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.     

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.     

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.     

Production and partial characterization of Duddingtonia flagrans (AC001) crude extract and its in vitro larvicidal action against trichostrongylid infective larvae

The production and partial characterization of Duddingtonia flagrans (AC001) crude extract and its in vitro larvicidal action against trichostrongylid infective larvae from sheep were studied. D. flagrans was grown in liquid medium with glucose, casein, bibasic potassium phosphate (K₂HPO₄), magnesium sulfate (MgSO₄), zinc sulfate (ZnSO₄), ferrous sulfate (FeSO₄), and copper sulfate (CuSO₄). The proteolytic activity was measured within varied pHs and temperatures. To determine the thermostability, the crude extract was incubated at 28°C for 72 h. To study the effect of different chemical compounds on the activity of the crude extract, the samples were incubated in solutions containing (10 mM): calcium chloride (CaCl₂), copper II sulfate (CuSO₄), zinc sulfate (ZnSO₄), magnesium sulfate (MgSO₄), inhibitor phenylmethylsulfonyl fluoride (PMSF), and 0.5% SDS. Results showed that the highest activity obtained (79.23 U/mL) was at pH 9.0, while the optimum temperature was 60°C (119.6 U/mL). The thermostability analysis demonstrated that after 72 h the activity was maintained or increased. It was found that the CuSO₄, ZnSO₄, and PMSF strongly inhibited the proteolytic activity. Moreover, the MgSO4 and SDS, caused a weak inhibition of the proteolytic activity. There was a significant (P<0.01) reduction in number of treated L₃ when compared to control (94.2%). The results suggest that the crude extract produced by D. flagrans (AC001) in liquid medium exerted larvicidal activity on trichostrongilid L₃ and therefore may contribute to a large-scale industrial production.     

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.     

Influence of the preservation period in silica-gel on the predatory activity of the isolates of Duddingtonia flagrans on infective larvae of cyathostomins (Nematoda: Cyathostominae)

The continued maintenance of nematophagous fungi predatory activity under laboratory conditions is one of the basic requirements for a successful biological control. The purpose of this study was to evaluate the influence of time on the preservation of the fungus Duddingtonia flagrans (AC001 and CG722) stored in silica-gel for 7 years and their subsequent predatory activity on cyathostomin L₃ larvae in 2% water-agar medium (2% WA). Samples of the isolates AC001 and CG722, originating from vials containing grains of silica-gel sterilized and stored for 7 years, were used. After obtaining fungal conidia, the predation test was conducted over 7 days on the surface of 9.0 cm Petri dishes filled with 2% WA. In the treated groups each Petri dish contained 500 cyathostomin L₃ and conidia of fungal isolates in 2% WA. In the control group (without fungi) the plates contained 500 L₃ in 2% WA. The experimental results showed that isolated AC001 and CG722 were efficient in preying on cyathostomin L₃ (p < 0.01) compared to control (without fungus). However, no difference was observed (p > 0.01) in the predatory activity of the fungal isolates tested. Comparing the groups, there was a significant reductions of cyathostomin L₃ (p < 0.01) of 88.6% and 78.4% on average recovered from the groups treated with the isolates AC001 and CG722, respectively, after 7 days. The results of this test showed that the fungus D. flagrans (AC001 and CG722) stored in silica-gel for at least 7 years maintained its predatory activity on cyathostomin L₃.     

Effect of refrigeration storage of nemathophagous fungi embedded in sodium alginate pellets on predatory activity against asinine gastrointestinal nematodes

ABSTRACT

The objective of this study was to evaluate the effectiveness of pelleted formulations of Duddingtonia flagrans and Monacrosporium thaumasium sodium alginate matrix stored for two and five years, by refrigeration of 2–8°C, on the predation of nematode infective larvae after passage of the gastrointestinal tract of asinines. Asinines were divided into seven groups, each group containing eight animals, in which each animal received a single dose of 100?g of pellets (containing 20?g of fungal mycelia) along with commercial feed to facilitate ingestion: GI – received D. flagrans pellets stored for five years; GII- received pellets of D. flagrans stored for two years; GIII – received newly produced D. flagrans pellets; GIV – received pellets of M. thaumasium stored for five years; GV – received pellets of M. thaumasium stored for two years; GVI – received pellets of newly-stocked M. thaumasium; and Control – received pellets without nematophagous fungi. It was observed that after passage of the pellets containing D. flangras (AC001) and M. thaumasium (NF34) by the gastrointestinal tract of the asinines, regardless of pellet storage time in assays A (Petri dishes) and B (coprocultures), there was a significant larval reduction (p?<?0.01) up to 72?h. It was concluded that the use of sodium alginate matrix pellets containing D. flagrans and M. thaumasium stored for two and five years were effective on the predation of infective nematode larvae after passage of the gastrointestinal tract from asinines.     

Duddingtonia flagrans: centrifugal flotation technique with magnesium sulphate for the quantification and qualification of chlamydospores in sheep faeces

Duddingtonia flagrans is a nematode-trapping fungus responsible for attacking larval stages of helminths in pasture, which has potential as a biological control method. The aim of this study was to test the magnesium sulphate centrifugal flotation technique for the quantification of D. flagrans chlamydospores in sheep faeces and to verify their morphological viability. In this experiment one sheep received an oral dose of 4.5 × 10⁶ chlamydospores/day during 20 days. Fecal samples were collected between days 15 and 20 and analyzed by the centrifugal flotation technique with magnesium sulphate. Densities of 1.23, 1.27 and 1.31 g mL⁻¹ recovered 1.45 × 10⁵, 3.87 × 10⁵ and 1.65 × 10⁵ chlamydospores from the faeces, respectively. Based upon the results it was concluded that this is an efficient technique for the chlamydospores quantification in ovine faeces. Moreover, it allowed more accurate visualization of chlamydospore morphology.     

海藻酸钠寡糖对小鼠免疫及抗氧化功能的影响

目的 研究海藻酸钠寡糖对小鼠免疫及抗氧化活性的影响。方法 采用不同浓度的海藻酸钠寡糖分别对小鼠连续灌胃15 d后,测量小鼠生长性能、小鼠胸腺和脾脏指数以及小鼠血浆中SOD和GSH的活性。结果 海藻酸钠寡糖能促进小鼠的生长性能,提高小鼠的胸腺和脾脏指数。其中给小鼠灌注高浓度的海藻酸钠寡糖15 d后,与对照组相比,小鼠的特定生长率和增重率分别提高了37.0%和48.5%（P<0.01）,小鼠胸腺和脾脏指数分别提高了18.8%和21.7%（P<0.01）。海藻酸钠寡糖还能增加小鼠的抗氧化活性,与对照组相比,灌注高浓度海藻酸钠组小鼠的SOD和GSH-Px活性分别提高了92.6%和35.9%（P<0.01）。结论 海藻酸钠寡糖能促进小鼠的生长性能,增强小鼠的免疫和抗氧化功能。     

Effects of three mealybug species on the development,survivorship and reproduction of the predatory lady beetle Cryptolaemus montrouzieri Mulsant

The lady beetle Cryptolaemus montrouzieri Mulsant (Coleoptera: Coccinellidae) is an important predator of mealybugs. The development, survivorship, longevity and reproduction of C. montrouzieri feeding on three different mealybug species [Dysmicoccus neobrevipes Beardsley, Ferrisia virgata Cockerell and Planococcus minor (Maskell)] were investigated in the laboratory at 26 ± 1°C, 75-–90% RH and 14:10 (L:D) h photoperiod. Results indicated that, when feeding on different mealybugs, no significant differences were observed between developmental periods and survivorship of C. montrouzieri (from egg to adult), but differences were recorded between the sex ratios, preovipositional periods, adult longevities and reproduction of the differently treated lady beetle populations. The highest sex ratio (0.56), the longest preovipositional period (6.6 days) and adult longevity (84.8 days for females and 93.9 days for males), and the maximum fecundity (659.0 eggs/female) of C. montrouzieri were recorded when feeding on F. virgata. Moreover, C. montrouzieri had a high net reproductive rate (313.66), intrinsic rate of increase (0.0816) and finite rate of increase (1.085) when feeding on F. virgata. Results indicated that the population growth of C. montrouzieri may increase faster when feeding on F. virgata than feeding on either of the other two mealybugs.     

Resporulation of Metarhizium anisopliae granules on soil and mortality of Tenebrio molitor: Implications for wireworm management in sweetpotato

In Australia, sweetpotato (Ipomoea batatas L.) is vulnerable to root feeding insect pests such as wireworms (e.g., Agrypnus spp.). The number of registered insecticides to control these insect pests is limited and often pest pressure, for example by wireworms, is severe close to harvest, further limiting what insecticides can be applied. Incorporating biological control agents such as entomopathogenic fungi (e.g., Metarhizium anisopliae) into integrated pest management programmes may be feasible in sweetpotato. M. anisopliae has been shown to be effective in controlling more than 200 insects and it is able to reside and grow in the rhizosphere and rhizoplane, suggesting that M. anisopliae could be a promising candidate against soil insect pests. In the study presented here, M. anisopliae was formulated into calcium alginate granules fortified with nutrients. The resporulation of the fungal granules was tested on four different soil types in the laboratory. The biocontrol efficacy of the resulting fungal growth was also examined using larval mealworms, Tenebrio molitor as a model insect in the laboratory and the glasshouse. Our results indicated that sterilised soil favoured optimal fungal resporulation, although different soil types did not have a significant effect on fungal resporulation. The resulting fungal resporulation and growth on sterilised soil caused high mortality (up to 76%) of larval mealworms in the glasshouse, whereas the fungal granules applied to non-sterile soil demonstrated poor resporulation that led to low mortality (13%) of larval mealworms. The result of this study indicates that the manipulation of microbial populations in field soil is required to enhance the fungal growth and potential insect control against wireworms in the field.     

Humidity‐controlled cold storage of Neoseiulus californicus (Acari: Phytoseiidae): effects on male survival and reproductive ability

Humidification can suppress water loss from an organism and has great potential for improving the cold storage of short‐lived arthropods, such as predatory mites. The effectiveness of humidity‐controlled cold storage was recently verified for Neoseiulus californicus (McGregor) females but was not examined for males. Combining both males and females in one storage protocol might increase the predator population because it would enhance the opportunity for multiple mating, which is necessary for females to maximize their egg production. Newly emerged adult males were stored at an air temperature of 5°C and relative humidity (RH) of 100% or 80%. The median survival time (LT₅₀) was 32 days at 100% RH and 14 days at 80% RH; the survival curves differed significantly. Males stored at 100% RH for 0, 10, 20 and 30 days were introduced to virgin females for mating at 25°C to evaluate their reproductive ability. The pre‐oviposition period was significantly prolonged in the females mated with males stored for ≥20 days. No negative effects of storage were observed on the oviposition period, total number of eggs or net reproduction rate (R₀) in the females mated with males stored for ≤20 days or on the mean generation time (T) for those stored for 30 days. A slight decrease in the intrinsic rate of increase (r_m) was observed in the females mated with males stored for ≥20 days. Our storage method can preserve N. californicus males for 20 days with only a minor reduction in their survival and reproductive ability.     

Effects of Metarhizium robertsii on the bloodsucking mosquito Aedes flavescens and non‐target predatory insects (Odonata)

The effects of different concentrations and methods of treatment with Metarhizium robertsii Bisch., Rehner & Humber conidia on the non‐target aquatic dragonfly larvae Lestes sponsa Hansemann, Lestes dryas Kirby and Aeshna affinis Vander Linden and on the target bloodsucking mosquito larvae Aedes (O.) flavescens (Muller) were analysed. We found that dragonflies are significantly less susceptible than mosquitoes to the fungus. Larvae of L. sponsa larvae were more susceptible to wet conidia than dry conidia. However, the mortality of the air‐breathing larvae of A. affinis was significantly higher after treatment with dry conidia relative to aqueous suspension. The results help to minimize the negative effects of entomopathogenic fungi on non‐target predator insects under the control of mosquito larvae.     

Effects of entomopathogenic nematodes on the spruce web‐spinning sawfly Cephalcia arvensis panzer and its parasitoids in the field

Field tests were conducted in the Asiago Forest, Venetian Prealps (Italy) to evaluate the efficacy of nematode strains (Heterorhabditis sp. HL 81 Steinernema carpocapsae IS 230 S. feltiae C = bibionis,) IS 389 S. kraussei SK) against the spruce web‐spinning sawfly Cephalcia arvensis. Soil applications of 100 juveniles cm^‐2 of S. feltiae and S. kraussei resulted in 56% and 36.4% reduction of emergences of sawfly, respectively, when performed before the mature larvae drop and enter the soil. The effectiveness of S. feltiae becomes 32.3% if the nematodes are applied when the larvae have already prepared their chambers. S. feltiae parasitized more females and long‐term diapausing individuals than S. kraussei. The two most effective strains (IS 389 and SK) seem to be well adapted to low temperature, which is likely to be the most important limiting factor for nematode activity in the mountain spruce forests. An ichneumonid parasitoid (Xenoschesis fulvipes,) was strongly affected by S. feltiae, resulting in 66.6% reduction of emergences. Another ichneumonid Ctenopelma lucifer, seems to be less affected than X. fulvipes by the nematode application.     

Intra-specific Variation in Virulence and In Vitro Production of Macromolecular Toxins Active Against Locust Among Beauveria bassiana Strains and Effects of In Vivo and In Vitro Passage on These Factors

Strains of Beauveria bassiana isolated from locust or from the soil varied considerably in their virulence and their ability to produce in vitro toxic metabolites against Locusta migratoria. Among the pathogenic isolates, only culture filtrates of 90/2-Dm, 92/11-Dm and 0023-Su were toxic by injection, a result which demonstrates that isolates of B. bassiana can be pathogenic for L. migratoria whether they secrete toxic metabolites in vitro or not. Toxic metabolites secreted by strains 90/2-Dm and 92/11-Dm were macromoleculer as they were retained by dialysis (cutoff of 6./8 kDa for globular proteins), whereas those secreted by 0023-Su were not. The effect of in vitro passage on virulence and on toxicogenic activity of isolate 90/2-Dm was dependent on the mycological media the inoculum was produced on. The virulence of isolate 90/2-Dm was significantly reduced after two passages through Sabouraud Dextrose Agar (SDA&rpar whereas two passages through Malt Agar (MA) increased its virulence and its toxicogenic activity. Nevertheless, the most aggressive conidia and the most toxic macromolecules were obtained after two passages of the isolate 90/2-Dm through the host. The bioactive macromolecules present in the crude filtrate of isolate 90/2-Dm were precipitated by 90% saturation of ammonium sulphate, and the insecticidal activity was exclusively detected in high molecular mass fraction after gel filtration on Sephadex G-25. In addition, the insecticidal effect of the Sephadex G-25 fraction was significantly reduced after exposure for 2 h at 608C and 20 min at 1208C, suggesting that the insecticidal metabolites in the culture filtrates of isolate 90/2-Dm were proteinaceous.