Culture medium characteristics on the predatory activity of the nematophagous fungus Duddingtonia flagrans

The influence of casein and pH on the activity of the nematophagous fungus Duddingtonia flagrans (AC001) on trichostrongylide larvae was evaluated. A ‘positive influence’ was observed contributing to the reduction of 63% in the average number of recovered L₃ in the media supplemented with casein and pH 7.0.     

An extracellular serine protease of an isolate of Duddingtonia flagrans nematophagous fungus

This study aimed to present a protease produced by Duddingtonia flagrans fungus (AC001), and to evaluate its activity in the biological control of cyathostomin infective larvae (L₃). The crude extract from D. flagrans grown in liquid medium was applied first to a DEAE-Sepharose? and later to a CM-Sepharose? ion exchange column. Protease activity was determined under different pHs and temperatures. Subsequently, the effects of metal ions and phenylmethylsulfonyl fluoride (PMSF) inhibitor on activity were evaluated. Next, the protease activity in the biological control of nematodes was tested. A new 38 kDa serine protease (Df1) was purified. Optimum activity was obtained at pH 8.0 and 60°C; CuSO₄, ZnSO₄ and PMSF strongly inhibited the activity. Df1 (AC001) showed an L₃ reduction rate of 58%. In conclusion, a serine protease produced by D. flagrans (AC001) has been isolated, which is effective in the in vitro destruction of cyathostomin L₃.     

Interaction of the nematophagous fungus Duddingtonia flagrans on Amblyomma cajannense engorged females and enzymatic characterisation of its chitinase

The present work aimed to evaluate the production and the characterisation of a chitinase from nematophagous fungus Duddingtonia flagrans (AC001) and observe the interaction of this fungus on engorged females of Amblyomma cajennense under laboratory conditions. In assay A, the engorged females of A. cajennense were separated and immersed for 5 seconds in a fungal suspension of 10⁶ conidia/ml of the fungus D. flagrans and placed in Petri dishes, in the dark. In assay B, wheat bran supplemented with 1% chitin and liquid minimal medium was used [K₂HPO₄ (5.0 g/l), MgSO₄ (0.10 g/l), ZnSO₄ (0.0050 g/l), FeSO₄ (0.001 g/l) e CuSO₄ (0.50 mg/l)], as a substrate for chitinase production. To demonstrate the presence of chitinase in the crude extract obtained after the enzymatic extraction, a purification process was developed using a specific adsorption technique. The results from assay A demonstrated the interaction of the D. flagrans conidia produced from chitin-agar on engorged females of A. cajennense. In the assay B, D. flagrans produced a chitinase successfully, with a high value for enzyme activity. The molecular mass of semi-purified enzyme was estimated at approximately 34 kDa. It was concluded that the fungus produced a chitinase and has some entomopathogenic activity, as demonstrated here for the first time; however, it is strongly suggested that further studies are needed to elucidate the molecular mechanism of infection of target organisms by this fungus.     

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.     

Effect of dung burial by the dung beetle Bubas bison on numbers and viability of Cryptosporidium oocysts in cattle dung

Cryptosporidium oocysts were inoculated into fresh dung (∼1.2 × 10⁴ oocysts per gram wet weight) and fed to dung beetles to assess the effect of dung burial by the dung beetle Bubas bison on the distribution of the oocysts in small cores of soil in the laboratory. The experiment consisted of five replicates of each of two treatments; controls (dung but no dung beetles) and the experimental treatment (inoculated dung and seven pairs of dung beetles). After 5 days, when approximately 90% of the dung was buried, the surface and buried dung was recovered and subsampled. The oocysts in the subsamples were recovered and enumerated using qPCR. Oocyst viability was evaluated using an assay based on the exclusion or inclusion of two fluorogenic vital dyes, 4′,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI). Results revealed that overall 13.7% of oocysts remained on the surface and 86.3% of oocysts were buried. The viability of oocysts in buried dung was only 10% compared to oocysts the surface dung (58%). Therefore, widespread dung burial by B. bison during the winter months could substantially reduce the numbers of Cryptosporidium oocysts available to be washed into waterways following winter rains.     

Interaction and ovicidal activity of nematophagous fungus Pochonia chlamydosporia on Taenia saginata eggs

The ovicidal activity of the nematophagous fungi Pochonia chlamydosporia (isolates VC1 and VC4), Duddingtonia flagrans (isolate AC001) and Monacrosporium thaumasium (isolate NF34) on Taenia saginata eggs was evaluated under laboratory conditions. T. saginata eggs were plated on 2% water-agar with fungal isolates and controls without fungus and examined after 5, 10 and 15 days. At the end of the experiment P. chlamydosporia showed ovicidal activity against T. saginata eggs (p < 0.05), mainly for internal egg colonization with results of 12.8% (VC1) and 2.2% (VC4); 18.1% (VC1) and 7.0% (VC4); 9.76% (VC1) and 8.0% (VC4) at 5, 10 and 15 days, respectively. The other fungi showed only lytic effect without morphological damage to the eggshell. Results demonstrated that P. chlamydosporia was effective in vitro against T. saginata eggs unlike the other fungi.     

蜣螂与壁虎刚毛的比较及改形对其功能的影响

脱附与粘附是工程作业的两个相互矛盾的难题,土壤和物料的粘附严重降低了机械的作业效率和质量,没有粘附又使某些机械无法工作或运行。目前世界流行的脱附技术,包括向界面注入空气的充气法、向界面注入溶液的充液法、利用机械或超声波的振动法、施加电场的电渗法、施加磁场的磁     

Dung beetle community (Coleoptera: Scarabaeidae: Scarabaeinae) in a tropical landscape at the Lachua Region,Guatemala

Biological diversity conservation within natural reserves has been prioritized, but conservation efforts outside protected areas (where most human activities take place) have been very little considered. In this scenario, an alternative agricultural practice that may reduce the impacts of fragmentation in outer landscapes is a perforation process, which involves conservation in agricultural fields surrounded by continuous forests. Such practices enhance the positive impact of ecological services on fields. In this study we analyzed the biological diversity state in perforation fields and their surrounding forests. The analysis was done using dung beetles as biological indicators. A nested pattern in dung beetles distribution was found, which ordered the surrounding continuous forest sites as the ones with the highest species richness, followed by the perforation fields, and placed the fragmentation practice fields (continuous agricultural fields surrounding forest patches) with the lowest one. Indicator species for perforation fields and surrounding continuous forests were chosen. In general, perforation practice fields differed in composition, based upon functional groups richness and identity; it also contained a higher species richness than the fragmentation practice. Agricultural practices that enhance biological diversity conservation such as perforation, should be recommended and considered in natural resource management by local communities in order to take advantage of ecological services that otherwise may be gradually lost.     

Effects of a juvenile hormone analogue on the eggs, post-embryonic development, metamorphosis and diapause induction of the Colorado potato beetle, Leptinotarsa decemlineata

The effect of a juvenile hormone analogue, S-71639, was tested on the eggs, four larval instars and adults of the Colorado potato beetle, Leptinotarsa decemlineata Say, by topical application or after treatment of the foodplant. The last larval instar is very sensitive to S-71639. Treatment of this instar delayed the onset of pupation and prevented adult emergence. Treated animals showed severe abnormalities, but they were not immediately killed at the doses used in this study. Treatment of larvae also interfered with the photoperiodic induction of diapause. Adults, kept under diapausing conditions, started to lay eggs after treatment with S-71639. The ovicidal effect of the compound was rather weak. The implications for practical use of S-71639 in control of the Colorado potato beetle are being discussed.

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Résumé L'effet d'un analogue de l'hormone juvénile, S-71639, a été testé sur les oeufs, les quatre stades larvaires et les adultes du doryphore, Leptinotarsa decemlineata Say, par application topique ou après traitement de la plante-hôte. Le dernier stade larvaire est très sensible au S-71639. Le traitement de ce stade retarde le début de la métamorphose et empêche l'émergence adulte. Les animaux traités montrent de graves anomalies, mais ne sont pas immédiatement tués par les doses utilisées dans cette étude. Le traitement des larves perturbe aussi l'induction photopériodique de la diapause. Les adultes placés dans des conditions de diapause, commencent à pondre après traitement au S-71639. L'effet ovicide de la substance est plutôt faible. Les implications pour l'utilisation pratique du S-71639 dans la lutte contre le doryphore sont discutées.

     

Asymmetric competition in cattle dung between two species of Onthophagus dung beetle and the bush fly, Musca vetustissima

Abstract.

• 1 Competition in cattle dung pads between two dung beetles, Onthophagus ferox Harold and Onthophagus binodis Thunberg, and the bush fly, Musca vetustissima Walker, was investigated in laboratory experiments, to determine why spring fly abundance in the field did not fall following the introduction of O. binodis.
• 2 At low beetle densities, the number of eggs laid by each species was reduced by the second species. A similar amount of dung was buried by each species alone or by both together.
• 3 At high beetle densities O. binodis egg production was substantially affected by each additional O.ferox, but O.ferox egg production was not affected by each additional O.binodis. Asymmetric competition occurred because O.ferox buried more dung than O.binodis, and a greater proportion in day 1 (pre-emptive dung burial).
• 4 O.ferox caused greater M. vetustissima egg-puparia mortality than O. binodis. Mortality mostly occurred in young M. vetustissima larvae less than 1 day old. Total egg-puparia fly mortality was correlated better with the dung buried on day 1 than dung buried on day 8 (pre-emptive dung burial). O.binodis did not add to fly mortality by O.ferox at high densities because of asymmetric competition between the beetles.

     

The effect of larval predators Thanasimus dubius (Coleoptera: Cleridae), produced by an improved system of rearing, against the southern pine beetle Dendroctonus frontalis (Coleoptera: Curculionidae)

The southern pine beetle, Dendroctonus frontalis (Coleoptera: Curculionidae) (SPB), is known to be a major bark beetle pest of pines throughout the southeastern United States. A common predator of bark beetles, Thanasimus dubius (Coleoptera: Cleridae), has been suggested to play a prevalent role on SPB dynamics. Evaluations of T. dubius have been limited by rearing methods; an artificial diet for larval T. dubius exists, and preservatives such as sorbic acid could help to maximize diet shelf-life and enhance the efficiency of the rearing system. The effects of sorbic acid at different concentrations (0%, 0.1% and 0.2%) in the larval diet for T. dubius were measured, and the effects of increased feeding time intervals (2-3 vs. 5 days) on predator performance evaluated. In addition, an experimental bioassay was conducted where newly hatched T. dubius larvae were released at four densities (0, 50, 100, and 200 per log) on pine logs infested by SPB. Sorbic acid in the diet reduced female fecundity (by 20-40%), but did not affect adult T. dubius size or longevity. However, using this preservative may not be necessary because it had no effect on the overall efficiency of the rearing system, while refreshing the larval diet every 5 days (compared with 2-3 days) did improve its efficiency, even without sorbic acid. The release of larval T. dubius resulted in a highly significant effect on the SPB ratio of increase (RI). This experiment was facilitated by the improvements in our rearing methods.     

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.     

In vitro characterization bioassays of the nematophagous fungus Purpureocillium lilacinum: Evaluation on growth,extracellular enzymes,mycotoxins and survival in the surrounding agroecosystem of tomato

The effects of water stress and temperature on in vitro growth and enzymatic activity of Purpureocillium lilacinum (Sordariomycetes, Hypocreales, Ophiocordycipitaceae) isolates with demonstrated capacity to control Nacobbus aberrans (Secernentea, Tylenchida, Pratylenchidae) were evaluated in this study. Also, saprophytic and endophytic colonization in tomato plants were determined. P. lilacinum was able to grow under the evaluated levels of osmotic and matric stress, but the increase in water stress caused reductions in radial growth rates. Moreover, the fungal isolates produced chitinases, proteases, and leucinostatins under inductive conditions. The nematophagous fungi were able to develop saprophytically (10⁴ CFU g^?1 of soil). Meanwhile, only P. lilacinum SR38 demonstrated endophytic capacity. The results suggest that P. lilacinum can be effectively applied as biocontrol agents of phytoparasitic nematodes in tomatoes under variable agroecological conditions.     

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.     

Independence of genetic variation between circadian rhythm and development time in the seed beetle, Callosobruchus chinensis

A positive genetic correlation between periods of circadian rhythm and developmental time supports the hypothesis that circadian clocks are implicated in the timing of development. Empirical evidence for this genetic correlation in insects has been documented in two fly species. In contrast, here we show that there is no evidence of genetic correlation between circadian rhythm and development time in the adzuki bean beetle, Callosobruchus chinensis. This species has variation that is explained by a major gene in the expression and period length of circadian rhythm between strains. In this study, we found genetic variation in development time between the strains. The development time was not covaried with either the incidence or the period length of circadian rhythm among the strains. Crosses between strains suggest that development time is controlled by a polygene. In the F₂ individuals from the crosses, the circadian rhythm is attributable to allelic variation in the major gene. Across the F₂ individuals, development time was not correlated with either the expression or the period length of circadian rhythm. Thus, we found no effects of major genes responsible for variation in the circadian rhythm on development time in C. chinensis. Our findings collectively give no support to the hypothesis that the circadian clock is involved in the regulation of development time in this species.     

Effect of Paenibacillus alvei, strain K165, on the germination of Verticillium dahliae microsclerotia in planta

Verticillium wilt is a devastating disease of a wide range of herbaceous and woody plant hosts, incited by the soilborne fungus Verticillium dahliae. In the present study, the effect of the potential biocontrol isolate Paenibacillus alvei, strain K165, on the germination of V. dahliae microsclerotia (msc) was investigated. Strain K165 was isolated from tomato root tips and its activity against V. dahliae has been shown in glasshouse and field experiments. In the present study, the application of K165 resulted in the reduction of msc germination of V. dahliae, in the root tips and the zone of elongation, of eggplants by 50% compared to the control treatment; whereas 10 and 12 cm away from root tips and in soil without plants the percentage of msc germination was reduced by 26% and 40%, respectively. However, K165 did not significantly affect the number and length of hyphae per germinated msc. In a split-root system, K165 triggered induced systemic resistance in eggplants against V. dahliae by reducing disease severity and msc germination by 27% and 20%, respectively. In addition, K165 colonised the rhizosphere of eggplants and soil in a population density of 5 and 3 log₁₀ cfu g⁻¹, 7 dpi, respectively. This is the first report of evaluating the direct/indirect effect of a rhizospheric bacterium on msc germination in the rhizosphere of eggplants, indicating that strain K165 reduces msc germination.