Trematodes enhance the development of the nematode-trapping fungus Arthrobotrys (Duddingtonia) flagrans

The capability of helminth (nematode and trematode) parasites in stimulating nematode trap and chlamydospore development of the nematophagous fungus Arthrobotrys (formerly Duddingtonia) flagrans was explored. Dead adult specimens of trematodes (the liver fluke Fasciola hepatica and the rumen fluke Calicophoron daubneyi) and nematodes (the ascarid Parascaris equorum and the strongylid Oesophagostomum spp.), as well as their secretory products, were placed onto corn meal agar plates concurrently inoculated with A. flagrans. Trapping organs were observed after 5 d and chlamydospores after 16 d, including in the control plates in the absence of parasitic stimulus. However, our data shows that both nematodes and trematodes increase trap and chlamydospore production compared with controls. We show for the first time that significantly higher numbers of traps and chlamydospores were observed in the cultures coinoculated with adult trematodes. We conclude that both the traps and chlamydospores formation are not only related to nematode-specific stimuli. The addition of secretory products of the trematode C. daubneyi to culture medium has potential for use in the large scale production of chlamydospores.     

Growth of the Fungus Duddingtonia flagrans in Soil Surrounding Feces Deposited by Cattle or Sheep Fed the Fungus to Control Nematode Parasites

The results reported in this paper represent work from two separate experiments, namely a plot trial using cattle feces conducted at Kungsãngen in Uppsala, Sweden and a plot trial using sheep feces undertaken at Tåstrup in Copenhagen, Denmark. In both trials, a technique was used to monitor the level of Duddingtonia flagrans propagules in soil surrounding feces. The feces were from animals fed or not fed D. flagrans fungal chlamydospores. Also presented are the numbers of soil nematodes in soil surrounding sheep feces. The results indicate that D. flagrans has little growth beyond the fecal environment into surrounding soil when chlamydospores are fed to either sheep or cattle. This is substantiated by the soil nematode data. No statistical differences in the number of nematode taxa identified, Shannon Weiner H′, proportion of various feeding groups, and B/B + F (B and F are the proportions of bacterial and fungal-feeding nematodes) were found when soil surrounding sheep feces containing chlamydospores and parasitic nematode eggs was compared to soil surrounding feces containing parasitic nematode eggs alone. It is unlikely that the application of D. flagrans as a biological control agent against the free-living stages of nematode parasites of these livestock will negatively affect populations of nontarget soil nematodes.     

Establishment of Arthrobotrys flagrans as biocontrol agent against the root pathogenic nematode Xiphinema index

Plant-parasitic nematodes cause devastating agricultural damage worldwide. Only a few synthetic nematicides can be used and their application is limited in fields. Therefore, there is a need for sustainable and environment-friendly alternatives. Nematode-trapping fungi (NTF) are natural predators of nematodes. They capture and digest them with their hyphae and are starting to being used as bio-control agents. In this study, we applied the NTF Arthrobotrys flagrans (Duddingtonia flagrans) against the wine pathogenic nematode Xiphinema index. A. flagrans reduced the number of X. index juveniles in pot cultures of Ficus carica, an alternative host plant for X. index, significantly. Sodium-alginate pellets with A. flagrans spores were produced for vineyard soil inoculation under laboratory conditions. The NTF A. conoides, A. musiformis and A. superba were enriched from several soil samples, showing their natural presence. Trap formation is an energy-consuming process and depends upon various biotic and abiotic stimuli. Here, we show that bacteria of the genus Delftia, Bacillus, Pseudomonas, Enterobacter and Serratia induced trap formation in NTF like A. conoides and A. oligospora but not in A. flagrans in the absence of nematodes. The application of NTF along with such bacteria could be a combinatorial way of efficient biocontrol in nematode-infested soil.     

A suitable model for the utilization of Duddingtonia flagrans fungus in small-flock-size sheep farms

Effective alternatives to anthelmintic treatment of nematode parasite infections of sheep are required because of the high prevalence of drug resistance. Within this context, the nematode-trapping fungus Duddingtonia flagrans has become a valuable component of various integrated control strategies. Toward this objective, a small quantity of lyophilized D. flagrans chlamydospores (10⁶ spores per animal) was administered to sheep in a one-year plot study. Animals grazing on native pasture were divided into two homogeneous groups and were kept in 1-ha paddocks in the southern region of Brazil. The oral administration of chlamydospores led to a significant reduction (p < 0.05) in the number of nematode eggs per gram of feces and in the larval availability on herbage (difference of 37.6%) in comparison to the control group. Control animals needed to be dewormed three times during the experiment, whereas the fungus-treated animals maintained a low parasite load, independent of seasonal variation. Although D. flagrans cannot serve as a panacea for nematode parasite control of livestock, it represents a significant advance toward rationalizing the use of endoparasitic drugs in small animals.     

Biocompatibility of Steinernema glaseri with a nematode trapping fungus Arthrobotrys superba on different nutrient media

Steinernema glaseri is known to be the most efficacious biocontrol agent against many soil insects belonging to various orders and of diverse habitats. In nature the efficacy of such entomogenous nematodes may be challenged by many micro pathogens. Keeping this in view, the biocompatibility of Steinernema glaseri with a nematophagous fungus Arthrobotrys superba was assessed on eight different nutrient media. The predatory activity of A. superba was evaluated in terms of trap formation, conidiophore formation, and number of adhesive cells formed in the presence and absence of nematodes. The fungus failed to form any trap on any of the culture media in the absence of nematodes. However, in the presence of nematodes, the trap formation by the A. superba was increased and found to be maximum on LNM (low nutrient mineral salt) medium and minimum at WA (water agar) of all the culture media tested. In the present investigation, the effect of CMA (2% and 5%) was not considerable; however, gradually higher dilutions of CMA induced greater number of traps. This less pronounced effect might be due to nutrient richness because low nutrient levels were found to be necessary for the formation of conidial traps. The number of conidiophores decreased with increase in dilution of Corn Meal Agar from 5% to 2%. Higher number of chlamydospores was observed in phenylalanine treated medium which indicates the inhibiting effect of phenylalanine on the growth of A. superba. Our results suggest that richness of soil has pronounced effect on the predatory activity of nematophagous fungus, Arthrobotrys superba which may further affect the biopotential of entomogenous nematode Steinernema glaseri, therefore, care should be taken while releasing entomogenous nematodes in an agroecosystem for managing various insect pests in a more efficient manner.     

Application of a formulation of the nematophagous fungus <Emphasis Type="Italic">Duddingtonia</Emphasis><Emphasis Type="Italic">flagrans</Emphasis> in the control of cattle gastrointestinal nematodiosis

The viability of a formulation of Duddingtonia flagrans was assessed in the control of parasite gastrointestinal nematodes of cattle. Two groups (A and B) of eight crossbred Holstein × Zebu cattle, approximately one year old, were placed in Brachiaria decumbens pasture. Each animal in group B (treated) received orally 20 g sodium alginate pellets containing mycelial mass of the D. flagrans fungus, while the animals in the group A (control) received pellets without fungus for seven months, starting in March 2005. The egg per gram of feces counting the gastrointestinal nematodes showed a difference (P < 0.05) in the treated group in June, July and August, with reductions of 58% (June), 47% (July) and 51% (August) compared to the control group. The infective larvae recovered in the pastures collected up to 20 cm from distance of the fecal dung in group B differed (P < 0.01) from the larvae recovered in group A. At the end of the experimental period, the animals in group B presented a greater weight gain (P < 0.01) compared to the untreated group (A). The treatment of cattle with pellets containing the D. flagrans nematophagous fungus, at the dose and duration used was effective in controlling the infective larvae of gastrointestinal nematodes of cattle.     

Über Entstehung und Keimung der Chlamydosporen von Botrytis cinerea Pers.

On the genesis and germination of the chlamydospores of Botrytis cinerea Pers. The chlamydospores of Botrytis cinerea are hyaline single cells of extremely variable form and size. They are formed under conditions unfavourable for growth as terminal or intercalary cells by transformation of vegetative mycelium parts and are liberated by hyphal disintegration. The chlamydospore genesis in vitro in aging malt agar cultures began about after one month. But the chlamydospore formation could also be initiated earlier by different conditions of culture. The chlamydospores germinated either with hypha or by microconidia — a herewith first described mode of germination. Intermediates of these both modes of chlamydospore germination could be regulated very differentiatedly by transferring the chlamydospores into malt solution (2%)and/or destilled water and by changing the duration of stay in the individual media. Under adverse external conditions no germination occurred. The three Botrytis cinerea-isolates did not show any differences in habitus, genesis and germination of their chlamydospores. Also in vivo on outdoor- and greenhouse-tomatoplants the occurrence of chlamydospores was no rarity. Since the chlamydospores are produced under very different adverse conditions of growth but cannot survive a period of drought lasting longer than three months without damage, they do not represent long-termed resistant perennating structures, but temporary stages of the fungus for intervening periods.     

Evaluation of a Peroxyacetic Acid Disinfectant in Hot-water Treatment for the Control of Basal Rot (Fusarium oxysporum f. sp. narcissi) and Stem Nematode (Ditylenchus dipsaci) in Narcissus

Formaldehyde is used routinely in the hot-water treatment (HWT) of narcissus bulbs for the control of stem nematode (Ditylenchus dipsaci) and basal rot (caused by Fusarium oxysporum f.sp. narcissi). Formaldehyde is unpleasant for operators to use, and does not kill all of the thick-walled chlamydospores of F. oxysporum and so less hazardous but more effective materials are being sought. Peroxyacetic acid (as Jet 5, a commercial disinfectant containing 5% peroxyacetic acid) was evaluated in vitro and in a field trial as a possible alternative to formaldehyde. In laboratory studies, peroxyacetic acid (as 1% Jet 5) was as effective as formaldehyde (as 0.5% commercial formalin containing 38 to 40% formaldehyde) in killing free-swimming stem nematodes and nematodes in the wool stage. Peroxyacetic acid (as 0.5% Jet 5) killed F. oxysporum chlamydospores within 1 h, whereas total kill was not achieved with formaldehyde (concentration as above) after 4 h. In a 2 year field trial, there was no evidence of detrimental effects on a healthy narcissus stock due to using peroxyacetic acid. In an infested, diseased stock, bulbs were virtually destroyed by stem nematode within 2 years when HWT was not given. The greatest reduction in nematode symptoms, and the highest bulb yields, were found when formaldehyde or the higher rates of peroxyacetic acid were used in combination with thiabendazole.     

Effects of times and storage conditions of Duddingtonia flagrans chlamydospores in sodium alginate pellets on its nematode predatory ability

This study aimed to determine the effects of Duddingtonia flagrans contained in sodium alginate pellets on trichostrongylide larvae under different storage conditions and durations. The in vitro predatory activity of D. flagrans in pellets against trichostrongylide larvae in sheep faeces were assessed at 0, 1, 3, 6, 9, and 12 months after the pellet was stored under four different conditions (i.e. ?20, 4°C, outdoors, and indoors). These results revealed that the numbers of larvae in faeces of sheep treated with pellets containing chlamydospores (treatment groups) were significantly lower than those in the control groups (without chlamydospores) for all trial months under four storage conditions for different durations (p?<?.05). The obtained reduction rates of the infective larvae (L3) in the four treatment groups ranged from 45.62% to 96.73% throughout the entire experiment. The overall mean L3 reduction percentages were 89.22%?±?3.74%, 88.97%?±?1.33%, 68.60%?±?14.31%, and 75.45%?±?13.18% for 4°C refrigeration, ?20°C refrigeration, indoor, and outdoor conditions, respectively. The pellets stored under these storage conditions for a year were provided to sheep for ingestion (in vivo test), and the results showed that the number of recovered larvae in sheep faeces at 24?h after ingestion were significantly lower than that before ingestion. For in vivo test, the L3 reduction percentage in the faeces was 90.99% (?20°C), 74.81% (outdoor), 83.53% (4°C), and 65.60% (indoor). Under the four storage conditions, D. flagrans spores contained in the pellets can maintain their survival ability to a varying degree in a year.     

Population dynamics of pinewood nematode and the endoparasitic fungus Esteya vermicola: interactions under experimental conditions

Esteya vermicola (Ophiostomataceae), an endoparasitic fungus, exhibits great potential as a biological control agent against pinewood nematodes (PWNs). The present study reports the interaction between PWNs and E. vermicola at different spore concentrations, number of PWNs and the time of culture. The addition of PWNs enhanced the sporulation of E. vermicola after 10 days of culture. The 5-day-old cultures of E. vermicola prior to addition of PWNs increased the highest amount of sporulation than that of 10- or 15-day-old cultures. The PWNs were completely killed by E. vermicola in the pine tree powder culture medium at the concentrations of 10⁷ and 10⁸ colony-forming-units (CFU) per ml. The interaction of the PWNs and E. vermicola was that PWNs provide nutrition to E. vermicola, however, the PWNs can also feed on mycelium of E. vermicola. The effect of E. vermicola on control of PWNs was determined by the population size, time of pest infection and the duration of co-infection.     

Synergistic effect of the herbicides glyphosate and MCPA on survival of entomopathogenic nematodes

The survival and infectivity of the infective juveniles of two species of entomopathogenic nematodes, Steinernema feltiae (Rhabditida: Steinernematidae) Heterorhabditis bacteriophora (Rhabditida: Heterorhabditidae), were determined after exposure for 72 h to two concentrations of the herbicides glyphosate and MCPA, as well as to the combination of the two herbicides (glyphosate + MCPA). For all herbicide treatments, concentrations and exposure times, S. feltiae was more tolerant to the herbicides than H. bacteriophora. The exposure of entomopathogenic nematodes to glyphosate + MCPA caused significantly higher mortality (26.33–57.33%) than glyphosate (0.67–15%) or MCPA (2.33–19%) alone. These results confirm the synergistic effect of the glyphosate + MCPA combination on the mortality in these nematodes. Nematode infectivity of Galleria mellonella larvae in response to the herbicides presence was evaluated in Petri dish assays containing sterile sand. Nematode infectivity was not significantly reduced by exposure to herbicides in S. feltiae but H. bacteriophora was less tolerant. Synergistic effect was obtained in the nematode mortality test but no synergistic effect was observed in the nematode infectivity assay. Our results suggest that possible synergistic effects of agrochemicals on survival of nematodes should be tested before mixing with entomopathogenic nematodes.     

Nematicidal activity of culture filtrate of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> against <Emphasis Type="Italic">Meloidogyne hapla</Emphasis>

Culture filtrates of Beauveria bassiana at different concentrations were evaluated for nematicidal activity against the northern root knot nematode (Meloidogyne hapla); bioassays included egg hatching, mortality and infectivity on tomato plants in pots under glasshouse conditions. The percentage mortality and inhibition of hatching of root-knot nematode were directly proportional to the concentration of culture filtrates of B. bassiana. Soil drenching with culture filtrate of B. bassiana significantly reduced nematode population densities in soil and in the roots and subsequent gall formation and egg-mass production by M. hapla under glasshouse conditions.     

Effectiveness of the entomopathogenic nematode <Emphasis Type="Italic">Steinernema feltiae</Emphasis> in agar gel formulations against larvae of the Colarado potato beetle, <Emphasis Type="Italic">Leptinotarsa decemlineata</Emphasis> (Say.) (Coleoptera: Chrysomelidae)

The entomopathogenic nematode Steinernema feltiae strain Ustinov Russia was used on potato foliage to control larvae of the Colorado potato beetle, Leptinotarsa decemlineata (Say.) (Coleoptera: Chrysomelidae). Nematodes were applied in formulations of agar at 4%, 2%, 1% and 0.5% concentrations and compared to a control application of nematodes in water for nematode survival. Agar formulation significantly improved efficacy by increasing nematode survival through reduction in desiccation when compared to water-based formulation. More than 70% of infective juvenile nematodes (IJs) died after being incubated in the highest concentration of agar for 12 h, while only 8% mortality was recorded at the 1% concentration. Suspension of nematodes in 1% agar gel was shown to be efficacious in both laboratory and greenhouse tests for extension of the nematodes’ survival. Agar formulation droplets dried slower than control droplets by 127.8 min. Leptinotarsa decemlineata mortality significantly increased when insects were exposed to infective juvenile nematodes for four hours after application. In conclusion, the agar formulation enhanced nematode survival by providing a suitable environment thereby delaying dryingand increasing the possibility for nematodes to invade their host on the foliage.     

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.     

The NADPH oxidase AoNoxA in <Emphasis Type="Italic">Arthrobotrys oligospora</Emphasis> functions as an initial factor in the infection of <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Reactive oxygen species (ROS) produced by NADPH oxidases can serve as signaling molecules to regulate a variety of physiological processes in multi-cellular organisms. In the nematophagous fungus Arthrobotrys oligospora, we found that ROS were produced during conidial germination, hyphal extension, and trap formation in the presence of nematodes. Generation of an AoNoxA knockout strain demonstrated the crucial role of NADPH oxidase in the production of ROS in A. oligospora, with trap formation impaired in the AoNoxA mutant, even in the presence of the nematode host. In addition, the expression of virulence factor serine protease P186 was up-regulated in the wild-type strain, but not in the mutant strain, in the presence of Caenorhabditis elegans. These results indicate that ROS derived from AoNoxA are essential for full virulence of A. oligospora in nematodes.     

Linoleic acid — The nematicidal principle of several nematophagous fungi and its production in trap-forming submerged cultures

Linoleic acid was shown to be the only detectable nematicidal agent in the mycelial extracts of several predacious fungi of the genus Arthrobotrys. Although the compound is present in saprophytic cultures, induction of trap formation by nematodes or phenylalnyl-valine caused a significant increase in its production. In submerged cultures, the number of traps formed by Arthrobotrys conoides and Arthrobotrys oligospora was directly correlated to the increase of the concentration of linoleic acid. In A. conoides, the ratio of ergosterol to linoleic acid decreased from 2.6 in saprophytic cultures to 1.1 in trap-forming cultures induced with nematodes. Linoleic acid exhibited nematicidal activities towards the free-living nematode Caenorhabditis elegans with an LD₅₀ value of 5 g/ml.     

In vivo-Untersuchungen zur Entstehung und Funktion der Chlamydosporen von Botrytis cinerea Pers. am Wirt-Parasit-System Fuchsia hybrida – B. cinerea

In vivo-investigations on the formation and function of chlamydospores of Botrytis cinerea Pers. in the host-parasite-system Fuchsia hybrida–B. cinerea On naturally with Botrytis cinerea Pers. infected and artificially inoculated outdoor- and greenhouse-plants of Fuchsia hybrida the extra- and intramatrical formation of the B. cinerea- chlamydospores was investigated. The chlamydospores served 1. as structures of survival, which were tested with regard to their tolerance of drought, nutrient- and oxygen-deficiency, attack by bacteria and pH-requirements. 2. The chlamydospores represented dispersal units, which were capable of germination. 3. The chlamydospores could function as structures of infection, because after chlamydospore germination the outgrowing mycelium – either directly or after production of macroconidia – could serve as secondary inoculum and start new infections.     

Use of statistical tools in the study of the conditions of predation of Duddingtonia flagrans versus Panagrellus sp

The objective of the present work was to study the conditions of predation of Duddingtonia flagrans conidia versus Panagrellus sp using response surface methodology. Conidia of D. flagrans (AC001) isolate were transferred into water-agar (WA) culture media at different pHs and different concentrations defined according to Central Composite Design (CCD). Five different concentrations of D. flagrans conidia: (1292, 500, 1000, 1500 and 1707) were used. For 2%WA media were used the following pHs were used: 6.29, 6.5, 7.0, 7.5 and 7.71. The response of the design corresponded to the number of larvae (transformed to square root scale) observed at the end of the experiment (10 days). At the tenth day, the non predated larvae were recovered in the Petri dishes. The results showed that 2%WA media at pH 7.0 contributed to improve the predatory activity of conidia of D. flagrans, and therefore this tool may be used in future studies under laboratory and natural conditions.     

Effect of temperature on the development of <Emphasis Type="Italic">Steinernema carpocapsae</Emphasis> and <Emphasis Type="Italic">Steinernema feltiae</Emphasis> (Nematoda: Rhabditida) in liquid culture

For commercial use of the entomopathogenic nematodes Steinernema carpocapsae and Steinernema feltiae in biological control of insect pests, they are produced in liquid culture on artificial media pre-incubated with their symbiotic bacteria Xenorhabdus nematophila and Xenorhabdus bovienii, respectively. After 1 day of the bacterial culture, nematode dauer juveniles (DJs) are inoculated, which recover development. The adult nematodes produce DJ offspring, which are harvested and can be sprayed. This study determined optimal temperatures to obtain high DJ progeny within a short process time. Temperatures assessed were 23°C, 25°C, 27°C, and 29°C for S. carpocapsae and 20°C, 23°C, 25°C, and 27°C for S. feltiae. The recovery of inoculated DJs was hardly affected and was reduced only in S. carpocapsae at 29°C. The fecundity (eggs in uterus) in S. carpocapsae reached a maximum at 27°C; whereas, maximum yields were recorded at 25°C. For both Steinernema spp., highest DJ densities were obtained after 15 days incubation at 25°C. Optimal culture temperature for both nematode species is 25°C. S. carpocapsae was more sensible to suboptimal temperature than S. feltiae. Results on total DJ density and DJ proportion of the total nematode population were more variable at non-optimal temperature condition for S. carpocapsae than for S. feltiae. Suboptimal culture temperature also reduced DJ infectivity.