The partial characterisation of endoproteases and exoproteases from three species of entomopathogenic entomophthorales and two species of deuteromycetes

The entomopathogenic entomophthoraceae (zygomycotina) Erynia rhizospora, Erynia dipterigena, and Erynia neoaphidis and the deuteromycete Aspergillus flavus produced single novel endoproteases (pI ca. 9) with activity against trypsin and chymotrypsin substrates. In contrast, the deuteromycete Paecilomyces farinosus produced a chymotrypsin (pI ca. 10).Inhibitor studies confirmed that the mixed activities (purified by isoelectric focusing) were derived from single endoproteases. The most potent inhibitor was Chicken ovoinhibitor. Little or no inhibition was observed for P. farinosus endoprotease by any of the chemicals tested.Although the different fungi possessed a broad spectrum of aminopeptidase activity, 3 species (E. rhizospora, E. dipterigena, and A. flavus) showed a preference for leucine at the N-terminal position and 2 species (E. neoaphidis and P. farinosus) showed maximal activity against arginine. Inhibitor studies confirmed these aminopeptidases as metallo-enzymes.     

Collagenolytic activity in several species of deuteromycetes under various storage conditions

The ability of deuteromycetes of the genera Penicillium, Aspergillus, and Botrytis to retain collagenolytic activity was studied after both 2 and 10 years of storage on a Czapek medium under a layer of mineral oil at 4°C, as well as in silica gel granules at 20 and ?60°C. The enzymatic activity of several species, including Botrytis terrestris, Penicillium janthinellum, Penicillium chrysogenum, and Penicillium citrinum, was retained under both conditions of storage. Aspergillus repens retained enzymatic activity only if stored under a layer of mineral oil. The viability of conidia and the collagenolytic activity of Botrytis terrestris, P. janthinellum, P. chrysogenum, and Penicillium citrinum, maintained on silica gel for 10 years, depended on the storage temperature. The viability of the test strains improved after storage on a silica gel at ?60°C. A strain of Aspergillus repens lost its ability to dissolve collagen at various storage tempeatures on the silica gel. The index of lysis for three strains of Penicillium deuteromycetes (Penicillium janthinellum, Penicillium chrysogenum, and Penicillium citrinum) increased after a 10-year storage on silica gel at ?60°C.     

Collagenolytic activity in several species of deuteromycetes under various storage conditions

The ability of deuteromycetes of the genera Penicillium, Aspergillus, and Botrytis to retain collagenolytic activity was studied after both 2 and 10 years of storage on a Czapek medium under a layer of mineral oil at 4 degrees C, as well as in silica gel granules at 20 and -60 degrees C. The enzymatic activity of several species, including Botrytis terrestris, Penicillium janthinellum, Penicillium chrysogenum, and Penicillium citrinum, was retained under both conditions of storage. Aspergillus repens retained enzymatic activity only if stored under a layer of mineral oil. The viability of conidia and the collagenolytic activity of Botrytis terrestris, P. janthinellum, P. chrysogenum, and Penicillium citrinum, maintained on silica gel for 10 years, depended on the storage temperature. The viability of the test strains improved after storage on a silica gel at -60 degrees C. A strain of Aspergillus repens lost its ability to dissolve collagen at various storage temperatures on the silica gel. The index of lysis for three strains of Penicillium deuteromycetes (Penicillium janthinellum, Penicillium chrysogenum, and Penicillium citrinum) increased after a 10-year storage on silica gel at -60 degrees C.     

Isolation and molecular characterization of five entomopathogenic nematode species and their bacterial symbionts from eastern Australia

BioControl - Entomopathogenic nematodes (EPNs) are used in biological control of pest insects but their potential may be limited by strain availability from different bioregions and effectiveness...     

Inhibition of trypsin-like enzymes on cells with rhodamine-aprotinin

Aprotinin, a polypeptide inhibitor of trypsin-like enzymes, has been labelled with rhodamine. Rhodamine-aprotinin inhibits trypsin in free solution in an identical manner to aprotinin. Rhodamine-aprotinin binds to trypsin-like enzymes on cells in formaldehyde fixed wax embedded sections. This technique has been used to locate cells possessing trypsin-like enzymes by means of fluorescent microscopy. In the present study we have used this technique to locate tumour cells.     

Cuticle hydrolysis in four medically important fly species by enzymes of the entomopathogenic fungus Conidiobolus coronatus

Entomopathogenic fungi infect insects via penetration through the cuticle, which varies remarkably in chemical composition across species and life stages. Fungal infection involves the production of enzymes that hydrolyse cuticular proteins, chitin and lipids. Host specificity is associated with fungus–cuticle interactions related to substrate utilization and resistance to host‐specific inhibitors. The soil fungus Conidiobolus coronatus (Constantin) (Entomophthorales: Ancylistaceae) shows virulence against susceptible species. The larvae and pupae of Calliphora vicina (Robineau‐Desvoidy) (Diptera: Calliphoridae), Calliphora vomitoria (Linnaeus), Lucilia sericata (Meigen) (Diptera: Calliphoridae) and Musca domestica (Linnaeus) (Diptera: Muscidae) are resistant, but adults exposed to C. coronatus quickly perish. Fungus was cultivated for 3 weeks in a minimal medium. Cell‐free filtrate, for which activity of elastase, N‐acetylglucosaminidase, chitobiosidase and lipase was determined, was used for in vitro hydrolysis of the cuticle from larvae, puparia and adults. Amounts of amino acids, N‐glucosamine and fatty acids released were measured after 8 h of incubation. The effectiveness of fungal enzymes was correlated with concentrations of compounds detected in the cuticles of tested insects. Positive correlations suggest compounds used by the fungus as nutrients, whereas negative correlations may indicate compounds responsible for insect resistance. Adult deaths result from the ingestion of conidia or fungal excretions.     

Inhibition of guanidinobenzoatase by a substrate for trypsin-like enzymes

Guanidinobenzoatase is a proteolytic enzyme capable of degrading fibronectin and is a tumour associated enzyme. Guanidinobenzoatase has been shown to be an arginine selective protease and is distinct from trypsin, plasmin and thrombin, the latter enzymes can be assayed with bis(carbobenzyloxycarbonyl-L-argininamido)-Rhodamine or BZAR. Guanidinobenzoatase is inhibited by BZAR when the enzyme is assayed in free solution and when the enzyme is cell-bound in frozen sections of tumour containing tissues. It is proposed that BZAR and its analogues may be of value in inhibiting tumour cell invasion in vivo and also that the selectivity of BZAR may be used to direct cytotoxic drugs to tumour cells possessing active guanidinobenzoatase.     

Distribution of entomopathogenic nematodes in Southern Cameroon

A first survey of entomopathogenic nematodes (EPN) was conducted in three agro-ecological zones of Southern Cameroon in 2007 and 2008. Entomopathogenic nematodes were recovered from 26 of 251 soil samples (10.4%). Three species, Heterorhabditis baujardi, Steinernema sp. A and Steinernema sp. B were found. The two steinernematids were considered unidentified species. Among the positive samples, 23 samples contained only H. baujardi (88.5%), two contained Steinernema sp. A co-occurring with H. baujardi (7.7%), and one sample contained Steinernema sp. B (3.9%). H. baujardi was frequent in forest and fruit crop (cocoa and oil palm plantations). Steinernema sp. A was found in a tree plantation of teak, Steinernema sp. B in a forest habitat. Nematodes were mostly present in acidic soils with pH ranging from 3.7 to 7.0. The highest EPN presence was recorded in sandy loam, sandy clay loam, sandy clay and clay soils. EPNs were not recovered in sand, loamy sand and clay loam soils. Using principal component analysis for elucidating the major variation patterns among sampling sites, four factors explaining for 73.64% of the overall variance were extracted. Factors were a combination of geographical (latitude, longitude, altitude), soil (pH, contents of sand, silt and clay, organic carbon, texture), and moisture (wilting point, field capacity) parameters as well as climatic parameters (mean annual rainfall, mean air temperature). Logistic regression and redundancy analyses (RDA) revealed that soil pH, longitude, available water and altitude were associated with presence and absence of EPN. Both logistic regression and RDA indicated that, increasing soil pH and longitude, associated with decreasing altitude, led to higher percentages of samples containing entomopathogenic nematodes.     

Starch gel electrophoresis of certain enzymes from five species of Fomes

The electrophoretic banding patterns of five enzymes (esterase, catechol oxidase, peroxidase, alkaline phosphatase, and acid phosphatase) from eleven isolates of five species of the wood-rotting basidiomycete, Fomes, were subjected to mathematical analysis in order to examine taxonomic relationships. Generally, greater similarity was observed between the banding patterns for isolates of the same species than between those for isolates of different species. The experimental and analytical technique used in this study could be an aid for the determination of taxonomic relationships among these and other fungi.     

Production of extracellular enzymes in the entomopathogenic fungus Verticillium lecanii

This study investigates the mechanisms as well as strategies for purification and characterization of potential enzymes involved inpathogenesis of entomopathogenic fungi. The test strain of Verticillium lecanii that was screened, during the present investigation,proved to be an efficient producer of protein and polysaccharide degrading enzymes (amylase, protease, and lipase), henceindicating versatility in biochemical mechanisms. Halo zones produced colony growth of V. lecanii on agar confirmed activity ofprotease, amylase and lipase enzyme by the V. lecanii isolate. Enzymatic Index (EI) observed were: Protease – 2.195, Amylase-2.196, Lipase- 2.147. Spectrophotometric analysis of enzymatic activity of V.lecanii at five different pH – 3, 5, 7, 9, 11 revealed thathighest proteolytic activity of the V. lecanii isolate was reported at pH 7 and 9 whereas proteolytic activity was minimum at acidicpH 3. Maximum amylolytic activity of V. lecanii on the 7^th day of inoculation was at pH 3 i.e. in an acidic environment in contrast toneutral pH 7. Maximum lipolytic activity of V. lecanii was found at pH 7. Since enzyme production in entomopathogenic fungi isspecific and forms an important criterion for successful development as well as improvement of mycoinsecticides, hence asignificant conclusion from the present analysis is the degree of variation in secretion of enzymes in test strain of Verticillium lecanii.     

Distribution and characterisation of entomopathogenic fungi from Korean soils

We investigated the occurrence of entomopathogenic fungi in 1080 soil samples representing multiple locations and conditions in Korea. Entomopathogenic fungi were isolated from soils using a selective medium containing dodine and antibiotics. Following an initial identification based on morphology, the fungal isolates were more precisely identified by the sequence of their nuclear ribosomal RNA (rRNA) internal transcribed spacer (ITS) regions. As a result, entomopathogenic fungi were found to occur in 32% (342 isolates) of the soil samples studied. The most abundant species were Beauveria spp. (125 isolates) and Metarhizium spp. (82 isolates). Entomopathogenic fungi were more often recovered from natural mountain and riparian soils than from agricultural habitats. The pathogenicity of isolated fungi was evaluated by using wax moth Galleria mellonella L. (Lepidoptera: Pyralidae) larvae. It was determined that 60% (207 isolates) of the isolates were pathogenic using this model. These entomopathogenic fungi may, therefore, have potential use against a variety of agricultural pests. This is the first study of the isolation and distribution of entomopathogenic fungi in representative sampling locations throughout Korea.     

Safety evaluation of four entomopathogenic nematode species against two silkworm species

Chinese oak silkworm (Antheraea pernyi) and mulberry silkworm (Bombyx mori) are economically important insects used for silk production and food resource. Entomopathogenic nematodes (EPNs) from the families of Steinernematidae and Heterorhabditidae are beneficial organisms currently considered in biological control. In this paper, we evaluated survival of two silkworm species exposed to four Steinernema species which are widely applied in pest control. The results showed that among four Steinernema species, S. bicornutum and S. feltiae did not have an effect on the larval survival to the two silkworm species, whereas S. carpocapsae and S. glaseri did have an effect. Each Steinernema species poses no threat to hatchability of eggs, pupation rate, larval durations and cocoon shell ratio.