In vitro lectin binding to the outer surface of Spirocerca lupi at different life-stages.

Spirocerca lupi is the esophageal nematode of dogs. Early, transient eosinophilia occurs in experimentally infected dogs, but is absent in advanced cases, suggesting that the nematode evades the dog's immune system. Lectins are proteins or glycoproteins of plant or animal origin, binding different saccharides, with varying specificities and avidities, used to characterize surface haptens in plant and animal parasitic helminths. This study investigated the in vitro binding of six lectins (Concanavalin A [ConA], wheat germ agglutinin [WGA], peanut agglutinin [PNA], soybean agglutinin [SBA], Dolichus biflorus agglutinin [DBA] and Ulex earopaeus agglutinin I [UEA]) to the surface of S. lupi nematodes at different life stages, the L2 and L3 larvae (dead and alive) and to dead adult worms, with negative controls, with and without addition of the six respective inhibitory sugar haptens. Con A moderately bound to surfaces of both live and frozen L3, to the stoma and excretory pores of adult worms, and to the outer surface nematode's eggs, within a female worm, but not to L2. PNA bound only to stoma and excretory pores surfaces in both frozen and live L3. WGA bound strongly to the outer surfaces of live and dead L2 and L3, which resulted in molting of live larvae. These results suggest that the nematode's surface content change during its development. Such changes may play roles in the nematode's interactions with the intermediate and definitive hosts' tissues, and in its ability to evade the immune response, its long survival within the host, and even induce neoplastic transformation.

Interactions between Escherichia coli and the plant-parasitic nematode Meloidogyne javanica.

AIMS: To determine the potential of the plant-parasitic nematode Meloidogyne javanica to serve as a temporary reservoir for Escherichia coli. METHODS AND RESULTS: The adhesion to and persistence of E. coli on the surface of M. javanica were evaluated at different times and temperatures. A pure culture of green fluorescent protein (GFP) tagged E. coli was mixed with ca. 1000 J2 M. javanica for 2 h at 25 degrees C. The nematodes were then washed and the rate of the adhesion of the bacteria to the nematodes was determined by counting the viable nematode-associated E. coli, and by fluorescence microscopy. A dose-dependent adhesion rate was observed only at a bacterium to nematode ratio of 10(4)-10(6) : 1. The adhesion of E. coli to the nematodes was also tested over a 24 h-period at 4 degrees C, 25 degrees C and 37 degrees C. At 4 degrees C and 37 degrees C, maximal adhesion was observed at 5 h; whereas at 25 degrees C, maximal adherence was observed at 8 h. Survival experiments showed that the bacteria could be detected on the nematodes for up to 2 weeks when incubated at 4 degrees C and 25 degrees C, but not at 37 degrees C. CONCLUSIONS: Under laboratory conditions, at 4 degrees C and 25 degrees C, M. javanica could serve as a temporary vector for E. coli for up to 2 weeks. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings support the hypothesis that, in the presence of high concentrations of E. coli, M. javanica might serve as a potential vehicle for the transmission of food-borne pathogens.

A Chemo Attractant in Onion Root Exudates Recognized by Ditylenchus dipsaci in Laboratory Bioassay.

ABSTRACT A quantitative bioassay that translates preferences of axenically cultured and field population of Ditylenchus dipsaci, observed in vitro, into relative attractiveness of sterile root exudates preparations and their components is described. Onion (Allium cepa cv. White Lisbon) root exudates (ORE) are consistently and significantly much more attractive than the buffer control in all these assays. Exudates from oat cv. Lodi, mustard cv. Albatross and tomato cv. Rehovot 13 are significantly more attractive than the buffer but less attractive than ORE; Arabidopsis sp. cv. Landsberg erecta, oil seed rape cv. Cetes and wheat cv. Bet Hashita are as attractive as the buffer, but canary grass and clover exudates are less attractive than the buffer and, therefore, are classified as repellent. No significant differences in relative attractiveness were detected among exudates from other two cultivars of onion (Texas Grano 502 and Granex Hybrid) and one cultivar of leek (Large American Flag), but exudates from one onion (cv. Evergreen Long White Bunching) and one leek (cv. Broad London) were less attractive than ORE. Relative attractiveness is linear in relation to dilution exponent and therefore log-linear in relation to ORE concentration. Host (onion) penetration study reveals that penetration preferences by D. dipsaci follow the same pattern as those predicted by relative attractiveness coefficients estimated in the bio-assays. Preliminary characterization of the chemo attractant from ORE, using the behavioral bioassay, demonstrated that it was stable to heat and to proteolytic enzymes, nonvolatile and water soluble with a molecular mass <700 kDa.

Characterization of Meloidogyne javanica surface coat with antibodies and their effect on nematode behaviour.

The surface coat of the 2nd-stage juveniles (J2) of plant-parasitic nematodes is considered to be involved in interactions with microorganisms in the soil and rhizosphere, as well as with the host plant. Characterization of surface antigens might be important in the development of new nematode control strategies. In this study, polyclonal and monoclonal antibodies raised against Meloidogyne javanica, M. incognita and other plant-parasitic nematodes were tested for their binding to the surface coat and secreted-excreted products of M. javanica. Some of the monoclonal and polyclonal antibodies raised against M. incognita showed cross-reactivity with the surface coat of M. javanica. Western blot analysis of M. javanica surface coat extracts revealed labelling of several polypeptides with a 48 kDa main band for the polyclonal antibody IACR-PC Mi 373, and a 55 kDa main band for PC Mj E2. Further characterization of the antigens recognized by the polyclonal antibody PC Mj E2, in planta, showed that they were present in the parasitic stages J2 and J3 and that the surface coat was shed during root penetration. The hypodermis of the infective juveniles was labelled by PC Mj E2 and the monoclonal antibody IACR-Misec 3F.4, suggesting that these surface antigens are produced in the hypodermis. Nematode behaviour was affected by all the antibodies that bound to the surface coat of the pre-parasitic J2, and we demonstrated that the movement pattern of the M. javanica J2 was affected by these antibodies. Continuous binding of the antibodies to the M. javanica surface inhibited the infection of Arabidopsis thaliana roots on agar plates.

Isolation of a novel collagen gene (Mj-col-5) in Meloidogyne javanica and analysis of its expression pattern.

Mj-col-5, isolated from the plant parasitic nematode Meloidogyne javanica, has a longer carboxy-terminus than other members of the Caenorhabditis elegans COL-6 subfamily of cuticle collagen, including an extra tyrosine residue, and may form altered nonreducible cross-linkages. By semiquantitative determination at different life stages, Mj-col-5 transcript was shown to be more abundant in eggs than in juveniles/young females and adult females. To characterize further this gene's contribution to the changing cuticle of the nematode, we expressed a fusion protein containing a nonconserved 58-amino-acid sequence from the putative Mj-col-5 gene product and raised rabbit antiserum against the fusion protein. The antiserum detected a strongly reacting band (36 kDa, designated MJE36) on western blots of M. javanica eggs extracted with beta-mercaptoethanol. MJE36 was sensitive to collagenase and was not detected on western blots of extracts from M. javanica second-stage juveniles or adult females. A band of the same molecular size was detected in Meloidogyne incognita egg extracts but not in those of Heterodera avenae. Immunoblot indicated that MJE36 is not present in egg shells of M. javanica.

Biological Control of the Root-Knot Nematode Meloidogyne javanica by Trichoderma harzianum.

ABSTRACT The fungal biocontrol agent, Trichoderma harzianum, was evaluated for its potential to control the root-knot nematode Meloidogyne javanica. In greenhouse experiments, root galling was reduced and top fresh weight increased in nematode-infected tomatoes following soil pretreatment with Trichoderma peat-bran preparations. The use of a proteinase Prb1-transformed line (P-2) that contains multiple copies of this gene improved biocontrol activity in the greenhouse experiments compared with the nontransformed wild-type strain (WT). All the Trichoderma strains showed the ability to colonize M. javanica-separated eggs and second-stage juveniles (J2) in sterile in vitro assays, whereas P-2 also penetrated the egg masses. This protease-transformed line presented the same nematicidal and overall proteolytic activity as the WT in in vitro tests in which concentrated soil extracts from Trichoderma-treated soils immobilized the infective J2. However, the J2 immobilization and proteolytic activities of both P-2 and the WT were higher than those obtained with strain T-203. Characterization of the activity of all Trichoderma strains soil extracts on J2 showed that it was heat resistant and restricted to the low-molecular-weight fraction (less than 3 kDa). It is suggested that improved proteolytic activity of the antagonist may be important for the biological control of the nematodes.

Nematicidal activity of essential oils and their components against the root-knot nematode.

ABSTRACT Nematicidal activity of essential oils extracted from 27 spices and aromatic plants were evaluated in vitro and in pot experiments. Twelve of the twenty-seven essential oils immobilized more than 80% of juveniles of the root-knot nematode Meloidogyne javanica at a concentration of 1,000 mul/liter. At this concentration, most of these oils also inhibited nematode hatching. Essential oils of Carum carvi, Foeniculum vulgare, Mentha rotundifolia, and Mentha spicata showed the highest nematicidal activity among the in vitro tested oils. These oils and those from Origanum vulgare, O. syriacum, and Coridothymus capitatus mixed in sandy soil at concentrations of 100 and 200 mg/kg reduced the root galling of cucumber seedlings in pot experiments. The main components of these essential oils were tested for their nematicidal activity. Carvacrol, t-anethole, thymol, and (+)-carvone immobilized the juveniles and inhibited hatching at >125 mul/liter in vitro. Most of these components mixed in sandy soil at concentrations of 75 and 150 mg/kg reduced root galling of cucumber seedlings. In 3-liter pot experiments, nematicidal activity of the essential oils and their components was confirmed at 200 and 150 mg/kg, respectively. The results suggest that the essential oils and their main components may serve as nematicides.

Local and Systemic Induced Resistance to the Root-Knot Nematode in Tomato by DL-beta-Amino-n-Butyric Acid.

ABSTRACT Chemical inducers of pathogenesis-related proteins and plant resistance were applied to tomato plants, with the aim of inducing resistance to the root-knot nematode Meloidogyne javanica. Relative to control plants, foliar spray and soil-drenching with dl-beta-amino-n-butyric acid (BABA) reduced root-galling 7 days after inoculation, as well as the number of eggs 30 days after inoculation. Other chemicals (alpha- and gamma-amino-n-butyric acid, jasmonic acid, methyl jasmonate, and salicylic acid) were either phytotoxic to tomato plants or did not improve control of root-knot nematodes. Fewer second-stage juveniles invaded BABA-treated tomato roots, and root-galling indices were lower than in control tomato plants. Resistance phenomena in seedlings lasted at least 5 days after spraying with BABA. Nematodes invading the roots of BABA-treated seedlings induced small, vacuolate giant cells. Postinfection treatment of tomato plants with BABA inhibited nematode development. It is speculated that after BABA application tomato roots become less attractive to root-knot nematodes, physically harder to invade, or some substance(s) inhibiting nematode or nematode feeding-site development is produced in roots.

Meloidogyne javanica surface proteins: characterization and lability.

Characterization of surface coat (SC) proteins including carbohydrate-binding proteins and glycoproteins of the plant-parasitic nematode Meloidogyne javanica 2nd-stage juvenile (J2) is reported. Extraction of surface proteins with sodium dodecyl sulfate (SDS) and separation by denaturing polyacrylamide gel electrophoresis (SDS-PAGE) results with bands at 6, 9, 14, 22, 26, 31, 46, 49, 58, 66, 80, 205 and 250 kDa. On Western blots, the neoglycoprotein, fucosylated-, mannosylated- and glucosylated-bovine serum albumin, reacted with the 14, 22, 26, 58 and 66 kDa bands. The lectins, Concanavalin A and wheat-germ agglutinin (WGA) labelled surface protein bands of 6, 9, 14, 31, 58 and 66 kDa; WGA also labelled the 22 and 26 kDA bands. Biotin reagents were used to specifically trace surface proteins on live J2. SDS-PAGE of biotinylated J2 extracts revealed only 2 specific biotin-protein bands at 46 and 49 kDa. The labile and transitory nature of Meloidogyne javanica SC was demonstrated by the dynamics of human red blood cells (HRBC) adherence to J2 of different ages. HRBC adherence was also used to demonstrate the SC recovery of detergent-treated J2, which was further exhibited in the SDS-PAGE profiles.

An immunoreactive protein to wheat-germ agglutinin antibody is induced in oat roots following invasion of the cereal cyst nematode Heterodera avenae, and by jasmonate.

A protein that cross-reacts to a wheat-germ agglutinin antibody was induced in oat roots following the invasion of second-stage juveniles (J2) of the cereal cyst nematode Heterodera avenae. This protein, designated ASP45, was acid soluble, and its molecular mass was about 45 kDa on a sodium dodecyl sulfate-polyacrylamide gel. ASP45 was induced in both compatible and incompatible interactions between the nematode and the plant, and also in roots by exposure to jasmonic acid (JA) or methyl jasmonate. However, ASP45 was not induced by elicitors of pathogenesis-related proteins, abscisic acid, or wounding. Lipoxygenase activity, which is involved in JA synthesis, was higher in nematode-infected and JA-treated roots than in their noninfected, untreated counterparts. Inhibition of lipoxygenase activity in roots abolished ASP45 induction in the nematode-infected roots. Amino acid sequences similar to that of ASP45 were found in chitinases of poplar tree and Arabidopsis, even though ASP45 showed no chitinase activity. Although the biological role of ASP45 in infected roots is not clear, JA is suggested to be involved in signal transduction after pathogen invasion of the plant.

The first isolated collagen gene of the root-knot nematode Meloidogyne javanica is developmentally regulated.

The nematode's surface comprises a multilayered cuticle, which consists mainly of collagen proteins. We identified, cloned and characterized the first cuticular collagen gene, Mjcol-3, of the plant-parasitic nematode Meloidogyne javanica. The gene putatively encodes a 32.4-kDa collagen protein, including a propeptide which possesses a subtilisin-like protease-cleavage site. Six introns were identified in the gene sequence, with three slightly different acceptor-splicing sites. The basic structure of the predicted MJCOL-3 protein sequence is highly similar to that of the Caenorhabditis elegans DPY-7, with 65.9% identity between the two amino acid sequences. Relative to DPY-7, the putative MJCOL-3 protein has a shorter carboxy-terminus. This non-conserved feature may indicate different contributions of DPY-7 and MJCOL-3 collagens to the structure of the cuticle. Mjcol-3 is developmentally regulated: transcripts were found mainly in preparasitic developing eggs, less in parasitic third- and fourth-stage juveniles and young females shortly after the fourth molt, and much less in females before egg-laying.

Are Pathogenesis-Related Proteins Induced by Meloidogne javanica or Heterodera avenae lnvasion?

Changes in root- and leaf-soluble proteins were investigated in tomato after invasion by the root-knot nematode Meloidogyne javanica, or in barley and wheat after invasion by the cereal cyst nematode Heterodera avenae. Infection of susceptible tomato plants by M. javanica did not cause any change in the soluble-protein composition of leaves or roots compared with uninoculated plants at an early infection stage. No pathogenesis-related proteins (chitinase, glucanase, or P-14) were induced in the leaf apoplast. Changes in leaf proteins were not observed after invasion of wheat cultivars by H. avenae, whereas, in barley, a few changes in intercellular leaf proteins were recorded in resistant cultivars. These changes, however, were not the same among different H. avenae-resistant cultivars. Protein changes were found at an early stage of infection in barley and wheat roots infected with H. avenae, but no difference was found between resistant and susceptible cultivars.

Attachment of Pasteuria penetrans Endospores to the Surface of Meloidogyne javanica Second-stage Juveniles.

Pasteuria penetrans spore adhesion to Meloidogyne javanica second-stage juveniles (J2) was examined following several different pretreatments of the latter. The detergents sodium dodecyl sulfate and Triton X-100, the carbohydrates fucose and alpha-methyl-D-mannoside, and the lectins concanavalin A and wheat germ agglutinin reduced spore attachment. Spores exposed to M. javanica surface coat (SC) extract exhibited decreased adherence to the J2 surface. Second-stage juveniles that had been treated with antibodies recognizing a 250-kDa antigen of J2 SC extract had fewer spores attached to their surfaces, as compared to nontreated J2, except in the head region. This inhibition pattern was similar to that of antibody-labelling on M. javanica J2 as observed by electron microscopy. It is suggested that several SC components, such as carbohydrate residues, carbohydrate-recognition domains, and a 250-kDa antigen, are involved in P. penetrans spore attachment to the surface of M. javanica.

Gold-conjugated Reagents for the Labeling of Carbohydrate-Recognition Domains and Glycoconjugates on Nematode Surfaces.

Various fluorescent conjugated lectins have been used for the detection of glycoconjugates on nematode surfaces under light microscopy. Several problems have been experienced with these reagents including penetration of the cuticle by fluorescent lectins, non-glycoconjugate specificity, strong nematode autofluorescence at the emission wavelength of the fluorescent dye, and prevention of persistent visualization due to rapid quenching of the fluorescent components. Gold-conjugated reagents combined with silver enhancement alleviated these difficulties when working with three phytonematode species (Heterodera avenae, H. latipons, and Meloidogyne javanica) and two entomopathogenic species (Steinernema carpocapsae and S. glaseri) under light-microscopy visualization of binding by fluorescent lectins and neoglycoproteins. Moreover, gold-conjugated reagents resulted in stable bindings that enabled long-term observations.

Carbohydrate-recognition domains on the surface of phytophagous nematodes.

Human red blood cells (HRBC) adhered to preparasitic second-stage juveniles (J2) of Heterodera avenae, Heterodera schachtii, Meloidogyne javanica, Pratylenchus mediterraneus, Rotylenchulus reniformis, and Tylenchulus semipenetrans over the entire nematode body. Binding was conspicuously confined to the head and tail of Longidorus cohni, Xiphinema brevicolle, and Xiphinema index. Binding was Ca2+ and Mg2+ dependent. In contrast, HRBC did not adhere to Anguina tritici, Aphelenchoides subtenius, Ditylenchus dipsaci, M. javanica females, and Panagrellus redivivus, even in the presence of these cations. Incubation of M. javanica J2 with fucose, glucose, N-acetylglucosamine, mannose, or trypsin decreased the intensity of subsequent HRBC binding, while galactose and N-acetylgalactosamine increased binding intensity. HRBC binding was diminished when nematodes were pretreated with trypsin and eliminated when pretreatments with detergents removed the surface coat. HRBC adhered to nylon fibers coated with surface coat extracted from M. javanica J2; binding was Ca2+ and Mg2+ dependent and diminished when the nylon fibers were coated with bovine serum albumin or preincubated with fucose and mannose. These results demonstrate that HRBC adhesion involves carbohydrate moieties of HRBC and corresponding carbohydrate-recognition domains (CRD) distributed in the nematode surface coat. To our knowledge this is the first report of a surface CRD in the phylum Nematoda.

The surface coat of plant-parasitic nematodes: chemical composition, origin, and biological role-a review.

Chemical composition, origin, and biological role of the surface coat (SC) of plant-parasitic nematodes are described and compared with those of animal-parasitic and free-living nematodes. The SC of the plant-parasitic nematodes is 5-30 nm thick and is characterized by a net negative charge. It consists, at least in part, of glycoproteins and proteins with various molecular weights, depending upon the nematode species. The lability of its components and the binding of human red blood cells to the surface of many tylenchid plant-parasitic nematodes, as well as the binding of several neoglycoproteins to the root-knot nematode Meloidogyne, suggest the presence of carbohydrate-recognition-domains for host plants and parasitic or predatory soil microorganisms (Pasteuria penetrans and Dactylaria spp., for example). These features may also assist in nematode adaptations to soil environments and to plant hosts with defense mechanisms that depend on reactions to nematode surfaces. Surface coat proteins can be species and race specific, a characteristic with promising diagnostic potential.

Rawlsian optimal population size.

"In this paper, I examine the implications of the Rawlsian maximin criterion for optimal population size and intergenerational allocation of resource when fertility is endogenous. I show that whenever children are better-off than their parents in laissez-faire, then the size of the population and parental bequests are also optimal according to the Rawlsian criterion....I then show that by using proper price-based corrective policies, society can achieve a Rawlsian optimal allocation."

Telluria mixta (Pseudomonas mixta Bowman, Sly, and Hayward 1988) gen. nov., comb. nov., and Telluria chitinolytica sp. nov., soil-dwelling organisms which actively degrade polysaccharides.

Pseudomonas mixta (type strain, ACM 1762 [= ATCC 49108], an actively dextranolytic species that possesses both lateral and polar flagella, was compared with the strictly aerobic, rod-shaped, chitinolytic bacterium "Pseudomonas chitinolytica" ACM 3522T (= CNCM I-804) (T = type strain), which has a similar flagellation pattern, by performing phenotypic characterization and DNA-DNA hybridization studies and by analyzing DNA base compositions and 16S rRNA sequences. Our results indicated that "P. chitinolytica" ACM 3522T was phenotypically and genotypically distinct from P. mixta and other phenotypically analogous Pseudomonas spp., Xanthomonas maltophilia, and other aerobic chitin degraders. The 16S rRNA sequences of strains ACM 1762T and ACM 3522T were found to be very similar (97%) to each other and indicated that these organisms are proteobacteria that belong to the beta subclass. The strains were deeply branched in the beta subclass and were distinct from other pseudomonads, including Pseudomonas cepacia, and from Comamonas testosteroni. On the basis of phenotypic, genotypic, and phylogenetic evidence, it is proposed that P. mixta and "P. chitinolytica" ACM 3522T represent two distinct species in a new genus called Telluria. Thus, the genus Telluria gen. nov. contains Telluria mixta comb. nov. and Telluria chitinolytica sp. nov., which are strictly aerobic, rod-shaped, soil-dwelling bacteria that are active polysaccharide degraders.

Ruscus hypophyllum: A New Host for Aphelenchoides fragariae.

Aphelenchoides fragariae was isolated from the phylloclades of the ornamental plant Ruscus hypophyllum (Liliaceae). Rotylenchus buxophilus, Scutellonema brachyurum, and Meloidogyne were identified as the most common plant-parasitic nematodes in the soil near the roots. The pathology and life history of A. fragariae were closely related to the climate. To our knowledge, this is the first report of R. hypophyllum as a host of plant-parasitic nematodes.