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PubMed2004

Specificity of association between Paenibacillus spp. and the entomopathogenic nematodes, Heterorhabditis spp.

Endospore-forming bacteria, Paenibacillus spp., have recently been isolated in association with insect pathogenic nematodes Heterorhabditis spp. Sporangia adhere to nematode infective juveniles (J3) and are carried with them into insects. Paenibacillus proliferates in the killed insect along with Heterorhabditis and its obligate bacterial symbiont, Photorhabdus, despite the antibiotic production of the latter. Nematode infective juveniles leave the insect cadaver with Paenibacillus sporangia attached. The specificity of the relationship between Paenibacillus and Heterorhabditis was investigated. Sporangia of nematode-associated Paenibacillus adhered to infective juveniles (but not other stages) of all Heterorhabditis species tested, and to infective juveniles of vertebrate parasitic Strongylida species, but not to a variety of other soil nematodes tested. Paenibacillus species that were not isolated from nematodes, but were phylogenetically close to the nematode-associated strains, did not adhere to Heterorhabditis, and they were also sensitive to Photorhabdus antibiotics in vitro, whereas the nematode-associated strains were not. Unusual longevity of the sporangium and resistance to Photorhabdus antibiotics may represent specific adaptations of the nematode-associated Paenibacillus strains to allow them to coexist with and be transported by Heterorhabditis. Adaptation to specific Heterorhabditis-Photorhabdus strains is evident among the three nematode-associated Paenibacillus strains (each from a different nematode strain). Paenibacillus NEM1a and NEM3 each developed best in cadavers with the nematode from which it was isolated and not at all with the nematode associate of the other strain. Differences between nematode-associated Paenibacillus strains in cross-compatibility with the various Heterorhabditis strains in cadavers could not be explained by differential sensitivity to antibiotics produced by the nematodes' Photorhabdus symbionts in vitro.

Animals↗
PubMed2006

Differences in penetration routes and establishment rates of four entomopathogenic nematode species into four white grub species.

We compared the penetration of the entomopathogenic nematodes Steinernema scarabaei (AMK001 strain), S. glaseri (NC1 strain), Heterorhabditis zealandica (X1 strain), and H. bacteriophora (GPS11 strain) into third-instars of the scarabs Popillia japonica, Anomala orientalis, Cyclocephala borealis, and Rhizotrogus majalis. When larvae were exposed to nematodes for 6-72 h larval mortality and nematode establishment rate and occasionally speed of kill often showed the same pattern within nematode-white grub combinations. But no two nematodes or white grub species had the same pattern for these observations for all white grub or nematode species, respectively. Mortality, establishment, and speed of kill followed a similar pattern for H. zealandica, S. glaseri, and S. scarabaei, but there was no clear relationship for H. bacteriophora. Significant nematode establishment was only observed after at least 48 h exposure in most nematode-white grub combinations. Faster establishment was observed only for H. zealandica in A. orientalis and R. majalis (after 24 h) and for S. scarabaei in P. japonica and R. majalis (after 12 h). Nematode establishment after 72 h in the different scarab species was generally low for S. glaseri (<1.5%) and H. bacteriophora (<3%), higher for H. zealandica (2-5%), and the highest for S. scarabaei (1-14%). However, in another experiment establishment was generally higher after 96h exposure. Nematode penetration sites were determined by comparing nematode establishment in larvae with mouth, anus, mouth+anus, or none sealed with glue. The trends for each nematode species were very similar in the different white grub species. H. zealandica and H. bacteriophora showed excellent cuticular penetration ability but may also penetrate through mouth and/or anus. S. glaseri also penetrated through the cuticle but lower establishment in larvae with mouth or mouth+anus sealed suggested that the mouth is an important penetration site. S. scarabaei showed a preference for the mouth as a penetration site, but it showed some cuticular penetration ability and may also use the anus as a penetration site. The methodology used cannot exclude that cuticular penetration also included penetration through the spiracles. To fully understand the effect of nematode and white grub species on nematode virulence, future studies will have to compare host immune response to the penetrating IJs and the role of the symbiotic bacteria in these interactions.

Animals↗
PubMed2005

A novel approach to identify plant parasitic nematodes using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Plant parasitic nematodes are difficult to identify because different species are morphologically similar, and this makes their control more difficult. The aim of this work was to develop a rapid, simple method to identify plant parasitic nematodes, based on analysis of protein profiles of nematodes generated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Two methods have been used: grinding and direct analysis of intact nematodes. Both methods were standardised using the nematode Anguina tritici (wheat seed-gall nematode) as a model. Development of the approach involved optimisation of experimental parameters to generate reproducible diagnostic protein profiles for plant parasitic nematodes. With alpha-cyano-4-hydroxycinnamic acid (CHCA) as the matrix, the most effective solvent extraction was with 90% acetone. With sinapinic acid (SA) as matrix, 90% ethanol was most effective. When intact nematodes were analysed directly by mixing with the matrix solution, 40 min extraction with CHCA matrix solution generated the best protein profiles. The standardised methods were applied to analyse the seed-gall nematodes A. tritici and A. funesta and to the root-knot nematode, Meloidogyne javanica, which infects many horticultural crops. Typical protein profiles and diagnostic peaks were identified for these nematode species and for mixtures of Anguina species. The results provide 'proof-of-concept' that these nematode species can be identified by protein profiling using MALDI-TOFMS. This new approach could be extended to identify other plant and non-plant parasitic nematodes.

Animals↗
PubMed2002

Enhancement of entomopathogenic nematode production in in-vitro liquid culture of Heterorhabditis bacteriophoraby fed-batch culture with glucose supplementation.

Nematode yield is a decisive factor for successful large-scale commercial production of entomopathogenic nematode. Various carbon sources were tested in in-vitro liquid culture to improve the yield of the entomopathogenic nematode Heterorhabditis bacteriophora. Canola oil was the optimal carbon source for nematode culture compared to carbohydrates when applied as a sole carbon source. However, when some of carbohydrates were applied together with canola oil, significant increases in nematode yield were observed. When 25 mg glucose ml(-1) was supplemented to 25 mg oil-based liquid culture medium ml(-1), the highest nematode yield, 3.62 x 10(5) infective juveniles, was achieved at 12 days, but nematode growth was suppressed at higher than 75 mg glucose ml(-1). A fed-batch culture process was introduced in nematode liquid culture consisting of two growth phases: bacteria and nematode. In the oil fed-batch culture, in which only glucose was initially added and oil was fed to the culture after the bacterial growth phase concurrent with nematode inoculation, nematode yield increased up to 4.25 x 10(5) infective juveniles ml(-1), while the batch culture resulted in 3.60 x 10(5) infective juveniles ml(-1). These results indicate that glucose is a superior carbon source for the bacteria, whereas canola oil is optimal for the nematode. The application of fed-batch culture provides significant enhancement of nematode yield in in-vitro liquid culture.

Animals↗
PubMedDate not supplied

Insect cellular and chemical limitations to pathogen development: the Colorado potato beetle, the nematode Heterorhabditis marelatus, and its symbiotic bacteria.

This research examines possible factors limiting pathogen development and reproduction in a novel host insect. The nematode Heterorhabditis marelatus and its symbiotic bacterium, Photorhabdus luminescens, kill 98% of nematode-treated Colorado potato beetle (CPB) prepupae, but the nematode reproduces in only 1-6% of beetles. We examined nematode/bacterial inhibition at each step of the normal developmental pathway to determine host feature(s) limiting nematode reproduction. We found that in vivo encapsulation of nematodes occurred in only 1.6% of CPB, and in 5% of in vitro hanging drops of hemolymph. Thus, the cellular defense system did not strongly limit nematode reproduction in the CPB. The symbiotic bacterium was negatively affected by a heat-labile factor found in the CPB's hemolymph which often caused the bacterium to switch from the primary form that produces antibiotics and nutrients necessary for the nematodes' development, to a secondary form that provides only limited nutrients. A 58 kDa protein was isolated and bioassayed for activity against P. luminescens, but caused a delay in bacterial growth rather than the primary-secondary form switch. Thus, the identity of the heat-labile factor could not be confirmed as being the 58 kDa protein. The heat-labile factor did not directly affect the nematode. The addition of lipids in the form of olive oil to heated CPB hemolymph allowed nematodes to reproduce in 17% of hanging drops, in contrast to zero reproduction in hemolymph without oil. Reproductive nematodes were smaller when grown in CPB hemolymph than in hemolymph of the highly susceptible Galleria mellonella. These data suggest that both the toxic heat-labile factor and a lack of appropriate nutrients alter the CPB-bacterium-nematode interaction. These factors preclude the use of this otherwise highly effective nematode-bacterial complex in the longterm control of the CPB.

Animals↗
PubMed2003

Susceptibility of the boll weevil to Steinernema riobrave and other entomopathogenic nematodes.

The susceptibility of the boll weevil (BW), Anthonomus grandis Boheman, to Steinernema riobrave and other nematode species in petri dishes, soil (Hidalgo sandy clay loam), and cotton bolls and squares was investigated. Third instar weevils were susceptible to entomopathogenic nematode (EN) species and strains in petri dish bioassays at 30 degrees C. Lower LC(50)'s occurred with S. riobrave TX- 355 (2 nematodes per weevil), S. glaseri NC (3), Heterorhabditis indicus HOM-1 (5), and H. bacteriophora HbL (7) than H. bacteriophora IN (13), S. riobrave TX (14), and H. bacteriophora HP88 (21). When infective juveniles (IJs) of S. riobrave were applied to weevils on filter paper at 25 degrees C, the LC(50) of S. riobrave TX for first, second, and third instars, pupae, and 1-day-old and 10-days-old adult weevils were 4, 5, 4, 12, 13, and 11IJs per weevil, respectively. The mean time to death, from lowest to highest concentration, for the first instar (2.07 and 1.27days) and second instar (2.55 and 1.39days) weevils were faster than older weevil stages. But, at concentrations of 50 and 100IJs/weevil, the mean time to death for the third instar, pupa and adult weevils were similar (1.84 and 2.67days). One hundred percent weevil mortality (all weevil stages) occurred 3days after exposure to 100IJs per weevil. Invasion efficiency rankings for nematode concentration were inconsistent and changed with weevil stage from 15 to 100% when weevils were exposed to 100 and 1IJs/weevil, respectively. However, there was a consistent relationship between male:female nematode sex ratio (1:1.6) and nematode concentration in all infected weevil stages. Nematode production per weevil cadaver increased with increased nematode concentrations. The overall mean yield of nematodes per weevil was 7680IJs. In potted soil experiments (30 degrees C), nematode concentration and soil moisture greatly influenced the nematode efficacy. At the most effective concentrations of 200,000 and 400,000IJs/m(2) in buried bolls or squares, higher insect mortalities resulted in pots with 20% soil moisture either in bolls (94 and 97% parasitism) or squares (92 and 100% parasitism) than those of 10% soil moisture in bolls (44 and 58% parasitism) or squares (0 and 13% parasitism). Similar results were obtained when nematodes were sprayed on the bolls and squares on the soil surface. This paper presents the first data on the efficacy of S. riobrave against the boll weevil, establishes the potential of EN to control the BW inside abscised squares and bolls that lay on the ground or buried in the soil.

Animals↗
PubMed1997

Efficacy of a dehydrated steinernematid nematode against black cutworm (Lepidoptera: Noctuidae) and diamondback moth (Lepidoptera: Plutellidae).

We compared the ability of in vitro-produced, commercially formulated with in vivo-produced, nonformulated Steinernema carpocapsae (Weiser) Poinar. All strain to infect and kill larvae of black cutworm, Agrotis ipsilon (Hufnagel), and diamondback moth, Plutella xylostella (L.). In vitro-produced nematodes formulated in wettable dispersible granules, which were stored dry, were rehydrated in water for 0-72 h before application. Against black cutworms, the efficacy of nematodes was (from most to least effective): in vivo > in vitro rehydrated for 72 h > in vitro rehydrated for 48 h > in vitro rehydrated for 24 h > dehydrated (0 h). Nematodes rehydrated for 72 h in water or moist soil were equally effective against black cutworm larvae, and both were significantly more effective than nematodes without rehydration. These results indicated that nematodes in the wettable dispersible granule formulation required time to rehydrate in the soil before infecting black cutworm larvae. Nematode treatments described above were applied to radish plants held at 100 or 75% RH and tested against diamondback moth larvae. At 100% RH, nematode efficacy was (from most to least effective): in vitro rehydrated for 72 h > in vivo > in vitro rehydrated 48 h > in vitro rehydrated 24 h > dehydrated (0 h). The efficacy of all treatments was lower at 75% than at 100% RH, and the ranking of in vivo and in vitro nematodes rehydrated for 72 h was reversed. The nematodes in the wettable dispersible granule formulation were effective for foliar treatments when humidity was high and nematodes were rehydrated for at least 48 h before application. The data show that nematode infectivity was reduced unless nematodes were rehydrated.

Animals↗
PubMed1999

Infectivity, distribution, and persistence of the entomopathogenic nematode Steinernema carpocapsae all strain (Rhabditida: Steinernematidae) applied by sprinklers or boom sprayer to dry-pick cranberries.

We evaluated infectivity, distribution, and persistence of commercially produced Steinernema carpocapsae (Weiser) All strain applied through solid set sprinkler irrigation or boom sprayer to 2 dry-pick cranberry farms on peat soil in British Columbia in 1993. Most infectivity assays used Galleria mellonella (L.) larvae. When possible, larvae of the target pest, Otiorynchus sulcatas (F.) were used as assay organisms. Nematodes in almost all samples of nematode suspensions diluted from shipping containers, from spray tanks, or collected in cups after passage through application equipment were infective to G. mellonella larvae. When O. sulcatus larvae were used as assay organisms, 93% (n = 14) of assays from the spray tank and 67% (n = 12) of assays after application showed infectivity. In the spring, sprinklers delivered nematodes to only 15 of 20 sample points on the 0.2-ha plot; delivery by the boom sprayer was better but 2 of 20 points on the 0.2-ha plot received approximately twice as many nematodes as the other points. In the fall, nematode delivery by both systems was more even. However, the average number of nematodes per milliliter of sprayed water collected from the 20 samples on each farm after each application did not correspond to the rates of nematodes applied. Persistence of nematodes in the soil was encouraging, but percentage of infectivity was lower than expected. After application in the spring, assays using G. mellonella larvae showed the presence of infective nematodes in soil samples (0-5 and 5-10 cm deep) on each sampling day (0, 3, 7, and 25) after application by boom sprayer, and on days 0, 3, and 7 after application through sprinklers. In the fall, G. mellonella assays showed infective nematodes in soil samples on each sampling day (0, 3, 7, and 25) after application by boom sprayer, and on days 0, 3, 7, 35, 60, 135, and 250 after application through sprinklers. In the spring, when assays lasted 4 d, percentage of infectivity rose to a maximum of 45% on the 3rd d after application by boom sprayer and declined thereafter. In the fall, when assays lasted 10 d, percentage of infectivity rose to a maximum of 58% on the 7th d after application through sprinklers and remained between 20 and 58% until day 135, declining thereafter; infectivity after boom application remained between 37 and 45% on days 3 and 7, and began to decline on day 25. Nematode infectivity was not compromised in peat soil, muck, or silty clay loam, but infectivity in loam (that may have contained nematicide residues) was very low. We suggest that the inconsistent control of O. sulcatus by S. carpocapsae on British Columbia cranberry farms may be partially explained by problems associated with application and factors related to nematode entry into the soil.

Agriculture↗
PubMed1999

EFFECTS OF PLANTS ON NEMATODE COMMUNITY STRUCTURE.

Plant-related inputs provide the resources for nematode communities. Sampling of nematode communities must be on appropriate temporal and spatial scales. Size, feeding types, food or host specificity, and chronology allow over 200 nematode species to coexist in a district. Relationships between nematode functional groups and ecological processes regulating decomposition processes have been found in field experiments. Pulse-labeling experiments have shown root-feeding nematodes to increase the flow of carbon from roots to soil microbial biomass. Soil texture is related to suitability for cropping and affects nematode communities through crop-specific infestations. Nematode diversity tends to be greatest in ecosystems with least disturbance, and bacterial-feeding nematodes make the greatest contribution to the decomposer food web in more intensively managed ecosystems. Indices of the nematode fauna reflect changes in the nematode community; these changes reflect soil and ecological processes. Understanding the role of nematodes in these processes is the key to understanding of the relationship between plants and soil nematode communities.

Journal Article↗
PubMed2002

Interaction between root lesion nematode Pratylenchus vulnus and two species of Fusarium on growth and development of maple seedlings.

Producing healthy seedlings and distributing them to far areas, is one of the most important factor for developing forests. Because of easy planting, rapid growth and good wood quality, Acer velutinum is the most useful species, among the softwood and hardwood trees. Growth and development of the nurseries were effected by different pests and diseases, the nematodes are one of these agents. They are not actively surveyed in the forest nurseries in Iran. On a survey of maple seedling in the forest nurseries Pratylenchus vulnus, Fusarium solani and F. oxysporum were identified. The interaction between these two fungi and nematode were studied in randomized complete block design with four replications and six treatments. For mass production and multiplication of the nematodes, they were sterilized with Streptomycin Sulfate, they were reared on carrot disc culture. The nematodes were then added to each pot. The results showed a highly significant difference (p = 0.01) between different treatments. In the treatment with nematodes alone growth of seedlings was minimum and population density of nematodes in soil and root tissue was maximum. In this treatments 75% of seedlings were died and seedling average height were 2.25 cm. In the treatments with nematode and fungi together the population of nematodes were decreased and the growth of seedlings were increased, in comparison with nematode alone. F. oxysporum showed more antagonistic effect on nematodes than F. solani. In the nematode + F. oxysporum treatment, 25% of seedlings were died and seedling average height was 12.75 cm. In the nematode + F. solani treatment, 50% of seedlings were died and the seedling average height was 5.5 cm. This is the first report of pathogenecity of Pratylenchus vulnus and its interaction whit fungi on maple seedlings in Iran.

Acer↗
PubMedDate not supplied

Phenotypic markers for selection of nematode-resistant sheep.

Nematode-resistance of an animal can be defined as an enhanced natural ability, relative to its peers, to both prevent establishment of larval nematodes and evict any that do establish. These parameters are not measurable in a practical sense and consequently nematode-resistance has usually been defined in terms of low faecal nematode egg counts (FEC). Studies in New Zealand and Australia have demonstrated that nematode-resistance, as measured by FEC, has a heritability of about 0.3 in Romney and Merino sheep. However as a selection trait FEC has practical limitations and its use may incur production penalties through withholding drench treatment for prolonged periods or from a need for artificial challenge. FEC is influenced by the level and composition of a natural nematode challenge and especially the expression of the immune response. Thus immunological parameters which reflect the underlying genetic resistance could potentially be used as phenotypic markers. Ideally, a useful phenotypic marker would be easy to sample and its assay would be inexpensive and able to be automated, in addition to being strongly correlated with nematode-resistance. Results from several New Zealand trials have indicated that antibody levels (particularly IgG1) to excretory/secretory antigens of L3 nematodes such as Trichostrongylus colubriformis may meet these criteria. Levels of antibody against L3 antigens are also independent of on-farm drenching strategies. Blood eosinophil count has also been considered for use as a selection parameter but a high degree of sample variability reduces its potential. Other immunological parameters associated with nematode-resistance which have potential as phenotypic markers include serum nematode-specific IgE and products of mucosal mast cells such as proteinases. It is likely that as the critical immune responses of sheep to nematodes become more clearly defined, new immunological parameters with potential for use as phenotypic markers will be found. The definition of these immune responses will also assist in the identification and characterization of genetic markers.

Animals↗
PubMed1997

Differential scanning calorimetry studies on an Antarctic nematode (Panagrolaimus davidi) which survives intracellular freezing.

Differential scanning calorimetry was used to characterize thermal events associated with freezing and melting of suspensions and extracts of Panagrolaimus davidi, an Antarctic nematode which can survive intracellular freezing. Nematode suspensions produced a single freezing exotherm with a shoulder on the peak representing the freezing of the nematodes. A shoulder on the peak of melting endotherms indicates the melting of the nematodes and of the water surrounding them. Exotherms were also detected from individual nematodes mounted in liquid paraffin. The freezing of nematodes was very rapid and in marked contrast to that of freezing-tolerant insects and vertebrates, which take hours or days to freeze. Eighty-two percent of the nematodes' body water froze. High levels of survival were obtained in nematodes exposed to temperatures down to -40 degrees C. No additional thermal events were observed after the freezing event and before the melting of samples cooled to -40 degrees C, indicating no changes in the proportion of body water frozen. Ice nucleating activity is present in nematode suspensions but not in supernatants from nematode extracts. No thermal hysteresis activity was detected in nematode extracts.

Animals↗
PubMed2002

Monoclonal antibodies reactive with secreted-excreted products from the amphids and the cuticle surface of Globodera pallida affect nematode movement and delay invasion of potato roots.

This paper describes Excreted-secreted proteins (ES) proteins that were immunolocalised in the cuticle, amphids and subventral glands of second stage juveniles of the two species of potato cyst nematodes (Globodera pallida and Globodera rostochiensis). Monoclonal antibodies reactive with these ES proteins were used in a bioassay to investigate their effect on nematode movement and on their ability to invade potato roots. Antibodies recognising the nematode cuticle surface and the amphids affected nematode movement and delayed nematode penetration of roots. These effects were temporary, since the nematodes were able to recover and infect potato roots. Movement of second stage juveniles treated with the antibodies was impaired for the first 30 min after inoculation: the juveniles remained close to the point of introduction and moved slowly and abnormally. They recovered normal movement after 1-2 h, possibly because the turnover rate of the secreted proteins meant that they were no longer blocked by the monoclonal antibodies. No effect was observed on second stage juveniles treated with an antibody reactive with secretions from the oesophageal glands. Nematodes treated with antibodies reactive with the nematode cuticle surface were notably more affected than those treated with other antibodies; nematodes failed to recover movement when in continuous contact with the antibodies. It is possible that the physical presence of the antibodies on the nematode surface affected their motility. Nematodes treated with antibodies reactive with secretions from the amphids were temporarily unable to move towards potato roots and their exploratory behaviour was greatly affected by the antibody treatment. Whether these antibodies were able to inhibit temporarily the function of the amphids or this effect was due to physical presence of the antibodies blocking the amphidial pore remains to be determined.

Animals↗
PubMedDate not supplied

Separation characteristics of liquid nematode cultures and the design of recovery operations.

Production of nematode-based pesticides involves the recovery of a viable nematode life stage known as the infective juvenile (IJ) from fermentation broth. Waste components to be separated from the IJs include non-IJ life stages, dead nematodes, nematode debris, spent media, and the nematode's associated bacteria. This paper reports separation characteristics of liquid cultures and suspensions of the nematodes Phasmarhabditis hermaphrodita, Steinernema feltiae, and Heterorhabditis megidis measured at small scale. Separation characteristics were determined for dead-end filtration, gravity settling and flotation. Results were used to identify large-scale recovery procedures. Separation of culture liquid by dead-end filtration of the crude fermentation broth was not possible due to rapid blinding of filters. However, nematode-water suspensions prepared by gravity settling could be concentrated using this separation method. Settling tests indicated that IJs could be efficiently separated from culture liquid by centrifugation but not by gravity settling. Examination of the effects of nematode concentration indicated an optimum concentration for gravity settling that may entail modest dilution of the fermentation broth. Flocculation of insoluble spent media in suspensions of P. hermaphrodita prevented its separation from nematodes by gravity settling. However, attachment of air bubbles to spent media allowed removal by flotation. Finally, adjustment of continuous phase density using sucrose allowed separation of non-IJ life stages, dead nematodes, and discarded cuticles from the IJs by flotation. The efficiency of this separation decreased with increasing nematode-solute contact time.

Animals↗