Linking the protease activity to the nematicidal action of edible mushroom.

Biological control using edible mushrooms as natural enemies is a sustainable alternative for pest management. Despite the well-established literature on toxins and secondary metabolites produced by these fungi in the biochemical control of nematodes, the nematicidal activity of proteases from different Pleurotus species is yet to be investigated. Therefore, this study aimed to correlate protease to the nematicidal activity of different mushrooms, Pleurotus sp., P. ostreatus (SB), P. ostreatus (Pearl), and P. djamor. For such a purpose, we performed motility assays of Panagrellus sp. at different time intervals, 6, 12, and 24 h for each of the mushrooms. In addition, the protease activity was measured using different pH (5, 7, and 9) and fermentation time intervals (45 and 75 days). Furthermore, we also evaluated the effect of this cell-free extract on Panagrellus sp. In response to these experiments, all edible mushrooms showed a reduction over 82% for the nematode-feeding activity (p < 0.01). The cell-free crude extract of each of the fungi studied showed nematocidal activity (p < 0.01). For the 45-day fermentation, P. djamor exhibited statistical significance (p < 0.01) compared with the others, reaching a reduction percentage of 73%. For the 75-day fermentation, Pleurotus sp. and P. ostreatus (Pearl) showed significant differences compared with the other fungi (p < 0.01), with reduction percentages of 64 and 62%, respectively. Herein, protease activity was associated with the nematicidal action of different Pleurotus species in controlling Panagrellus sp.

Crude Extracts of Pleurotus Spp. and the Presence of Their Proteins in the Ovicidal and Larvicidal Activity of Haemonchus contortus.

Mushrooms of the genus Pleurotus have shown nematophagous activity as it produces many chemical compounds and enzymes affecting parasitic nematodes. This study aimed to extract the inhibitory activity of the five strains of the fungus Pleurotus spp. It was evaluated against eggs and larvae of Haemonchus contortus. The extract of P. ostreatus obtained the highest level of inhibition of eggs at 97.6% (1341 µg/mL) followed by P. pulmonarius (EPP) at 81.2% (774 µg/mL). The extract selected for evaluation against larvae was P. pulmonarius, showing no effect for L3 larvae, but for L4 larvae an immobility effect of 56.93% was observed at 900 µg/mL. The protein profile showed the presence of 23 protein bands in the extract. The crude extract of P. pulmonarius showed degradation of tissues both inside the eggs and larvae L1. Metabolites produced by Pleurotus mushrooms can consider using in agriculture sustainable by utilizing in producing of ovicidal and larvicidal against H. contortus instead of chemical compounds.

Nematophagous fungus Pochonia chlamydosporia to control parasitic diseases in animals.

The control of gastrointestinal parasites in animals has become more challenging every year due to parasite resistance to conventional chemical control, which has been observed worldwide. Ovicidal or opportunistic fungi do not form traps to capture larvae. Their mechanism of action is based on a mechanical/enzymatic process, which enables the penetration of their hyphae into helminth eggs, with subsequent internal colonization of these. The biological control with the Pochonia chlamydosporia fungus has been very promising in the treatment of environments and prevention. When used in intermediate hosts of Schistosoma mansoni, the fungus promoted a high percentage decrease in the population density of aquatic snails. Secondary metabolites were also found in P. chlamydosporia. Many of these compounds can be used by the chemical industry in the direction of a commercial product. This review aims to provide a description of P. chlamydosporia and its possible use as a biological parasitic controller. The ovicidal fungus P. chlamydosporia is effective in the control of parasites and goes far beyond the control of verminosis, intermediate hosts, and coccidia. It can also be used not only as biological controllers in natura but also as their metabolites and molecules can have chemical action against these organisms. KEY POINTS: • The use of the fungus P. chlamydosporia is promising in the control of helminths. • Metabolites and molecules of P. chlamydosporia may have chemical action in control.

The Possible Biotechnological Use of Edible Mushroom Bioproducts for Controlling Plant and Animal Parasitic Nematodes.

The present paper reviewed publications on the nematocidal activity of edible mushrooms (EM) and their potential use as sustainable tools for the control of parasitic nematodes affecting agriculture and livestock industry. Nematodes are organisms living in the soil and animals' guts where they may live as parasites severely affecting economically important crops and farm animals, thus causing economic losses to worldwide agriculture. Traditionally, parasitic nematodes have been controlled using commercial pesticides and anthelmintic (AH) drugs. Over the years, nematodes developed resistance to the AH drugs, reducing the usefulness of many commercial drugs. Also, the use of pesticides/anthelmintic drugs to control nematodes can have important negative impacts on the environment. Different EM have been not only used as food but also studied as alternative methods for controlling several diseases including parasitic nematodes. The present paper reviewed publications from the last decades about the nematocidal activity of EM and assessed their potential use as sustainable tools for the control of nematodes affecting agriculture and livestock industry. A reduced number of reports on the effect of EM against nematodes were found, and an even smaller number of reports regarding the potential AH activity of chemical compounds isolated from EM products were found. However, those studies have produced promising results that certainly deserve further investigation. It is concluded that EM, their fractions and extracts, and some compounds contained in them may have biotechnological application for the control of animal and plant parasitic nematodes.

Effect of silver nanoparticles (AgNP's) from Duddingtonia flagrans on cyathostomins larvae (subfamily: cyathostominae).

The in vitro effect of silver nanoparticles of the Duddingtonia flagrans filtrate enriched with chitin was evaluated on infective larvae of cyathostomins (L3). After biosynthesis, an assay was carried out with two experimental groups in microtubes, for a period of 24 h: G1 (AgNP's-D. flagrans (43.4 µg/mL) + 120 L3) and G2 (distilled water + 120 L3). At the end of this period, AgNP's-D. flagrans (G1) demonstrated an effect on L3 with a 43% reduction (p < 0.01) in relation to G2. Thus, the authors suggest new designs with AgNP's-D. flagrans for the control of cyathostomins.

Combined use of ivermectin, dimethyl sulfoxide, mineral oil and nematophagous fungi to control Rhabditis spp.

Rhabditis spp., is a nematode known to cause otitis externa, an infection difficult to control, in cattle reared within tropical regions. The objective of this study was to assess the combined use of ivermectin 1%, dimethyl sulfoxide 1% and mineral oil 100% containing nematophagous fungi of both Duddingtonia flagrans (AC001) and Monacrosporium thaumasium (NF34) species to control in vitro Rhabditis spp. Thus, 12 experimental groups were designed with eight replicates each: G1 (nematodes + AC001); G2 (nematodes + NF34); G3 (nematodes + ivermectin 1%/positive control); G4 (nematodes + dimethyl sulfoxide 1%/positive control); G5 (nematodes + mineral oil 100%/positive control); G6 (nematodes + AC001 + ivermectin 1%); G7 (nematodes + NF34 + ivermectin 1%); G8 (nematodes + AC001 + mineral oil 100%); G9 (nematodes + NF34 + mineral oil 100%); G10 (nematodes + AC001 + dimethyl sulfoxide 1%); G11 (nematode + NF34 + dimethyl sulfoxide 1%); G12 (nematode + distilled water/negative control). The results demonstrated that all experimentally treated groups differed statistically (p < 0.01) from the control group. In the present study, the use of dimethyl sulfoxide 1% and mineral oil 100% in conjunction with conidia fungi portrayed noteworthy outcomes, which represents a future premise for the combined use of nematophagous fungi within these vehicles in both controlling Rhabditis spp.

Fungi predatory activity on embryonated Toxocara canis eggs inoculated in domestic chickens (Gallus gallus domesticus) and destruction of second stage larvae.

The objective of this study was to evaluate the infectivity of Toxocara canis eggs after interacting with isolated nematophagous fungi of the species Duddingtonia flagrans (AC001) and Pochonia chlamydosporia (VC4), and test the predatory activity of the isolated AC001 on T. canis second stage larvae after 7 days of interaction. In assay A, 5000 embryonated T. canis eggs previously in contact with the AC001 and VC4 isolated for 10 days were inoculated into domestic chickens (Gallus gallus domesticus), and then these animals were necropsied to collect material (digested liver, intestine, muscles and lungs) at 3-, 7-, 14-, and 21-day intervals after inoculation. In assay A, the results demonstrated that the prior interaction of the eggs with isolated AC001 and VC4 decreases the amount of larvae found in the collected organs. Difference (p < 0.01) was observed in the medium larvae counts recovered from liver, lung, intestine, and muscle of animals in the treated groups when compared to the animals in the control group. At the end of assay A, a percentage reduction of 87.1 % (AC001) and 84.5 % (VC4) respectively was recorded. In the result of assay B, the isolated AC001 showed differences (p < 0.01) compared to the control group, with a reduction of 53.4 % in the recovery of L2. Through these results, it is justified to mention that prior interaction of embryonated T. canis eggs with the tested fungal isolates were efficient in reducing the development and migration of this parasite, in addition to the first report of proven predatory activity on L2.

Proteolytic activity of the nematophagous fungus Arthrobotrys sinensis on Angiostrongylus vasorum larvae.

BACKGROUND: The predatory nematophagous fungus Arthrobotrys sinensis (SF53) produces three proteases with nematicidal activity when grown on solid media culture. However, the proteolytic profile produced by this fungus, when grown in liquid culture medium remains unknown. FINDINGS: Thus, the objective of this work was to evaluate the production of proteases from nematophagous fungus Arthrobotrys sinensis in liquid medium and its nematicidal activity on first stage larvae of A. vasorum. Proteases were obtained in its crude form, using Whatman no.1 filter paper, followed by centrifugation for 5 min at 10 × g and 4°C. A zymogram was performed with co-polymerized casein in an acrylamide gel as substrate. An in vitro assay to evaluate the nematicidal action of the proteases of A. sinensis (SF53) produced in liquid medium on A. vasorum L1 was conducted. By the analysis of the zymogram, it was observed a single halo at the beginning of digestion of the gel, suggesting that the three proteases of SF53 are produced in an enzymatic complex of large molecular weight. Regarding nematicidal activity, within 24 hours, the proteases produced in liquid medium of A. sinensis (SF53) showed a percentage reduction of 64% on the number of L1 of A. vasorum. CONCLUSION: In the present work, it is suggested that the three proteases of SF53 are produced in an enzymatic complex and was also demonstrated that these enzymes were effective in destroying A. vasorum L1.

Statistical experimental design to assess the influence of enzymes of nematophagous fungi versus helminths.

The present work used Plackett-Burman experimental design to assess the influence of enzymes of nematophagous fungi versus Strongyloides westeri and trichostrongylides larvae and Platynosomum fastosum eggs. The variables studied in the Plackett-Burman design were the proteases and chitinases of AC001 or VC4 as destructive agents of S. westeri and trichostrongylides larvae, and P. fastosum eggs. All tested enzymes had a significant effect (P < 0.05) on the destruction of S. westeri larvae. Furthermore, only VC4 and AC001 proteases showed a significant effect (P < 0.05) on the destruction of trichostrongylides larvae. On the other hand, chitinases of VC4 showed the highest significance (P < 0.05) on the destruction of P. fastosum eggs. It is proposed that statistical planning for the use of enzymes derived from nematophagous fungi is a viable way to elucidate some questions about their mechanism of action.

Effect of the fungus Pochonia chlamydosporia on Echinostoma paraensei (Trematoda: Echinostomatidae).

Echinostoma paraensei is a trematode of the genus Echinostoma that causes echinostomiasis in humans. The objectives of this study were to: evaluate the ovicidal activity of the nematophagous fungus Pochonia chlamydosporia (VC1 and VC4) on a solid medium 2% water-agar (2% WA) against E. paraensei eggs (assay A); evaluate ovicidal effect (destruction of eggs) of the isolate VC4 in supplemented culture media (assay B); and evaluate the ovicidal ability of the crude extract (VC4) on E. paraensei eggs (assay C). Eggs of E. paraensei (assay A) were placed in Petri dishes containing 2% WA with an isolate of the fungus P. chlamydosporia (VC1 and VC4) grown for 10 days, and without fungus as a control and evaluated regarding their destruction. In assay B, eggs of E. paraensei were placed in Petri dishes with different supplemented culture media and with VC4 isolate and the destruction of eggs was examined at the end of 25 days of interaction. In assay C, effects of the crude extract of P. chlamydosporia (VC4) on eggs were evaluated at the end of 7 days. In assay A, there was no difference (p>0.05) in ovicidal activity among the tested isolates (VC1 and VC4); however, the highest percentage for ovicidal activity (type 3 effect) was demonstrated by the isolate VC4. In assay B, the culture medium starch-agar showed the best results for the destruction of the eggs, with a percentage of 46.6% at the end of the assay. In assay C, the crude extract of VC4 was effective in the destruction of E. paraensei eggs, with a percentage reduction of 53%. The results of this study demonstrate that a rich culture medium with a greater availability of carbon and nitrogen may interfere directly in the predatory characteristics of ovicidal fungi.