Free-living Nematode Panagrellus Research Articles

Compatibility study of Duddingtonia flagrans conidia and its crude proteolytic extract

This study aimed to evaluate the concomitant use of the nematophagous fungus Duddingtonia flagrans (AC001) and its protease-rich crude extract for the in vitro control of Panagrellus sp., Haemonchus spp., and Trichostrongylus spp. The nematicidal tests were carried out on larvae of the free-living nematode Panagrellus sp. and infective larvae of the gastrointestinal parasitic nematodes of domestic ruminants (Haemonchus spp. and Trichostrongylus spp). Five experimental groups were set: (1) one control group (G1) and (4) four treated groups –G2 – active crude extract; G3 – denatured crude extract; G4 – fungus, and G5 – fungus + active extract. Plates were incubated at 28 ºC for 24 h followed by the recovery of the larvae using the Baermann technique. The results showed a lower recovery of Panagrellus sp. larvae in the experimental groups compared to the control group, as follows: 52 % (G2), 16 % (G3), 46 % (G4), and 77 % (G5). An even greater reduction (77 ± 5 %) occurred in the group (G5). In addition, the authors observed lower averages of L3 of Haemonchus spp. and Trichostrongylus spp. in the experimental groups compared to the control group, as follows: 59 % (G2), 0 % (G3), 86 % (G4), and 76 % (G5). In turn, there was a difference (p < 0.01) between (G5) and (G2). The results this study indicate a positive effect from the compatible use of the D. flagrans fungus and its enzymatic crude extract (protease), which has been demonstrated here for the first time and with potential field applications for further designs.

Bacillus toyonensis biovar Thuringiensis: A novel entomopathogen with insecticidal activity against lepidopteran and coleopteran pests

The Bacillus cereus group includes eight species: Bacillus anthracis, B. cereus, Bacillus cytotoxicus, Bacillus mycoides, Bacillus pseudomycoides, Bacillus thuringiensis, Bacillus weihenstephanensis and Bacillus toyonensis, which are highly related at the phylogenetic level. In this work, we performed the isolation and characterization of a novel Bacillus sp. strain exhibiting parasporal crystals with insecticidal activity that was initially classified as a Bacillus thuringiensis strain. Its genome encoded three genes showing homology to known pesticidal proteins from B. thuringiensis and Lysinibacillus sphaericus proteins: Cry7Ga1 (a crystal protein), a Mpp2Aa3 (ETX/Mtx2 family) homolog and a mosquitocidal-like protein (NPP1). However, since its genome sequence shared > 98% ANI with several Bacillus toyonensis genomes, the strain has subsequently been renamed as Bacillus toyonensis biovar Thuringiensis and designated Bto-UNVM_94. Bioassays demonstrated that this novel strain exhibited toxicity against Cydia pomonella (Lepidoptera: Tortricidae) and Anthonomus grandis (Coleoptera: Curculionidae), a low toxicity against Aedes aegypti (Diptera: Culicidae) and Alphitobius diaperinus (Coleoptera: Tenebrionidae) whereas no toxicity was shown to the free-living nematode Panagrellus redivivus (Rhabditida: Panagrolaimidae).

Taxonomic and biological characterization and predatory activity of four nematophagous fungi isolates of Arthrobotrys species from Tapachula, Chiapas, Mexico

ABSTRACT A batch of nematophagous fungi (NF), isolated from soil and animal faeces at Tapachula, Chiapas, Mexico, was identified through morphological methods and molecular tools. Additionally, the fungi were biologically characterised through their in vitro predatory capability against the free-living nematode Panagrellus redivivus. Molecular identification of nine isolates was achieved by amplifying the 18S, 5.8S and 28S regions using ITS5 and ITS4 oligonucleotides, followed by sequencing and aligning through BLAST. Only species of Arthrobotrys genus: A. oligospora, A. conoides, A. arthrobotryoides and A. musiformis were found. The in vitro bioassay revealed a large variability in the predatory capabilities of the different isolates. The isolate Z1-1-2 of A. arthrobotryoides showed the highest nematode reduction (NR) from 822.8 (± 169.5) to only 51.4 (± 35.2) = 93.7% NR; in contrast A. oligospora (isolate Z3-2-2) showed the lowest NR from 548.5 (± 66.6) to 234.2 (± 123.6) = 57.2% Genotyping analysis corroborated the morphological identification. The comparison of sequences with the NCBI sequences showed a close phylogenetic relationship. A phylogenetic tree was generated to compare its phylogenetic relationship with other strains reported in the NCBI. The NF isolated could be potential candidates for future studies on the biological control of nematodes.

Butlerius butleri (Nematoda: Diplogasteridae) Feeds on Haemonchus contortus (Nematoda: Trichostrongylidae) Infective Larvae and Free-Living Nematodes in Sheep Faecal Cultures Under Laboratory Conditions: Preliminary Report.

This study assessed the feeding activity of the predatory nematode Butlerius butleri on the infective larvae of the sheep parasitic nematode Haemonchus contortus (Hc-L3) and on two free-living nematodes Panagrellus redivivus (Pr) and Rhabditis sp. (R) in sterile sheep faecal cultures. A bioassay was carried out in sheep faecal cultures, where predatory and prey nematodes were put together to assess the predatory behaviour on the different nematodes. Faecal cultures (n = 9) were set up in different treatments as follows: Treatments 1, 2 and 3 contained 20 specimens of B. butleri and 2000 Hc-L3, Pr and R, respectively; treatments 4, 5 and 6, contained only 2000 Hc-L3, Pr and R, respectively, as control groups. Treatment 7 contained 20 B. butleri as control. All treatments were incubated at room temperature for 30days. The average number of nematodes recovered from each treatment was obtained and the reduction rate with respect to their corresponding control was estimated. Data were analysed using an ANOVA test using a completely randomised design in the SAS programme. Butlerius butleri reduced the Hc-L3, population by 91.8%, whereas its population increased 131.6 times. The interactions between B. butleri and Pr and R resulted in 80.9% and 62.5% nematode reduction and B. butleri increased its populations 268.3 and 82.7 times, respectively. This experiment was performed only once, so this can be considered a preliminary report at this time. This study provides evidence for the first time that B. butleri feeds on one of the most economically important nematodes of small ruminants, Hc-L3.

Antioxidant activity of crude extracts obtained from endophytic fungi isolated from Otoba gracilipes of dry tropical forest in Colombia

The nematode-trapping fungus Duddingtonia flagrans has been studied as a possible control method for gastrointestinal nematodes of livestock animals. These fungi capture and infect the nematode by cuticle penetration, immobilization, and digestion of the internal contents. It has been suggested that this sequence of events occurs by a combination of physical and enzymatical activities. The aim of this study was to investigate the participation of proteolytic enzymatic activity during the interaction of the nematophagous fungus D. flagrans with infective larvae of trichostrongylides and the free-living nematode Panagrellus spp. Protease inhibitors used interfered in the predatory activity of D. flagrans. However, only PMSF significantly reduced the mean number of Panagrellus spp. captured by D. flagrans in comparison with the control. The experiment with fluorogenic substrate showed that maximum urokinase activity during the interaction of the fungus with the infective larvae of trichostrongylides or Panagrellus spp. occurred within 7 or 1 h of incubation, respectively. The protease activity, especially of the serine class, may be important during the interaction between the fungus and nematodes.

Effects of Zinc Oxide Nanoparticles on Panagrellus redivivus (Nematoda) and Folsomia candida (Collembola) in Various Test Media

The toxic effects of zinc oxide (ZnO) nano- and non-nanoparticles on two species of soil organisms, the free-living nematode Panagrellus redivivus and the springtail Folsomia candida, were investigated. The toxicity of ZnO particles (with 15 and 140 nm average particle size) and ZnCl2 (used as a positive control for zinc ion dissolution) was tested in two different kinds of test media. Milli-Q water vs. soil solution was used for the tests involving P. redivivus, and plaster of paris vs. artificial soil for F. candida. For P. redivivus, application of the soil solution decreased the toxic effect. It is considered that this may be due to the less dissolved zinc ions, the organic matter content and ZnO aggregation. Furthermore, all of the compounds caused concentration-dependent mortality to the tested nematode species. In the tests with F. candida, the toxic effects of both ZnO and ZnCl2 were significantly lower in the tests involving plaster of paris. This could have resulted from the avoidance of the contaminated foods by the springtails. A decreasing effect on reproduction was observed only in the tests using artificial soil. Additionally, the ZnO particles of 140 nm significantly increased mortality in the soil solution test with P. redivivus and in the artificial soil in the tests with F. candida. Possibly, this is a consequence of a greater aggregation of the nanoparticle-sized ZnO at the end of the experiment, which resulted in a lower level of toxicity.

Hirsutella thompsonii and Pochonia chlamydosporia (Syn. Verticillium chlamydosporium) Mycelia Growth and Predation on Panagrellus redivivus

This research aimed to evaluate the nematophagous ability of 4077-Verticillium chlamydosporium var. chlamydosporium and 4466-Hirsutella thompsonii isolates and relate mycelia growth to the influence provoked by movement of nematodes. Each fungus grew in PDA (potato, dextrose, agar) medium end up to pure colonization. Then, ten mycelia plugs of 8 mm diameter were removed from colony borders and transferred to the center of ten Petri plates containing water-agar 2% medium. These plates were previously divided into four quadrants that received a number of 25 individuals of free-living nematodes (Panagrellus redivivus), composing a total of 100 nematodes per plate. Evaluations started after 24 hours of interaction, considering predation percentage and mycelia growth as stimuli of nematodes presence. Results showed growing predation performance to both isolates, being higher for V. chlamydosporium var. chlamydosporium since from first evaluation time, controlling more than 50% of nematode population initially added. Its predation potential was 39.2%, 38.4% and 48.35% higher than H. thompsonii at first, second and third evaluation day, respectively. Generally, nematodes did not stimulate mycelia growth, unless for H. thompsonii at 72 hours of interaction compared to control plates (without nematodes). Stress resulting from isolates transference from PDA to water-agar 2% resulted in sparse mycelia growth and it could have affected the predation performance of H. thompsonii that controlled nematodes in low levels throughout experiment. Independently of predation level, pictures revealed that both isolates has ability to control P. redivivus through hyphae penetration.

Yellow Pigment Aurovertins Mediate Interactions between the Pathogenic Fungus Pochonia chlamydosporia and Its Nematode Host

Nematophagous fungi are globally distributed soil fungi and well-known natural predators of soil-dwelling nematodes. Pochonia chlamydosporia can be found in diverse nematode-suppressive soils as a parasite of nematode eggs and is one of the most studied potential biological control agents of nematodes. However, little is known about the functions of small molecules in the process of infection of nematodes by this parasitic fungus or about small-molecule-mediated interactions between the pathogenic fungus and its host. Our recent study demonstrated that a P. chlamydosporia strain isolated from root knots of tobacco infected by the root-knot nematode Meloidogyne incognita produced a class of yellow pigment metabolite aurovertins, which induced the death of the free-living nematode Panagrellus redivevus. Here we report that nematicidal P. chlamydosporia strains obtained from the nematode worms tended to yield a total yellow pigment aurovertin production exceeding the inhibitory concentration shown in nematicidal bioassays. Aurovertin D was abundant in the pigment metabolites of P. chlamydosporia strains. Aurovertin D showed strong toxicity toward the root-knot nematode M. incognita and exerted profound and detrimental effects on the viability of Caenorhabditis elegans even at a subinhibitory concentration. Evaluation of the nematode mutation in the β subunit of F1-ATPase, together with the application of RNA interference in screening each subunit of F1FO-ATPase in the nematode worms, demonstrated that the β subunit of F1-ATPase might not be the specific target for aurovertins in nematodes. The resistance of C. elegans daf-2(e1370) and the hypersensitivity of C. elegans daf-16(mu86) to aurovertin D indicated that DAF-16/FOXO transcription factor in nematodes was triggered in response to the aurovertin attack. These findings advance our understanding of the roles of aurovertin production in the interactions between nematodes and the pathogen fungus P. chlamydosporia.

Serine proteases activity is important for the interaction of nematophagous fungus Duddingtonia flagrans with infective larvae of trichostrongylides and free-living nematodes Panagrellus spp.

The nematode-trapping fungus Duddingtonia flagrans has been studied as a possible control method for gastrointestinal nematodes of livestock animals. These fungi capture and infect the nematode by cuticle penetration, immobilization, and digestion of the internal contents. It has been suggested that this sequence of events occurs by a combination of physical and enzymatical activities. The aim of this study was to investigate the participation of proteolytic enzymatic activity during the interaction of the nematophagous fungus D. flagrans with infective larvae of trichostrongylides and the free-living nematode Panagrellus spp. Protease inhibitors used interfered in the predatory activity of D. flagrans. However, only PMSF significantly reduced the mean number of Panagrellus spp. captured by D. flagrans in comparison with the control. The experiment with fluorogenic substrate showed that maximum urokinase activity during the interaction of the fungus with the infective larvae of trichostrongylides or Panagrellus spp. occurred within 7 or 1 h of incubation, respectively. The protease activity, especially of the serine class, may be important during the interaction between the fungus and nematodes.

Effects of catechin polyphenols and preparations from the plant–parasitic nematode Heterodera glycines on protease activity and behaviour in three nematode species

Protease activities in preparations from the plant-parasitic nematodes Heterodera glycines and Meloidogyne incognita and the free-living nematode Panagrellus redivivus were inhibited by exposure to a series of eight catechin polyphenol analogues, (+)-catechin, (-)-epicatechin (EC), (-)-gallocatechin (GC), (-)-epigallocatechin (EGC), (-)-catechin gallate (CG), (-)-gallocatechin gallate (GCG), (-)-epicatechin gallate (ECG) and (-)-epigallocatechin gallate (EGCG) (1 mm each), and by a preparation from H. glycines cysts. General protease activity detected with the FRET-peptide substrate QXL520-KSAYMRF-K(5-FAM)a and proteasome chymotrypsin-like (CTL) activity detected with succinyl-LLVY-AMC were each inhibited significantly more (P < 0.05) by the gallated form of the polyphenol than by the corresponding non-gallated form. Species differences in response to inhibition across all analogues were revealed with the CTL substrate, but CG was a consistently potent inhibitor across all three species and with each substrate. A heat-stable component (CE) from H. glycines cysts inhibited M. incognita CTL activity by 92.07 ± 0.68%, significantly less (P < 0.05) in H. glycines (52.86 ± 2.77%), and by only 17.24 ± 0.55% (P < 0.05) in P. redivivus preparations. CTL activity was, however, inhibited more than 60% in all preparations by the proteasome-specific inhibitor MG-132. Hatching of M. incognita infective juveniles exposed to 1 mm CG, ECG, GCG or EGCG was reduced by 83.88 ± 4.26%, 69.98 ± 9.14%, 94.93 ± 1.71% and 87.93 ± 2.89%, respectively, while hatching of H. glycines was reduced less than 25% by each analogue. CE had no effect on nematode hatch, but did cause a 60% reduction in mobility of H. glycines infective juveniles exposed overnight to CE in vitro, which was more (P < 0.05) than the reduction of M. incognita infective juvenile mobility (20%).

Uso de productos derivados de bacillus thuringiensis como alternativa de control en nematodos de importancia veterinaria. revisión

Bacillus thuringiensis is an entomopathogenic bacterium that produces crystal-proteins with cytotoxic activity against insects and nematodes. B. thuringiensis toxicity on agriculture pests is widely recognized, however, there is little information about the lethal action of B. thuringiensis against nematodes. Recently, the nematicide activity of B. thuringiensis proteins was showed against Haemonchus, Teladorsagia, Nippostrongylus, and Ancylostoma genera of mammals, and against the plant parasitic nematodes, Globodera and Meloidogyne. From a list of B. thuringiensis strains with nematicide effect, selected proteins from the IB-16 isolate showed 50 to 100 % nematocidal efficacy against different developing stages of H. contortus. Additionally the in vitro assay showed evidence about the lethal activity of this strain against the free-living nematodes Panagrellus redivivus and Caenorhabditis elegans. The Cry5B protein of B. thuringiesis caused tissue damage on the C. elegans mid-gut and its action probably involves specific gut-receptor, similar to reporte with nematodes of importance in agriculture. Also, Cry5B protein-receptors link appears to involve carbohydrate moieties on intestinal cells, and cause tissue damage and nematodes death. Through these studies, selected B. thuringiensis proteins could be considered in future trials as potential alternative tools of control against parasitic nematodes of domestic animals, such as ruminants and other pathogens of mammals and even from agriculture crops.

Kinetics of capture and infection of infective larvae of trichostrongylides and free-living nematodes Panagrellus sp. by Duddingtonia flagrans

Duddingtonia flagrans, a nematode-trapping fungus, has been investigated as an agent for biological control against infective larvae of gastrointestinal nematode parasites of production animals. The initial process of nematode-trapping fungi infection is based on an interaction between the trap structure of the fungus and the surface of the nematode cuticle. This report investigates by light and scanning electron microscopy the kinetics of capture and infection during the interaction of D. flagrans with the infective larvae (L(3)) of trichostrongylides and the free-living nematode Panagrellus sp. D. flagrans was cultivated for 7 days in a Petri dish containing agar-water. L(3) and Panagrellus sp. were inoculated in the Petri dishes and the samples consisting of agar-L(3)-fungi and agar-Panagrellus sp.-fungi were collected after 10, 20, 30, 40, 50, 60, and 70 min and 3, 4, 5, 10, 15, 20, and 25 h of interaction. All samples were observed by light microscopy. The samples with 1, 5, 15, and 25 h of interaction were also analyzed by scanning electron microscopy. The interaction was monitored up to 25 h. An initial differentiation of predation structures was observed after 30 min of interaction. The presence of traps and of captured L(3) or Panagrellus sp. occurred after 70 min. The live captured nematodes were observed up to 3 h of interaction. However, after 4 h, all Panagrellus sp. were dead. It took 15 h of interaction for the fungus to invade the L(3), and the presence of hyphae inside the nematode near the region of penetration was evident. At this time, the hyphae had filled the whole body of Panagrellus sp. The complete occupation of the body of L(3) occurred at 20 h of interaction and with 25 h the nematode was completely damaged except for the cuticle. Although the double cuticle of L(3) slows the penetration of D. flagrans, it was possible to verify that the process of trap formation and capture occurs quickly when both nematodes were tested, suggesting that the organisms would eventually be killed once in contact with the fungi encouraging the use of the fungus as a biological control agent.

Overexpression of the Key Virulence Proteases Bace16 and Bae16 in Bacillus nematocida B16 to Improve Its Nematocidal Activity

Proteases Bace16 and Bae16, an alkaline serine protease and a neutral protease, respectively, in the nematocidal bacterium Bacillus nematocida B16, have been identified as two key virulence factors and shown to have remarkable nematotoxic activities against the free-living nematode Panagrellus redivius and the plant parasite nematode Bursaphelenchus xylophilus. To facilitate the successful biological control application of this organism in the field, we genetically altered the strain B. nematocida B16 and optimized its growth condition to overexpress these two pathogenic proteases. The recombinant integration vectors of pAX01-Bace16 and pAX01-Bae16 for overexpressing the two proteases were constructed and successfully transformed into competent cells of the bacterium B. nematocida B16. The optimal induction condition for overexpressing Bace16 is 2% xylose at 37°C for 48 h. Our analyses showed that the proteolytic activity and nematocidal activity of the strain overexpressing Bace16 increased by about 62 and 80%, respectively, over the wild-type strain. However, our tested induction conditions could not significantly improve either the proteolytic activity or the nematocidal activity of the Bae16 overexpression mutant.

Nematodetoxic Aurovertin-Type Metabolites from a Root-Knot Nematode Parasitic FungusPochonia chlamydosporia

Chemical investigation of one fungal strain P. chlamydosporia YMF 1.00613 isolated from root knots of tobacco infected by Meloidogyne incognita led to the isolation and identification of four aurovertin-type metabolites, which include a new compound, aurovertin I (A1), and three known metabolites, aurovertins E, F and D (A2-A4). Their structures were established by spectroscopic studies such as 1D- and 2D-NMR and MS analysis. Aurovertin I (A1) is the first natural product with an aurovertin skeleton with one less carbon. Compounds A3 and A4 showed the toxicity to the worms of the free-living nematode Panagrellus redivevus with the LC(50) values 88.6 and 41.7 microg/mL at 48 h, respectively. All four aurovertins did not show obvious inhibitory effects on egg hatch of root knot nematode Meloidogyne incognita. The results suggested that the aurovertin-type metabolites produced by P. chlamydosporia might be one of the pathogenic factors involved in the suppression of nematodes.

Acid phosphatase activity during the interaction of the nematophagous fungus Duddingtonia flagrans with the nematode Panagrellus sp

The use of Duddingtonia flagrans, a nematode-trapping fungus, has been investigated as a biological control method against free living larvae of gastrointestinal nematodes of livestock animals. This fungus captures and infects the nematode by cuticle penetration, immobilization and digestion of the internal contents. It has been suggested that this sequence of events occurs by a combination of physical and enzymatical activities. This report characterizes the acid phosphatase activity during the interaction of D. flagrans with the free-living nematode Panagrellus sp. The optimum pH for the hydrolysis of the acid phosphatase substrate p-nitrophenyl phosphate was 2.2, 2.8 and 5.4 from D. flagrans alone and 2.2 and 5.4 for Panagrellus sp alone, fungus–nematode interaction in liquid medium and fungus–nematode interaction in solid medium. Different acid phosphatase activity bands were detected by SDS–PAGE. Maximum acid phosphatase activity of the fungus or nematode alone and of the fungus–nematode interaction occurred within 70 min of incubation in the presence of the substrate 4-methylumbelliferyl phosphate. The activity of this enzyme was significantly higher for the fungus–nematode interaction when compared to the organisms alone, indicating a synergistic response. Furthermore, structures appeared in the hyphae after 30 min, nematodes were observed adhered after 40 min and many were captured by the typical fungus traps after 70 min of interaction. The participation of acid phosphatase activity and its importance during the interaction of the fungus with the nematode were discussed.

Digestion of invertebrate neuropeptides by preparations from the free-living nematodePanagrellus 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.

Evaluation and identification of potential organic nematicidal volatiles from soil bacteria

Naturally occurring volatile compounds with nematicidal activities (NAs) are of significant economical importance in agriculture and forestry. In this study, volatile organic compounds (VOCs) produced by 200 isolates of soil bacterial were evaluated in in vitro experiments. Our results identified that among the 200 bacterial isolates, 149 (74.5%) and 165 (82.5%) exhibited a greater than 20% NA against the free-living nematode Panagrellus redivivus and the pinewood nematode Bursaphelenchus xylophilus, respectively. Among them, 22 isolates showed 100% NA against P. redivivus and seven isolates showed 100% NA against B. xylophilus. When exposed to nematicidal volatiles, nematodes gradually reduced their movements within 1–12 h after treatment, and most stopped moving completely after 24 h. Our analysis indicated significant variation in nematicidal capability of the VOCs not only among bacterial species but also among isolates of the same species. Volatiles of representative isolates were extracted using solid-phase micro-extraction (SPME) and their structures identified using gas chromatography–mass spectrometry (GC–MS). The detected so volatile compounds included alcohols, aldehydes, ketones, alkenes and ethers. Of the 20 VOCs with strong NA (⩾80%), nine (phenol, 2-octanol, benzaldehyde, benzeneacetaldehyde, decanal, 2-nonanone, 2-undecanone, cyclohexene and dimethyl disulfide) displayed 100% NA to both model nematodes. Furthermore, five compounds (terpineol, benzeneethanol, propanone, phenyl ethanone and nonane) showed different NA to B. xylophilus (75–100%) and P. redivivus (17.02–100%).

Digestion and Assimilation of the Free-living Nematode Panagrellus redivivus Fed to First Feeding Coregonid Larvae: Evidence from Histological and Isotopic Studies

The free-living nematode Panagrellus redivivus is a potential source of live food for first feeding fish. The digestion and assimilation of nematodes by larval fish were investigated with the aid of a histological and stable isotope approach. Larvae of whitefish Coregonus lavaretus were reared for 8 d with nematodes and compared with an unfed control. Nematodes were readily ingested by 3-d-old larvae. Different stages of nematode digestion could be observed in transverse sections of fish larvae sampled on Day 6 at regular intervals after feeding. Nematodes were produced on corn medium. In this way nematodes with a stable carbon isotope signature clearly different from the isotopic pattern of the fish larvae could be obtained. Stable carbon isotope signatures for lipids and lipid-free matter of fish larvae sampled on Days 2 and 8 after first feeding were clearly influenced by the stable isotopic pattern of the nematodes. The high acceptance of the nematodes by Coregonus lavaretus larvae and the early onset of digestion and nutrient retention positively confirm the potential of PanagreUus redivivus as a live food for first feeding fish larvae.

Cloning and characterization of an extracellular serine protease from the nematode-trapping fungus Arthrobotrys conoides

An extracellular serine protease (Ac1) with a molecular mass of 35 kDa was purified from the nematode-trapping fungus Arthrobotrys conoides. The optimum activity of Ac1 is at pH 7.0 and 53.2 degrees C (over 20 min). Ac1 can degrade a broad range of substrates including casein, gelatin, bovine serum albumin, collagen, and nematode cuticles. Moreover, the enzyme can immobilize the free-living nematode Panagrellus redivivus and the pine wood nematode Bursaphelenchus xylophilus, indicating Ac1 may be involved in infection against nematodes. The encoding gene of Ac1 contains one intron of 60-bp and two exons encoding a polypeptide of 411 amino acid residues. The deduced polypeptide sequence of Ac1 showed a high degree of similarity to two previously reported serine proteases PII and Mlx from other nematode-trapping fungi (81% aa sequence identity). However, three proteases Ac1, Aoz1 and Mlx showed optimum temperatures at 53.2, 45 and 65 degrees C, respectively. Compared to PII, Ac1 appears to have a significantly higher activity against gelatin, bovine serum albumin, and non-denatured collagen. Moreover, our bioassay experiments showed that Ac1 is more effective at immobilizing P. redivivus than B. xylophilus.

Purification and cloning of a novel serine protease from the nematode-trapping fungus Dactylellina varietas and its potential roles in infection against nematodes

From the culture filtrate of the fungus Dactylellina varietas (syn. Dactylella varietas), an extracellular protease (designed Dv1) was purified by cation exchange and hydrophobic interaction chromatography. The purified protease showed a molecular mass of approximately 30 kDa and displayed an optimal activity at pH 8 and 60.5 degrees C (more than 20 min). This protease could degrade a broad range of substrates including casein, gelatin, BSA (bovine serum albumin), and nematode cuticle. However, its proteolytic activity was highly sensitive to the serine protease inhibitor Phenylmethylphonylfuoride (1 mM), indicating that it belongs to the serine-type peptidase group. This protease could immobilize the free-living nematodes Panagrellus redivivus and Caenorhabditis elegans and hydrolyze the purified cuticle of P. redivivus, suggesting it may play a role in infection against nematodes. The encoding gene of Dv1 and its promoter sequence were cloned using degenerate primers and the DNA walking technology. Its open-reading frame contains 1,224 base pairs and without any intron. The deduced amino-acid sequence shared low identity to serine proteases from other nematode-trapping fungi. Our report identified a novel pathogenic protease from the nematode-trapping fungus D. varietas, and the three-dimensional structure of this protease was predicted using the Swiss-Prot method.