Aminopeptidases in Caenorhabditis elegans and Panagrellus redivivus: detection using peptide and non-peptide substrates

Aminopeptidase activities were detected in extracts of the free-living nematodes Caenorhabditis elegans and Panagrellus redivivus using the aminoacyl substrate L-alanine-4-nitroanilide. The activities exhibited similarities in Km (C. elegans = 2.22 mM; P. redivivus = 2.09 mM) and specific activity (C. elegans = 1.38 +/- 0.43 mAU min(-1) x g(-1); P. redivivus, 1.23 +/- 0.18m AU min(-1) microg(-1). Each is inhibited competitively by amastatin (C. elegans IC50 = 0.46 microM; P. redivivus IC50 = 15.90 microM) and non-competitively by leuhistin (C. elegans IC50 = 3.00 microM; P. redivivus IC50 = 37.35 microM). The bioactive peptides adipokinetic hormone and substance P decrease the apparent aminopeptidase activities of each extract suggesting that the peptides compete with the Ala-pNA as substrates. With each extract, adipokinetic hormone appeared to be the more effective substrate. Digestion of adipokinetic hormone by C. elegans and P. redivivus extracts in the presence and absence of 1 mM amastatin produced distinct chromatographic profiles that suggest different digestion patterns for the two species. However, amastatin had clear effects on chromatographic profiles from each species indicating that an aminopeptidase is involved in the digestion of the peptide substrates. The data presented indicate that extracts of free-living nematodes are capable of metabolizing peptide hormones, and that this metabolism involves substrate-selective aminopeptidases.     

Responses of Heterodera glycines and Meloidogyne incognita to exogenously applied neuromodulators

Biogenic amines regulate important behaviours in nematodes and are associated with pharyngeal activity in plant-parasitic nematodes. A robust behavioural assay based upon nematode body movements was developed to expand the study of these and other neuroregulators in plant-parasitic nematodes. Dopamine, octopamine and serotonin each had significant but differing effects on the behaviour of soybean cyst nematode Heterodera glycines and root-knot nematode Meloidogyne incognita juveniles. Body movement frequency was increased twofold in H. glycines by 5 mM dopamine (P = 0.0001), but decreased by 50 mM dopamine in H. glycines (88%) and M. incognita (53%) (P < 0.0001). Movement frequency in both species was increased by 50-70% (P < 0.0001) by 50 mM octopamine, and 5 mM octopamine increased M. incognita movement frequency more than twofold (P < 0.0001). Movement frequency in each species was reduced by more than 90% by 5 mM serotonin (P < 0.0001). While amplitude of body movement in H. glycines was unaffected by any amine, it was significantly reduced in M. incognita by all amines (P < 0.0006). Stylet pulsing frequencies in either species were unaffected by dopamine or octopamine, but 5 mM serotonin stimulated pulsing in H. glycines by nearly 13-fold (P < 0.0001) and in M. incognita by more than 14-fold (P < 0.0001). The invertebrate neuropeptide FLRFamide (N-Phe-Leu-Arg-Phe) increased M. incognita body movement frequency 45% (P = 0.02) at 1 mM but did not affect stylet activity. Finally, H. glycines egg hatch was completely suppressed by 50 mM serotonin, and partially suppressed by 50 mM dopamine (75%; P < 0.0001) and 50 mM octopamine (55%; P < 0.0001).     

Toxicity of 2,4-diacetylphloroglucinol (DAPG) to Plant-parasitic and Bacterial-feeding Nematodes

The antibiotic 2,4-diacetylphloroglucinol (DAPG) is produced by some isolates of the beneficial bacterium Pseudomonas fluorescens. DAPG is toxic to many organisms, and crop yield increases have been reported after application of DAPG-producing P. fluorescens. This study was conducted to determine whether DAPG is toxic to selected nematodes. The plant-parasitic nematodes Heterodera glycines, Meloidogyne incognita, Pratylenchus scribneri and Xiphinema americanum, and the bacterial-feeding nematodes Caenorhabditis elegans, Pristionchus pacificus, and Rhabditis rainai, were immersed in concentrations ranging from 0 to 100 μg/ml DAPG. Egg hatch and viability of juveniles and adults were determined. DAPG was toxic to X. americanum adults, with an LD(50) of 8.3 μg/ml DAPG. DAPG decreased M. incognita egg hatch, but stimulated C. elegans hatch during the first hours of incubation. Viability of M. incognita J2 and of C. elegans J1 and adults was not affected. There were no observed effects on the other nematodes. The study indicated that DAPG is not toxic to all nematodes, and did not affect the tested species of beneficial bacterial-feeding nematodes. Augmentation of DAPG-producing P. fluorescens populations for nematode biocontrol could be targeted to specific nematode species known to be affected by this compound and by other antibiotics produced by the bacteria, or these bacteria could be used for other possible effects, such as induced plant resistance.     

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.     

Coprinus comatus damages nematode cuticles mechanically with spiny balls and produces potent toxins to immobilize nematodes

We reported recently a unique fungal structure, called the spiny ball, on the vegetative hyphae of Coprinus comatus (O. F. Müll.:Fr.) Pers. Although some observations regarding the role of this structure were presented, its function remained largely unknown. In this study, we showed that purified (isolated and washed) spiny balls could immobilize and kill the free-living nematode Panagrellus redivivus Goodey highly efficiently. Scanning electron microscopy studies illustrated that the spiny structure damaged the nematode cuticle, suggesting the presence of a mechanical force during the process of nematode immobilization. Severe injuries on nematode cuticles caused the leakage of inner materials of the nematodes. When these structures were ground in liquid nitrogen, their killing efficacy against nematodes was lost, indicating that the shape and the complete structure of the spiny balls are indispensable for their function. However, extraction with organic solvents never lowered their activity against P. redivivus, and the extracts showed no obvious effect on the nematode. We also investigated whether C. comatus was able to produce toxins which would aid in the immobilization of nematodes. In total, we identified seven toxins from C. comatus that showed activity to immobilize the nematodes P. redivivus and Meloidogyne incognita (Kofoid et White) Chitwood. The chemical structures of these toxins were identified with nuclear magnetic resonance, mass spectrometry, infrared, and UV spectrum analysis. Two compounds were found to be novel. The toxins found in C. comatus are O-containing heterocyclic compounds.     

Morphological variability and molecular phylogeny of the nematophagous fungus Monacrosporium drechsleri

An isolate of the nematode-trapping fungus Monacrosporium drechsleri was collected from cultures of the root-knot nematode Meloidogyne arenaria that had been maintained on tomato roots in greenhouse pots in Beltsville, Maryland. The plant-parasitic nematodes Heterodera glycines, Meloidogyne incognita and Pratylenchus zeae and the free-living nematodes Caenorhabditis elegans and Panagrellus redivivus were placed on colonies of M. drechsleri grown in Petri dishes to study ability of the isolate to trap various nematode hosts. None of the nematodes placed near adhesive knobs were motile within 1 d. To determine where M. drechsleri fits within the existing phylogeny of nematode-trapping fungi, the ITS1-ITS2 regions of rDNA and the nuclear gene EF1-alpha were sequenced for the new isolate of M. drechsleri, for the species M. parvicolle and M. lysipagum, and for an isolate of M. ellipsosporum distinct from the one listed in GenBank. Parsimony trees were constructed showing the closest molecular relative of M. drechsleri to be the newly sequenced isolate of M. ellipsosporum; the latter had a highly divergent sequence from the sequence recorded in GenBank for a different isolate of M. ellipsosporum. Unique, consistent and discrete morphological characters are absent in these related taxa, so an independent molecular character should be considered essential for their accurate identification.     

FMRFamide-like immunoreactivity within the nervous system of the nematodes Panagrellus redivius, Caenorhabditis elegans and Heterodera glycines

A polyclonal antiserum raised against the molluscan neuromodulatory peptide Phe-Met-Arg-Phe-amide (FMRFamide) reacts with nervous tissue in the free-living nematodes Panagrellus redivius and Caenorhabditis elegans and in infective juveniles (J₂) of the soybean cyst nematode Heterodera glycines . Sectioning of the nematodes was unnecessary but penetration of the antibody was improved by cutting the animals or by use of plasma etching to breach the cuticle before incubation in antiserum. Both procedures required subsequent exposure to detergents or partial digestion with protease K for optimum immunoreactivity. A positive reaction was observed for all three nematodes in the longitudinal nerve cords, nerve ring and ventral and lateral ganglia. Neurones were also visualized in association with the vulva in the free-living species and the spicules of male P. redivius . In H. glycines , neurones innervating the pharynx reacted positively, as did two loops of neural tissue on either side of the ventral nerve cord slightly anterior to the anus. Immunoreactivity was also noted in the amphidial pouches of H. glycines after prolonged (15 min) protease treatment. Further work is needed to establish the structure of the peptide antigen localized in these nematodes.     

Digestion of invertebrate neuropeptides by preparations from the free-living nematode Panagrellus redivivus

Proteases in the soluble fraction of homogenates prepared from the free-living nematode Panagrellus redivivus hydrolysed the amidated invertebrate neuropeptides FMRFa and FLRFa, and nematode FMRFa-like peptides (FLPs) KPNFLRFa (FLP-1-H), APKPKFIRFa (FLP-5-A), KNEFIRFa (FLP-8), KPSFVRFa (FLP-9), RNKFEFIRFa (FLP-12) and KHEYLRFa (FLP-14) in vitro. Results were assessed by analysing reaction components with RP-HPLC, UV detection at 210 nm and peak integration. Based upon substrate peak size, more than 90% of most of the peptide substrates was consumed after 1 h at 27 degrees C, but digestion was not complete even with a crude protease mixture. Two peptides, FLP-12 and FLP-14, were significantly less susceptible to digestion than the others. FLP-12 was the least susceptible of all sequences (71% loss; P < 0.0001), while FLP-14 was digested less (84% loss; P < 0.0004) than all but FLP-12. Product peak digestion patterns of FLP-12, a second nonapeptide (FLP-5-A), and FMRFa, incubated with aminopeptidase (amastatin) and serine endoprotease (AEBSF) inhibitors, demonstrated highly specific behaviours of each sequence to protease cleavage. Amastatin significantly (P < 0.03) reduced digestion of FLP-12 (54% loss) and FMRFa (61% loss; P < 0.0005), but had no effect on FLP-5-A. AEBSF had no protective effect on FMRFa but significantly decreased hydrolysis of FLP-5-A (77% loss; P < 0.0001) and FLP-12 (59% loss; P < 0.03). The combination of both inhibitors had additive effects only for FMRFa (34% loss; P < 0.0005). Further analysis of FMRFa digestion using peptides with D-amino acid substitutions demonstrated nearly complete protection of FdMRFa (2% loss; P < 0.0001) from all proteolytic digestion, whereas digestion of FMRdFa was complete. Results suggest that in addition to aminopeptidase and serine proteases, both deamidase and aminopeptidase P participate in neuropeptide metabolism in P. redivivus.     

Coprinus comatus: A basidiomycete fungus forms novel spiny structures and infects nematode

Nematophagous basidiomycete fungi kill nematodes by trapping, endoparasitizing and producing toxin. In our studies Coprinus comatus (O.F.Müll. : Fr.) Pers. is found to be a nematode-destroying fungus; this fungus immobilizes, kills and uses free-living nematode Panagrellus redivivus Goodey and root-knot nematode Meloidogyne arenaria Neal. C. comatus produces an unusual structure designated spiny ball. Set on a sporophore-like branch, the spiny ball is a burr-like structure assembled with a large number of tiny tubes. Purified spiny balls exhibit moderate nematicidal activity. Experiments show that spiny balls are not chlamydospores because of the absence of nuclei in the structures and quick formation within 3 d in a young colony. Nematodes added to C. comatus cultures on potato-dextrose agar (PDA) and cornmeal agar (CMA) become inactive in hours. Infection of nematodes by the fungus occurs only after the nematodes are immobilized (feeble or dead), probably by a toxin. Electron micrographs illustrate that C. comatus infect P. redivivus by producing penetration pegs with which hyphae colonize nematode bodies. An infected nematode is digested and consumed within days and hyphae grow out of the nematode.     

Impact of the nematophagous fungus Pochonia chlamydosporia on nematode and microbial populations

The microbial and nematode populations associated with two plants (tomato and cabbage) inoculated with the nematophagous fungus, Pochonia chlamydosporia var. chlamydosporia or root knot nematode (Meloidogyne incognita), or both, were compared with those in unplanted controls. The dominant factor affecting culturable microbial populations was found to be the presence or absence of tomato plants. Generally microbial colony counts were lowest in unplanted soil, small increases were associated with cabbage and significantly greater numbers with tomato plants. Differences in microbial diversity (estimated from community profiles of carbon substrate utlisation, using Biolog) were observed between planted and unplanted soils, however, there were few differences between soils with either of the two plants. The presence of P. chlamydosporia was associated with a reduction in the numbers of plant parasitic nematodes (51%-78%) including the migratory ectoparasites, whereas free-living nematodes, culturable bacteria and bacterial populations assessed by Biolog were unaffected by the application of fungus.     

Sterol biosynthesis in the free-living nematode Panagrellus redivivus

The fate of the known sterol precursor squalene 2,3-oxide was investigated in the free-living nematode Panagrellus redivivus. The nematodes were cultured axenically in the presence of [4-(3)H]squalene 2,3-oxide. Radioactivity was found in the total lipids of the isolated nematodes. Essentially all of the radioactivity encountered in the total lipids was found in the non-saponifiable fraction. The components present in the non-saponifiable fraction were separated and isolated by t.l.c. Three labelled components were identified by a combination of t.l.c., g.l.c. and mass spectroscopy. It is established that P. redivivus has the capacity for biosynthesis of lanosterol. No labelled C(27) sterols could be detected.     

Purification and cloning of an extracellular serine protease from the nematode-trapping fungus Monacrosporium cystosporium

An extracellular protease (Mc1) was isolated from the nematode-trapping fungus Monacrosporium cystosporium by gel filtration, anion-exchange, and hydrophobic interaction chromatographies. This protease had a molecular mass of approximately 38 kDa and displayed an optimal activity at pH 7-9 and 56 degrees (over 30 min). Its proteolytic activity was highly sensitive to the serine protease inhibitor PMSF (phenylmethylsulfonylfluoride, 0.1 mM), indicating that it belonged to the serine-type peptidase group. The Michaelis constant (Km) and Vmax for substrate N-Suc-Ala-Ala-Pro-Phe-pNA were 1.67x10-4 M and 0.6071 OD410 per 30 s, respectively. This protease could degrade a broad range of substrates including casein, gelatin, BSA (bovine serum albumin), and nematode cuticle. Moreover, the enzyme could immobilize the free-living nematode Panagrellus redivivus and the pine wood nematode Bursaphelenchus xylophilus, suggesting that it might play a role in infection against nematodes. The encoding gene of Mc1 was composed of one intron and two exons, coding for a polypeptide of 405 amino acid residues. The deduced amino acid sequence of Mc1 showed 61.4-91.9% identity to serine proteases from other nematode-trapping fungi. Our results identified that Mc1 possessed biochemical properties including optimal reaction condition and substrate preference that are different from previously identified serine proteases.     

Integrated management of root-knot nematode, Meloidogyne incognita infestation in tomato and grapevine

An integrated approach with the obligate bacterial parasite, Pasteuria penetrans and nematicides was assessed for the management of the root-knot nematode, Meloidogyne incognita infestation in tomato and grapevine. Seedlings of tomato cv. Co3 were transplanted into pots filled with sterilized soil and inoculated with nematodes (5000 juveniles/pot). The root powder of P. penetrans at 10 mg/pot was applied alone and in combination with carbofuran at 6 mg/pot. Application of P. penetrans along with carbofuran recorded lowest nematode infestation (107 nematodes/200 g soil) compared to control (325 nematodes/200 g soil). The rate of parasitization was 83.1% in the carbofuran and P. penetrans combination treatment as against 61.0% in the P. penetrans treatment only. The plant growth was also higher in the combination treatment compared to all other treatments. A field trial was carried out to assess the efficacy of P. penetrans and nematicides viz., carbofuran and phorate in the management of root-knot nematode, M. incognita infestation of grapevine cv. Muscat Hamburg. A nematode and P. penetrans infested grapevine field was selected and treatments either with carbofuran or phorate at 1 g a.i/vine was given. The observations were recorded at monthly interval. The results showed that the soil nematode population was reduced in nematicide treated plots. Suppression of nematodes was higher under phorate (117 nematodes/200 g soil) than under carbofuran (126.7 nematodes/200 g soil) treatment. The number of juveniles parasitized was also influenced by nematicides and spore load carried/juvenile with phorate being superior and the increase being 17.0 and 29.0% respectively over the control. The results of these experiment confirmed the compatibility of P. penetrans with nematicides and its biological control potential against the root-knot nematode.     

Inheritance of resistance to the root-knot nematode Meloidogyne javanica in lettuce

Resistance to the root-knot nematodes Meloidogyne spp. would be a valuable attribute of lettuce Lactuca sativa L. cultivars grown in tropical regions. The looseleaf lettuce 'Grand Rapids' is resistant to both M. incognita and M. javanica. Resistance to M. incognita has a high heritability, under the control of a single gene locus, in which the 'Grand Rapids' allele, responsible for resistance (Me), has predominantly additive gene action, and has incomplete penetrance and variable expressivity. We studied the inheritance of the resistance of 'Grand Rapids' (P(2)) to M. javanica in a cross with a standard nematode-susceptible cultivar Regina-71 (P(1)). F(1)(Regina-71 x Grand Rapids) and F(2) seed were obtained, and the F(2) inoculated, along with the parental cultivars, with a known isolate of M. javanica to evaluate nematode resistance. A high broad sense heritability estimate (0.798) was obtained for gall indices. Class distributions of gall indices for generations P(1), P(2), and F(2) were in agreement with theoretical distributions based on a monogenic inheritance model for the range of assumed degrees of dominance between approximately -0.20 and 0.20. M. javanica resistance appears to be under control of a single gene locus, with predominantly additive gene action. Whether or not the Grand Rapids allele imparting resistance to M. javanica is the same Me allele imparting resistance to M. incognita remains to be determined.     

Two FMRFamide-like peptides from the free-living nematode Panagrellus redivivus.

Peptides of the FXRFamide family, where X = M, I or L, are broadly distributed among invertebrates. Two such peptides were purified and sequenced from the free-living nematode, Panagrellus redivivus. Immunohistochemical techniques localized FMRFamide-like material in several regions of these organisms, including the nerve cords and, most prominently, in paired groups of cells located caudally to the base of the pharynx. RIA determinations gave an estimate of 2.8 nmol immunoreactive peptide/g of an acetone extract of P. redivivus. Four sequential HPLC purification steps, followed by sequencing by automated Edman degradation and FAB-MS, led to the identification of Ser-Asp-Pro-Asn-Phe-Leu-Arg-Phe-amide (SDPNFLRFamide) and Ser-Ala-Asp-Pro-Asn-Phe-Leu-Arg-Phe-amide (SADPNFLRFamide) as members of the FXRFamide family in this nematode.     

Characterization of a neutral serine protease and its full-length cDNA from the nematode-trapping fungus Arthrobotrys oligospora

A neutral serine protease (designated Aoz1) was purified to homogeneity from a strain of Arthrobotrys oligospora, obtained from soil in Yunnan Province. The purified protein showed a molecular mass of approximately 38?000 Dalton, pI 4.9 and displayed optimal activity at 45 C and pH 6-8. The protein could hydrolyze gelatin, casein and the chromogenic substrate azocoll, and it could immobilize nematodes in vitro (Panagrellus redivivus L. [Goodey]). The level of activity in culture medium was found to increase with increasing gelatin concentration. Scanning electron micrographs demonstrated dramatic structural changes in nematode cuticle treated with the purified protease. A partial peptide sequence obtained by N-terminal sequence analysis was used to design degenerate primers for the isolation of a cDNA gene encoding the mature protease. Analysis of the cDNA and corresponding genomic sequence revealed 97% identity with PII, a gene previously described from A. oligospora, and we conclude that this gene is likely a PII ortholog.     

A fungal lectin and its apparent receptors on a nematode surface

Abstract A lectin was isolated from the nematode-trapping fungus Arthrobotrys oligospora . This carbohydrate-binding protein was developmentally regulated and was found only on trap-bearing mycelia. The lectin receptor on the surface of the nematode Panagrellus redivivus has, furthermore, been investigated using homogenates from whole nematodes or nematode cuticle. The ability of macromolecules from nematodes, fractionated according to M _r and lectin affinities, to inhibit the capture of nematodes by A. oligospora was tested using an inhibition assay, based on a simple dialysis membrane technique. One major lectin-binding glycoprotein, with apparent M _r of 65 000, was isolated from the nematode cuticle.     

Development of a low-cost technology for mass production of the free-living nematode <Emphasis Type="Italic">Panagrellus redivivus</Emphasis> as an alternative live food for first feeding fish larvae

The free-living nematode Panagrellus redivivus is a suitable food source for first feeding fish. In the present report, a new method for the mass production of P. redivivus is presented. The technique involves multiplication of the nematode in monoxenic (single microorganism: Saccharomyces cerevisiae) solid culture (fluid media supported by 1- to 4-cm(3) sponge cubes) in autoclavable plastic bags (size range: 50 x 30 cm to 75 x 67 cm). Two growing media were tested: oat-meal medium (OM), which is an oat-based medium (16.7% oat-meal flour in 0.8% saline solution), and purified ingredient medium (PIM), a semi-synthetic medium (1.64% meat peptone, 0.94% yeast extract, 12.6% corn starch, 0.24% glucose, 1.48% sunflower oil, in 0.8% saline solution). The bags were inoculated with 350 nematodes/g medium. After an average period of 12 days (11-13 days) at 25 degrees C, the average yield (number of nematodes/g medium) was 241 x 10(3) for OM and 333 x 10(3) for PIM in 12-l bags (50 x 30 cm). The production scale has currently reached a bag volume of 50 l (75 x 67 cm); using PIM and the conditions described above, it was possible to harvest more than 1.3 x 10(9) nematodes/bag (291 x 10(3) nematodes/g medium). In PIM, when sun flower oil was replaced with the same amount of fish oil or cod liver oil, yields of 259 x 10(3) and 290 x 10(3) nematodes/g medium, respectively, were attained. The technology for mass production and formulation of P. redivivus should enable fish-hatchery operators to rely on a cheap, standardised, and permanently available live food product for first feeding fish larvae.