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.

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.

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.

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.

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.

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.

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.

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.

Infection of Narcissus Roots by Aphelenchoides subtenuis.

The widespread destruction of commercially grown bulbs of Narcissus tazetta papyraceus (Paper White) has been reported in Israel. This phenomenon is usually characterized by a premature yellowing of the foliage, accompanied by root rot and dark, sunken basal plates. This study confirmed thatAphelenchoides subtenuis is the main cause of the basal plate disease of Narcissus. In contrast to other Aphelenchoides species, which feed on stems or leaves, A. subtenuis penetrates Narcissus roots. In our experiments, in winter (6 to 8 weeks after penetration), nematodes laid their eggs in the root parenchymal cells without inducing obvious symptoms on foliage or roots. Toward spring, juveniles became numerous throughout the parenchymal cells of the root cortex. Consequently, the root system collapsed rapidly, at the usual peak of bulb and foliage production. Bulbs of infected plants were small and weighed less than those of uninfected plants, and foliage became necrotic prematurely. At that time, in field conditions, secondary elements like Fusarium penetrate the bulb and cause it to rot, given this syndrome the common name of basal plate disease. To our knowledge, this is the first report of an Aphelenchoides species as a root pathogen.

Glycoprotein Characterization of the Gelatinous Matrix in the Root-knot Nematode Meloidogyne javanica.

Proteinaceous components of freshly formed gelatinous matrix (GM) of the root-knot nematode Metoidogyne javanica were analyzed. Under reducing conditions, the prominent protein fragments had molecular weights of 26 to 66 kDa and 150 to >200 kDa, and most were glycosylated. Most of the fragments were digested by proteinase K, and fewer by trypsin. The lectins soybean agglutinin (SBA), Ulex europaeus agglutinin, and wheat germ agglutinin labeled the higher molecular weight bands (i.e., >200 kDa). SBA labeled additional protein fractions between 26 and 66 kDa. Although Bandeiraea simplicifolia lectin and Concanavalin A did not label bands on the Western blot, they did label the GM in the dot blot technique. Analysis of amino acids and amino sugars in the GM revealed an unusually high amount of ammonia and galactosamine moieties.

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.

Stage-specific Differences in Lectin Binding to the Surface of Anguina tritici and Meloidogyne incognita.

The occurrence and distribution of several lectin binding sites on the outer surfaces of eggs, preparasitic second-stage juveniles (J2), parasitic second-stage juveniles (PJ2), females, and males of two tylenchid nematodes, Anguina tritici and Meloidogyne incognita race 3, were compared. In both species, a greater variety of lectins bound to the eggs than to other life stages; lectin binding to eggs was also more intense than it was to other life stages. Species-specific differences also occurred. More lectins bound to the amphids or amphidial secretions of M. incognita J2 than to the amphids or amphidial secretions of A. tritici J2. Lectins also bound to the amphids or amphidial secretions of adult male and female A. tritici, but binding to the cuticle occurred only at the head and tail and was not consistent in all specimens. Canavalia ensiformis and Ulex europaeus lectins bound specifically to the outer cuticle of M. incognita. Several other lectins bound nonspecifically. Oxidation of the cuticle with periodate under mild conditions, as well as pretreatment of the nematodes with lipase, markedly increased the binding of lectins to the cuticle of A. tritici J2 but not, in most cases, to M. incognita J2 or eggs of either species.

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.

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.

Characterization of sialyl and galactosyl residues on the body wall of different plant parasitic nematodes.

The plant parasitic nematodes Helicotylenchus multicinctus, Meloidogyne javanica, Tylenchulus semipenetrans, and Xiphinema index, differing in their host specificity and parasitic habits, were analyzed as to their cuticle surface sialyl, galaclosyl, and/or N-acetylgalactosaminyl residues. The procedure involved the selective oxidation of sialic acid and galactose/N-acetylgal-actosamine residues using periodate and galactose oxidase, respectively, to form reactive aldehyde groups. These functional groups were coupled directly with a new hydrazide-containing compound, the fluorescent reagent lissamine rhodamine-beta-alanine hydrazide, or they were utilized to introduce DPN-groups to the nematode cuticle. The distribution of the DNP-tagged glycoconjugates was visualized by treating the nematodes with rabbit anti-DNP antibody and staining with fluorescein isothiocyanate (FITC)-labeled goat antirabbit IgG. Sialo residues were observed along the entire outer body wall of the first three aforementioned nematodes, but there were some differences in reaction among the various life stages within the species. In X. index, sialo residues were sited in the tail and head areas, mainly on the lips, oral opening, amphid apertures and stylet. Galactose oxidase treatments revealed galactose on N-acytylgalactosamine residues on T. sentipenetrans and X. index, but there were no indications that their presence was dependent on the developmental stage. Trypsin, pronase, and neuraminidase pretreatment completely abolished the fluorescence in T. semipenetrans but did not alter the sialo residue binding reaction in H. multicinctus or M. javanica, indicating possible differences in the outer body wall saccharide structure and composition between these nematodes. The existence and nature of sugar residues on the cuticle surface of nematodes could contribute to an understanding of the specific recognition by phytophagous nematodes of their host, and perhaps also of the virus transmission mechanism in those nematodes which serve as vectors.

Use of Chitin for Controlling Heterodera avenae and Tylenchulus semipenetrans.

The nematicidal effect of chitin, relative to other pesticides, was evaluated against two plant-parasitic nematodes, Heterodera avenae and Tylenchulus semipenetrans. Wheat seedlings, grown in soils artificially or naturally infested with H. avenae, were treated with 0.4% (w/w) ClandoSan (CLA) prepared from crustacean chitin, aldicarb (Temik 15G), or ethylene dibromide (EDB 90EC). The CLA treatment significantly increased wheat straw, ear, and average grain dry weights of nematode-infected plants, compared with the other two treatments. In an experiment covering two consecutive seasons, all three treatments reduced the number of cysts in the soil by 60%. In a one-season experiment, CLA reduced the number of cysts by 51% and aldicarb or EDB reduced cyst number by about 40%. A reduction of 50-90% in T. semipenetrans population densities on roots of two citrus rootstocks was recorded following an application of 0.2% (w/w) CLA to the soil.

A Collagenolytic Fungus, Cunninghamella elegans, for Biological Control of Plant-parasitic Nematodes.

The root-galling index of tomatoes inoculated with Meloidogyne javanica was decreased 70% when collagen was used as a soil amendment (0.1% w/w) and 90% when the amendment was supplemented with the collagenolytic fungus Cunninghamella elegans. The root-galling index was reduced 80% when the fungus was homogenized in collagen culture medium and added to soil without collagen supplement. Culture filtrates of the fungus C. elegans, grown on collagen as a single source of carbon and nitrogen, immobilized M. javanica second-stage juveniles and inhibited egg hatch. Root galling was reduced when tomato plants were inoculated with filtrate-treated juveniles. Culture filtrates reduced the motility of Rotylenchulus reniformis and Xiphinema index, but they had less effect on Anguina tritici and almost no effect on Ditylenchus dipsaci. Cunninghamella elegans had collagenolytic, elastolytic, keratinolytic, and nonspecific proteolytic activities when grown on collagen media, but only chitinolytic activity when grown on chitin media.