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Extracellular proteases are an important virulence factor for the nematophagous fungi Monacrosporium. The objective of this study was to optimize, purify, partially characterize, and to evaluate the nematicidal activity of the proteases produced by the nematophagous fungus Monacrosporium sinense (SF53) by solid-state fermentation. Wheat bran was used as substrate for protease production. The variables moisture, pH, incubation time, temperature, glucose, yeast extract, and the number of conidia were tested for their influences on protease production by SF53. To determine the optimal level of the selected variables the central composite design was applied. The crude extract obtained was purified in two steps, an ion exchange chromatography and a gel excision. SDS-PAGE and zymogram were performed for analysis of the purification process. Proteolytic activity was also tested at different pHs and temperatures. In the in vitro assay, the nematicidal activity of the three proteases was evaluated. pH and incubation time showed a significant effect (p<0.05) on production of protease. The highest value of activity was 38.0 (U/ml) under the conditions of pH 5.0 and incubation time of 211 h. SF53 produced three different proteases (Ms1, Ms2, and Ms3) which were directly purified from the zymogram. Ms1, Ms2, and Ms3 showed the following percentage of reduction (p<0.05) on the number of Panagrellus redivivus compared to control after 24 h: 76.8, 68.1, and 92.1%. This is the first report of the use of proteases of the isolate SF53 on a phytonematode, which may be a research tool in future works.

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The aims of this study were to test the action of the fungal extract of Pochonia chlamydosporia (VC4) on the hatching of cyathostomin eggs plated in Petri dishes containing 2% water-agar (2% WA) and its enzymatic activity in fecal cultures, in two experimental assays (A and B). The fungus P. chlamydosporia (VC4) was cultured in Erlenmeyer flasks (250ml) containing 50ml of liquid minimal medium supplemented with 0.2% gelatin for production of the crude enzymatic extract. Approximately 1kg of fresh feces was collected directly from the rectum of crossbred horses naturally infected with cyathostomins. The fecal material was used to obtain eggs and prepare fecal cultures. For assay A, one thousand eggs were plated on 4.5cm diameter Petri dishes together with 5ml of VC4 fungal filtrate and incubated at 26 degrees C in the dark for 24h. The control group consisted of 1000 eggs in Petri dishes containing 10ml of distilled water, which were incubated under the same conditions. After 24h, the total number of cyathostomin larvae present in each plate of the treated and control groups was counted. For assay B, about 20g of feces were added with 10ml of fungal extract of P. chlamydosporia (VC4) and incubated at 26 degrees C for 8 days. Third stage larvae (L(3)) were recovered at the end of this period. Significant difference (p<0.01) was found for the number of larvae between the treated group and the control at end of assay A. A 72.8% reduction in the hatching of cyathostomin eggs was found in the plates of the treated group compared with the control group. At the end of 8 days, the fungal extract of P. chlamydosporia (VC4), in assay B, was effective in reducing the number of L(3) cyathostomins in the treated group by 67.0% compared with the control group. Significant difference (p<0.01) was found between the means of L(3) recovered from the treated group and the control group. The results of this work showed that crude enzymatic extract of P. chlamydosporia (VC4) was effective in reducing hatching of cyathostomin eggs and therefore could be used as a biological control agent of this nematode.

Background: Helminth parasites cause morbidity and mortality in both humans and animals. Most anthelmintic drugs used in the treatment of parasitic nematode infections act on target proteins or regulate the electrical activity of neurons and muscles. In this way, it can lead to paralysis, starvation, immune attack, and expulsion of the worm. However, current anthelmintics have some limitations that include a limited spectrum of activity across species and the threat of drug resistance, which highlights the need for new drugs for human and veterinary medicine. Purpose: Present study has been conducted to determine the anthelmintic activity of silver nanoparticles (AgNPs) synthesized from the extract of nematophagous fungus, Duddingtonia flagrans, on the infecting larvae of Ancylostoma caninum (L 3 ). Methods: The nanoparticles were characterized by visual, ultraviolet, Fourier-transform infrared spectroscopy, transmission electron microscopy (TEM) analysis, and X-ray diffraction. The in vitro study was based on experiments to inhibit the motility of infective larvae (L 3 ), and the ultrastructural analysis of the nematode was performed by images obtained by TEM. Results: The XRD studies revealed the crystalline nature of the nanoparticles, and FTIR results implied that AgNPs were successfully synthesized and capped with compounds present in the extract. The results showed that the green synthesis of AgNPs exhibited nematicidal activity, being the only ones capable of penetrating the cuticle of the larvae, causing changes in the tegmentum, and consequently, the death of the nematode. Conclusion: The extract of the fungus D. flagrans is able to synthesize AgNP and these have a nematicidal action.

A serine protease from the nematophagous fungus Monacrosporium thaumasium (NF34a) was purified, partially characterized and tested in vitro in control of the first larval stage of Angiostrongylus vasorum. NF34a grew in liquid culture medium, producing its crude extract that was purified by ion exchange chromatography. The fractions with high protease activity were collected in a pool, and elution of proteases was monitored by enzymatic assay and protein content. Purification steps were monitored by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Protease activity was determined under different pH and temperature conditions, and the inhibitor effects of metal ions and phenylmethylsulfonyl fluoride (PMSF) were assessed. In an experimental test, the infection process of NF34a on first-stage larvae of A. vasorum was investigated. A purified serine protease (Mt1) was identified, with an approximate molecular mass of 40 kDa and apparent homogeneity in SDS-PAGE, having optimal activity at pH 7.0 to 8.0 and temperature of 60°C. Mg(2+) and Zn(2+) partially inhibited the activity of Mt1 while PMSF inhibited it completely. Mt1 production was observed when NF34a was grown using first-stage larvae of A. vasorum as the only source of carbon and nitrogen. These results show that the enzyme may have a possible role in the infection process of the larvae. In the in vitro test of applicability against A. vasorum L(1), we observed a reduction in the number of larvae of 23.9% (p < 0.05) in the group treated with Mt1 compared with the control group. However, even this low reduction demonstrates that the Mt1 is important in the infection process.

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