the highest mortality (6 days posttreatment) of CPB when applied to the surface of a soil column containing mature CPB larvae 5 cm below. Mortality ranged from 80 to 90% at rates of 79-158 nematodes/cm². Similar results were seen in a field microplot study with all four nematodes; S. feltiae (Mexican strain) and H. heliothidis were most effective. Adult CPB emergence was reduced 86.5-100% after application of 31-93 H. heliothidis/cm² and 88.4-100% with 93-155 S. feltiae (Mexican strain)/cm². The All strain of S. feltiae was moderately effective (ca. 80% reduction at 93-155 nematodes/cm²), while the Breton strain was ineffective (< 40% reduction at 155 nematodes/cm²). In small plots of potatoes enclosed in field cages, application of H. heliothidis and S. feltiae (Mexican strain) at rates of 93-155 nematodes/cm² before larval CPB burial in the soil resulted in 66-77% reduction in adult CPB emergence. Soil applications of these nematodes show potential for biological control of CPB.     

Seed Treatment as a Means of Preventing Nematode Damage to Crop Plants

A procedure for treating crop seeds with aqueous solutions of the systemic nematicide oxamyl is described. Seedlings from treated seeds were more resistant to attack by parasitic nematodes. Leachate from treated seeds reduced the number of free-living nematodes in the surrounding soil.     

Influence of Lysobacter enzymogenes Strain C3 on Nematodes

Chitinolytic microflora may contribute to biological control of plant-parasitic nematodes by causing decreased egg viability through degradation of egg shells. Here, the influence of Lysobacter enzymogenes strain C3 on Caenorhabditis elegans, Heterodera schachtii, Meloidogyne javanica, Pratylenchus penetrans, and Aphelenchoides fragariae is described. Exposure of C. elegans to L. enzymogenes strain C3 on agar resulted in almost complete elimination of egg production and death of 94% of hatched juveniles after 2 d. Hatch of H. schachtii eggs was about 50% on a lawn of L. enzymogenes strain C3 on agar as compared to 80% on a lawn of E. coli. Juveniles that hatched on a lawn of L. enzymogenes strain C3 on agar died due to disintegration of the cuticle and body contents. Meloidogyne javanica juveniles died after 4 d exposure to a 7-d-old chitin broth culture of L. enzymogenes strain C3. Immersion of A. fragariae, M. javanica, and P. penetrans juveniles and adults in a nutrient broth culture of L. enzymogenes strain C3 led to rapid death and disintegration of the nematodes. Upon exposure to L. enzymogenes strain C3 cultures in nutrient broth, H. schachtii juveniles were rapidly immobilized and then lysed after three days. The death and disintegration of the tested nematodes suggests that toxins and enzymes produced by this strain are active against a range of nematode species.     

Plant-parasitic Nematode Communities and Their Associations with Soil Factors in Organically Farmed Fields in Minnesota

A survey was conducted to determine the assemblage and abundance of plant-parasitic nematodes and their associations with soil factors in organically farmed fields in Minnesota. A total of 31 soil samples were collected from southeast (SE), 26 samples from southwest (SW), 28 from west-central (WC), and 23 from northwest (NW) Minnesota. The assemblage and abundance of plant-parasitic nematodes varied among the four regions. The soybean cyst nematode, Heterodera glycines, the most destructive pathogen of soybean, was detected in 45.2, 88.5, 10.7, and 0% of organically farmed fields with relative prominence (RP) values of 10.3, 26.5, 0.6, and 0 in the SE, SW, WC, and NW regions, respectively. Across the four regions, other common genera of plant-parasitic nematodes were Helicotylenchus (42.6, RP value, same below), Pratylenchus (26.9), Tylenchorhynchus and related genera (9.4), Xiphinema (5.6), and Paratylenchus (5.3). Aphelenchoides, Meloidogyne, Hoplolaimus, Mesocriconema, and Trichodorus were also detected at low frequencies and/or low population densities. The similarity index of plant-parasitic nematodes between two regions ranged from 0.44 to 0.71 and the similarity increased with decreasing distance between regions. The densities of most plant-parasitic nematodes did not correlate with measured soil factors (organic matter, pH, texture). However, the densities of Pratylenchus correlated negatively with % sand, and Xiphinema was correlated negatively with soil pH.     

Suppression of Cyst Nematode by Natural Infestation of a Nematophagous Fungus

Penetration of cabbage roots by Heterodera schachtii was suppressed 50-77% in loamy sand naturally infested with the nematophagous fungus Hirsutella rhossiliensis. When Heterodera schachtii was incubated in the suppressive soil without plants for 2 days, 40-63% of the juveniles had Hirsutella rhossiliensis spores adhering to their cuticles. Of those with spores, 82-92% were infected. Infected nematodes were killed and filled with hyphae within 2-3 days. Addition of KCl to soil did not increase infection of Heterodera schachtii by Hirsutella rhossiliensis. The percentage of infection was lower when nematodes were touched to two spores and incubated in KCl solution than when nematodes naturally acquired two spores in soil.     

Problems and Strategies Associated with Long-term Use of Nematode Resistant Cultivars

Plant-parasitic nematodes are obligate parasites, and planting cultivars that are highly resistant to these organisms places extensive selection pressure on the target species and affects nontarget nematodes as well. Problems encountered with long-term planting of cultivars resistant to nematodes include shifts in nematode races or species and the occurrence of multiple species of nematodes within the same field. These problems can be alleviated to some extent when crop management is used to lessen the selection pressure for change on the nematode populations. Race shifts within populations and possibly shifts between nematode species can be delayed by rotating susceptible cultivars and nonhost crops with resistant cultivars. Nematicides in conjunction with resistant cultivars may be used to limit damage by multiple species of nematodes. Some cultivars have resistance to multiple species of nematodes, but greatly increased research effort is needed in this area. More intensive plant breeding effort will be required to make nematode resistant cultivars competitive in quality and yield with more productive, susceptible cultivars.     

Evaluation of an Antibiotic-Producing Strain of Pseudomonas fluorescens for Suppression of Plant-Parasitic Nematodes

The antibiotic 2,4-diacetylphloroglucinol (DAPG), produced by some strains of Pseudomonas spp., is involved in suppression of several fungal root pathogens as well as plant-parasitic nematodes. The primary objective of this study was to determine whether Wood1R, a D-genotype strain of DAPG-producing P. fluorescens, suppresses numbers of both sedentary and migratory plant-parasitic nematodes. An experiment was conducted in steam-heated soil and included two seed treatments (with Wood1R and a control without the bacterium) and six plant-nematode combinations which were Meloidogyne incognita on cotton, corn, and soybean; M. arenaria on peanut; Heterodera glycines on soybean; and Paratrichodorus minor on corn. Wood 1R had no effect on final numbers of M. arenaria, P. minor, or H. glycines; however, final numbers of M. incognita were lower when seeds were treated with Wood1R than left untreated, and this reduction was consistent among host plants. Population densities of Wood1R were greater on the roots of corn than on the other crops, and the bacterium was most effective in suppressing M. incognita on corn, with an average reduction of 41%. Despite high population densities of Wood1R on corn, the bacterium was not able to suppress numbers of P. minor. When comparing the suppression of M. incognita on corn in natural and steam-heated soil, egg production by the nematode was suppressed in natural compared to steamed soil, but the presence of Wood1R did not result in additional suppression of the nematodes in the natural soil. These data indicate that P. fluorescens strain Wood1R has the capacity to inhibit some populations of plant-parasitic nematodes. However, consistent suppression of nematodes in natural soils seems unlikely.     

Com Yield Increases Relative to Nonfumigant Chemical Control of Nematodes

Corn yields were measured after application of nematicides in 16 experiments, mostly in medium-to-heavily textured soil, at 12 locations in Iowa during 1973-1976. The average maximum yield increase in plots treated with nematicides was 21% over yields in untreated plots. Yields were correlated negatively with nematode numbers or nematode biomass in nearly all comparisons. Correlations of nematode numbers in the soil with yield averaged -0.56 for Helicotylenchus pseudorobustus, -0.45 for Hoplolaimus galeatus, -0.51 for Pratylenchus spp., and -0.64 for Xiphinema americanum. Correlation coefficients for numbers of nematodes in the roots and yield averaged -0.63 for Pratylenchus spp. and -0.56 H. galeatus. Correlation coefficients for yield and total number of nematodes averaged -0.65 in roots and -0.55 in soils. Negative correlations also were greater for comparisons of yield with total parasitic-nematode biomass than with numbers of individual nematodes of a species or total numbers of parasitic nematodes.