Detection of an estrogen receptor in two nematode species and inhibition of binding and development by environmental chemicals.

The presence of estrogen receptors or binding proteins was demonstrated in the free-living nematode species Panagrellus redivivus and Caenorhabditis elegans by radioimmunoassay. Twenty-five nanomolar concentrations of toxaphene, dieldrin, and dieldrin plus nonylphenol significantly inhibited estrogen binding to the receptor in P. redivivus. Binding was inhibited but not significantly by 25 nM nonylphenol, toxaphene plus dieldrin, or toxaphene plus nonylphenol. The current research supports the hypothesis that dieldrin, nonylphenol, and toxaphene may mimic estrogen, altering the normal pathways of estrogen metabolism. Based on observations of secondary sex structures, estrogenic chemicals had no effect on sex ratios or growth in Panagrellus redivivus, but caused a reduction of fecundity in this nematode.

Efficacy of Bacillus thuringiensis, Paecilomyces marquandii,and Streptomyces costaricanus with and without Organic Amendments against Meloidogyne hapla Infecting Lettuce.

Chitin, wheat mash, or brewery compost were incorporated into unfumigated and methyl bromide-fumigated organic soils placed in microplots formed from cylindrical drainage tiles (0.25 m-diam. clay tile). After 3 weeks, Meloidogyne hapla and cell or spore suspensions of Bacillus thuringiensis, Paecilomyces marquandii, and Streptomyces costaricanus were individually added to the soils of designated microplots. A B. thuringiensis + S. costaricanus combination was also tested. Lettuce seedlings, cv. Montello, were transplanted into the soils 3 to 4 days later. All the bacterial and fungal antagonists applied without a soil amendment, except the B. thuringiensis + S. costaricanus treatment, reduced root galling and increased lettuce head weight in the unfumigated organic soil, but not in the fumigated soil. All three amendments were also effective against M. hapla and reduced root galling in fumigated and unfumigated soils. Wheat mash amendment increased lettuce head weight in the unfumigated soil. In general, no antagonist x amendment interaction was detected. Soil populations of B. thuringiensis were maintained at >/=4.0 log10 colony-forming units/g organic soil during the first 14 days after planting. However, viable cells of B. thuringiensis were not detected after 49 days.

Suppression of Meloidogyne hapla and Its Damage to Lettuce Grown in a Mineral Soil Amended with Chitin and Biocontrol Organisms.

Chitin was used as soil amendment in fiberglass field microplots, alone or with one or a combination of two to three species of Hirsutella rhossiliensis, Paecilomyces marquandii, Verticillium chlamydosporium, Bacillus thuringiensis, and Streptomyces costaricanus. Sudangrass and rapeseed were planted as cover crops and incorporated into soil as green manure amendments. Chitin amendment alone increased the marketable yield of lettuce in 1995 and reduced root-galling ratings and the reproduction of Meloidogyne hapla in both 1995 and 1996. Green manure amendments of sudangrass and rapeseed increased total and marketable yields of lettuce, and decreased root-galling ratings and the reproduction of M. hapla in 1996. Hirsutella rhossiliensis in combination with chitin increased total yield of lettuce over the chitin amendment alone in 1995. The combination of B. thuringiensis, S. costaricanus, and chitin either with or without P. marquandii increased total yield of lettuce over the chitin amendment alone in 1996. In most cases, however, the nematode-antagonistic organisms did not improve lettuce yield or further suppression of M. hapla compared to the chitin amendment alone. The introduced fungi were recoverable from the infested soil. The rifampicin-resistant mutant of B. thuringiensis was not isolated at the end of the season.

Effects of Monoclonal Antibodies, Cationized Ferritin, and Other Organic Molecules on Adhesion of Pasteuria penetrans Endospores to Meloidogyne incognita.

The incidence of adhesion of Pasteuria penetrans endospores to Meloidogyne incognita second-stage juveniles (J2) was studied after pretreatment of the latter with monoclonal antibodies (MAb), cationized ferritin, and other organic molecules in replicated trials. Monoclonal antibodies developed to a cuticular epitope of M. incognita second-stage juveniles gave significant reductions in attachment of P. penetrans endospores to treated nematodes. MAb bound to the entire length of J2 except for the area of the lateral field, where binding was restricted to the incisures. Since reductions in attachment with MAb treatment were modest, it is uncertain if these results implicated a specific surface protein as a factor that interacted in binding of the endospore to the nematode cuticle. Endospore attachment was decreased following treatment of the nematode with the detergents sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB). Endospore attachment to live nematodes was significantly greater than attachment to dead nematodes. Attachment rates of three P. penetrans isolates to M. incognita race 3 varied between isolates. The effects of neuraminidase, pronase, pepsin, trypsin, lipase, and Na periodate on endospore attachment were inconsistent. The cationic dye alcian blue, which binds sulfate and carboxyl groups on acidic glycans, had no consistent effect on endospore attachment. The incidence of endospore attachment was significantly lower but modest, at best, for nematodes that were treated with cationized ferritin alone or cationized ferritin following monoclonal antibody. The lack of consistency or extreme reduction in most experiments suggests that attachment of P. penetrans spores to M. incognita is not specified by only one physico-chemical factor, but may involve a combination of at least two physico-chemical factors (including surface charge and movement of the J2). This points to a need for analysis of combined or factorial treatment effects.

Streptomyces costaricanus sp. nov., isolated from nematode-suppressive soil.

A new bacterial strain, strain CR-43T (T = type strain), which was isolated from tropical soil and was previously shown to have antinematodal and antibiotic properties, is described. The name Streptomyces costaricanus is proposed for this organism. The generic placement of strain CR-43T was based on its typical morphology, its production of LL-diaminopimelic acid, and its fatty acid composition. To clarify the taxonomic position of strain CR-43T, it was compared with the type strains of similar Streptomyces species. The results of a number of biochemical tests and a profile analysis of the hydrolyzable fatty acids indicated that CR-43T differs from previously described species. Strain CR-43 (= ATCC 55274 = NRRL B-16897) is the type strain of S. costaricanus sp. nov.

Control of plant-parasitic nematodes by a nematicidal strain ofAspergillus niger.

An isolate ofAspergillus niger (designated PD-42) was evaluated in laboratory, greenhouse, and field trials for efficacy in controlling plant-parasitic nematodes. In greenhouse experiments, PD-42 drenches containing spores of PD-42 on oatmeal significantly reduced galling on tomato due to root-knot nematode as compared to untreated controls. In a one-half acre field experiment, PD-42 incorporated in seed coats was associated with significantly increased yield and decreased root-knot galling on pepper. In a second one-half acre field experiment, PD-42 drenches significantly reduced tomato and pepper root galling due toMeloidogyne incognita, and nonsignificant yield increases occurred. In each field experiment, treatment with PD-42 reducedRotylenchulus reniformis populations. The nematicidal components of theA. niger culture filtrates include citric acid, oxalic acid, and undetermined molecules larger than 8000 MW.

A novelstreptomyces species for controlling plant-parasitic nematodes.

A novel species ofSteptromyces isolated from nematode suppressive soils in Costa Rica was evaluated for efficacy in controlling plant-parasitic nematodes. This isolate, designated CR-43, was shown to inhibit reproduction ofCaenorhabditis elegans in a laboratory assay. Greenhouse trials utilizing three different methods of treatment with CR-43 gave significant reductions of tomato root galling due toMeloidogyne incognita. In a field experiment in Puerto Rico, in soil naturally infested withM. incognita, CR-43-treated pepper showed significant reductions in root galling and significant increases in yield as compared to untreated controls. In a second experiment in Puerto Rico, a significant reduction in tomato root galling and a slight reduction in root galling on pepper occurred. In this trial, yields on both tomato and pepper were higher in CR-43 treatments, but these differences were not statistically significant. In both experiments populations ofRotylenchulus reniformis were reduced by CR-43 treatment. In a field trial on strawberry in Massachusetts, CR-43-treated plants had lower numbers ofPratylenchus penetrons within roots and showed a significant decrease in black root rot disease. Studies on sterile filtrates from CR-43 cultures indicated that a major determinant of CR-43 antinematodal activity was mostly thermostable macromolecules of molecular weight higher than 6000. Culture filtrates of CR-43 exhibited antifungal activity in vitro.

Studies onPaecilomyces marquandii from nematode suppressive chinampa soils.

Two applications of isolates ofPaecilomyces marquandii from suppressive chinampa soils or P. lilacinus from Peru, fungi that parasitize nematode eggs, generally gave better control of tomato root-knot due toMeloidogyne incognita than did a single application. The effects on root galling by each of thePaecilomyces isolates varied between experiments; however, the ovicidal potential of the three isolates did not differ significantly. Proteins specific for each of the isolates were demonstrated by SDS gel electrophoresis. The results indicate thatP. marquandii is one of the natural soil organisms that contribute to nematode suppression in the chinampa agricultural soils.

Panagrellus redivivus and Caenorhabditis elegans: evidence for the absence of sialic acids.

Complementary experiments were performed to indicate the presence or absence of sialic acids in axenically cultured Panagrellus redivivus and Caenorhabditis elegans. Competitive displacement experiments with radiolabeled Limax flavus agglutinin demonstrated the presence of sialic acid in nematodes grown in medium which contained liver extract as a growth factor but the absence of sialic acid when heme was substituted for liver extract. This finding suggested that sialic acid present in the liver medium was responsible for conflicting results of other studies. Transmission electron microscopy of thin sections from nematodes labeled with an LFA-ferritin conjugate revealed no label to the surface area of the cephalic chemosensilla. Fluorometric analysis with a modification of the thiobarbituric acid assay was negative for sialic acid. Analyses by gas chromatography-mass spectrometry, sensitive to the high picomole range, were also negative for sialic acid. Taken together the results provide evidence for the absence of sialic acid in P. redivivus and C. elegans using the most sensitive and diagnostic technique currently available.

Studies on the surface coat (glycocalyx) of the dauer larva of Anguina agrostis.

Imprints of the surface coat (glycocalyx) from the cuticles of living second stage dauer larvae (DL2) of Anguina agrostis (syn. A. funesta) have been examined using incident light fluorescence microscopy and scanning electron microscopy. These surface coats contain residues of N-acetyl-D-glucosamine which were detected by treatment with wheat germ agglutinin labelled with either fluorescein or rhodamine. They also contain protein which was demonstrated by treatment with either pepsin or trypsin. These enzymes inhibited the attachment of the coryneform bacterium Clavibacter sp. to the surface coat, indicating that proteins play a crucial role in the adhesion of these bacteria to the nematode. This inhibition of attachment was reversed within 18 h after removal of the DL2 from the enzymes, indicating that the nematode was capable of renewing its surface proteins.

Suppression of plant parasitic nematodes in the chinampa agricultural soils.

Soil from the chinampa agricultural system in the Valley of Mexico suppressed damage by plant-parasitic nematodes to tomatoes and beans in greenhouse and growth chamber trials. Sterilization of the chinampa soil resulted in a loss of the suppressive effect, thereby indicating that one or more biotic factors were responsible for the low incidence of nematode damage. Nine organisms were isolated from chinampa soil, which showed antinematodal properties in culture. Naturally occurring populations of plant-parasitic nematodes were of lower incidence in chinampa soil than in Chapingo soil.

Morphological changes in Schistosoma mansoni caused by the mutagen ethyl methane sulphonate.

Three week old Schistosoma mansoni, cultured for up to sixty days after exposure to ethyl methane sulphonate, showed extensive ballooning which often involved most of the worm's body. Other changes included tegumental breakdown which was expressed in varying degrees from mild hernia to total disruption of body shape. Increase in gut size involved stretching of the epithelium and inclusions ranging from masses of disrupted tissue to large, dark opaque masses. Masses were also occasionally seen in the fluid filled spaces of worms. Evidence of hyperplasia was not observed in any of the histological examinations performed.

Lectin studies of surface carbohydrates and induction of gland secretion in the free-living stages ofSchistosoma mansoni.

Secretion from glands was observed when cercariae ofSchistosoma mansoni were exposed to certain lectins. Lectins fromMaclura pomifera, Pisum sativum, andTriticum vulgaris, were effective at 7.5 µ/ml. The effects of cercarial gland secretion caused byT. vulgaris agglutinin andArachis hypogaea agglutinin were blocked by pretreatment with the inhibiting glycan. Discharge of glands was not visualized after exposure of miracidia to lectins. The distribution of five labeled lectins was determined on live miracidia and cercariae. OnlyT. vulgaris agglutinin generally labeled the cercarial and miracidial bodies. Specific labeling occurred with the other lectins on the anterior, in glands or on their secretions, in flame cells in both stages, and on an unidentified ring of cells in miracidia. The possible mechanisms involved in changes caused by the lectins are discussed.

Control of Root-knot Nematodes on Tomato by Lectins.

Significant control of tomato root knot was achieved by applications of the lectins Concanavalin A (Con A) and Limax flavus agglutinin in greenhouse, growth chamber, and microplot trials. Four consecutive weekly applications at lower concentrations of Con A yielded better control than single applications at a higher total concentration. The present state of knowledge on binding of Con A to soil nematodes and the in vitro effect of this lectin in chemotactic behavior are discussed. The mode of action of Con A on root-knot control is unknown.

Caenorhabditis elegans: comparisons of chemotactic behavior from monoxenic and axenic culture.

Significant differences in chemotactic response of Caenorhabditis elegans were demonstrated for nematodes from monoxenic culture as compared to nematodes from axenic culture. These results support those of a previous study in which large differences in growth, development, behavior, and longevity were shown for C. elegans in comparative assays of the monoxenic and axenic regimes.

Fluorescent and Ferritin Labelling of Cuticle Surface Carbohydrates of Caenorhabditis elegans and Panagrellus redivivus.

Caenorhabditis elegans and Panagrellus redivivus were investigated for surface carbohydrates using fluorescent-labelled and ferritin-labelled lectins. Rhodamine-labelled Concanavalin A was specifically located in the cephalic region of both species. Rhodamine-labelled wheat germ agglutinin was located over the entire cuticle of P. redivivus but was absent on C. elegans. Rhodamine-labelled peanut agglutinin and Limax flavus agglutinin did not label nematodes of either species. Galactose and sialic acid were not detected on either species, whereas mannose-glucose residues were specifically localized in the head areas of both species. No detectable N-acetylglucosamine occurred on C. elegans, but it was evenly distributed over the cuticle surface of P. redivivus.

Differential Adhesion and Infection of Nematodes by the Endoparasitic Fungus Meria coniospora (Deuteromycetes).

The conidia of the endoparasitic fungus Meria coniospora (Deuteromycetes) had different patterns of adhesion to the cuticles of the several nematode species tested; adhesion in some species was only to the head and tail regions, on others over the entire cuticle, whereas on others there was a complete lack of adhesion. After adhesion, the fungus usually infected the nematode. However, adhesion to third-stage larvae of five animal parasitic nematodes, all of which carry the cast cuticle from the previous molt, did not result in infection. M. coniospora infected animal parasitic nematodes when this protective sheath was removed. Seven preparations of sialic acid (N-acetylneuraminic acid) gave three types of response in adhesion-infection of nematodes: (i) a significant reduction in conidial adhesions; (ii) no interference with adhesion, but a 10-day delay in infection; and (iii) a delay in infection by 2 to 3 days. The current results support previous findings indicating involvement of sialic acids localized on nematode cuticles in recognition of prey by M. coniospora.