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Extracellular enzyme production by nematophagous fungi in the presence and absence of nematodes
... On this regard is important to consider that cuticular wall of nematodes is composed by coats from different nature that include coats of lipids and proteins (de Freitas et al., 2023). Nematophagous fungi have developed a system of protein production with different enzymatic activities, including lipases and proteases, serine proteases, chitinases, among others (Mendoza-de Gives et al., 2003;Hastuti et al., 2022). In the present study a band of approximately 10 kDa was visualized in the fungal LCF obtained in CzeDoxB that did not appear on the control with only the medium. ...
... Other strategies contribute to the process of attraction, adhesion and cuticular degradation, penetration, and the invasion of nematode bodies, as well as eventually the fungal nutrition from the nematode internal tissues [46]. These mechanisms are developed in a sequence of biological, physiological and biochemical steps as follows: (a) production of attractants molecules that mimic sexual and food olfactory clues to lure nematodes [47]; (b) production of adhesive extracellular polymers that attach to the nematode cuticular surface contributing to the trapping process [48]; (c) nematode paralysing substances or nematotoxins [49]; (d) an enzymic system specially directed to degrade the nematode cuticle components to traverse and penetrate the cuticular wall [50,51]; and (e) production of nematocidal metabolites [16,52,53]. The results obtained in the present study showed differences in the nematocidal activity of FCF of A. musiformis growing in different media. ...
Haemonchus contortus (Hc) is a parasite affecting small ruminants worldwide. Arthrobotrys musiformis (Am) is a nematode-trapping fungi that captures, destroys and feeds on nematodes. This study assessed the predatory activity (PA) and nematocidal activity (NA) of liquid culture filtrates (LCF) of Am against Hc infective larvae (L3), and additionally, the mycochemical profile (MP) was performed. Fungal identification was achieved by traditional and molecular procedures. The PA of Am against HcL3 was performed in water agar plates. Means of non-predated larvae were recorded and compared with a control group without fungi. LCF/HcL3 interaction was performed using micro-tittering plates. Two media, Czapek–Dox broth (CDB) and sweet potato dextrose broth (SPDB) and three concentrations, were assessed. Lectures were performed after 48 h interaction. The means of alive and dead larvae were recorded and compared with proper negative controls. The PA assessment revealed 71.54% larval reduction (p < 0.01). The highest NA of LCF was found in CDB: 93.42, 73.02 and 51.61%, at 100, 50 and 25 mg/mL, respectively (p < 0.05). Alkaloids and saponins were identified in both media; meanwhile, coumarins were only identified in CDB. The NA was only found in CDB, but not in SPDB. Coumarins could be responsible for the NA.
... showing the greatest versatility for different enzymatic substrates. Other studies involving strains of the genus Pochonia have shown that this genus is also a producer of extracellular proteases in the presence of gelatine [79][80][81][82]. ...
Entomopathogenic fungi can regulate insect populations. They have extracellular enzymes that degrade cuticle components, mainly hydrocarbons, used as an energy source. The increase in insecticidal activity of fungi in a medium supplemented with cuticular hydrocarbons was assayed and the hydrolytic enzyme profiles of two strains of Purpureocillium lilacinum were evaluated. A spore suspension of P. lilacinum was inoculated in Petri plates with different values (0.99–0.97–0.95) of water activity (Aw) using the substrates gelatin, starch and tween-20. Growth rate on the different substrates and the enzymatic activity index for proteases, amylases and lipases at different incubation times, pH and Aw, was evaluated. Moreover, the insecticidal efficiency of strains grown in media supplemented with n-hexadecane and n-octacosane was analyzed. LT50 was calculated against adults of Tribolium confusum and showed that mortality increased about 15% when the strains grew in amended culture medium. High amylolytic activity was detected, but proteases were the main enzymes produced. Optimal protease production was observed in a range of acid and alkaline pH and lower Aw. The greatest growth rate was obtained in presence of gelatin. Lipase and amylase production was detected in small amounts. Fungal growth in media with hydrocarbon mixtures increased the pathogenicity of the two strains of P. lilacinum, with the strain JQ926223 being more virulent. The information obtained is important for achieving both an increase in insecticidal capacity and an understanding of physiological adaptation of the fungus.
... regarding the physical, chemical, and biological processes need to be elucidated. The pathogenic activity of nematophagous fungi against nematodes involves a complex process dependent on recognition of molecular structures of the surface of the cuticle of the nematodes and the participation of enzymes that together contribute to the immobilization, penetration, and subsequent colonization and digestion of the nematode by fungi (Mendoza-de-Gives et al. 2003). Thus, the investigation of nematophagous fungi which are more effective in preying nematodes in fecal material and the characterization of the fungusenematode interaction process may be an important aspects in the selection process of more aggressive fungi strains to improve biological control programs (Mendoza-de-Gives 1999). ...
The nematode-trapping fungus Duddingtonia flagrans has been studied as a possible control method for gastrointestinal nematodes of livestock animals. These fungi capture and infect 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. The aim of this study was to investigate the participation of proteolytic enzymatic activity during the interaction of the nematophagous fungus D. flagrans with infective larvae of trichostrongylides and the free-living nematode Panagrellus spp. Protease inhibitors used interfered in the predatory activity of D. flagrans. However, only PMSF significantly reduced the mean number of Panagrellus spp. captured by D. flagrans in comparison with the control. The experiment with fluorogenic substrate showed that maximum urokinase activity during the interaction of the fungus with the infective larvae of trichostrongylides or Panagrellus spp. occurred within 7 or 1 h of incubation, respectively. The protease activity, especially of the serine class, may be important during the interaction between the fungus and nematodes.
Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
... En los medios suplementados con gelatina se detectó la producción de proteasas por las dos cepas de Pochonia estudiadas, lo cual coincide con los resultados descritos por numerosos investigadores para este hongo nematófago (4,5,8,12). ...
DETECTION OF PROTEASES PRODUCED BY Pochonia chlamydosporia IN SOLID
MEDIUM
ABSTRACT: The detection of protease production is an important aspect for selecting some biological
control agents, because of the role these enzymes play in the infectious process. The aim of this work
was to modify a method for detecting proteases in solid medium supplemented with gelatine, using
different dyes and measuring the gelatine degradation halo, the colony growth and the protease activity
(PA) indicator. Trypan blue at 0.03% was the most efficient alternative with a good visualization of the
degradation halo and the least effect on the fungal colony growth.
(Key words: proteases; solid medium; Pochonia chlamydosporia)
... Nevertheless, variation in enzymatic activity was detected among the isolates, and it was possible to perceive a degree of relationship with nutrient availability. Variability of fungal enzymatic activity is thought to be dependent on nutrient availability (Mendoza de Gives et al., 2003). Only isolate Pc2 had a strong activity of esterases, proteases and oxidases when grown in a low nutrient medium. ...
Pochonia chlamydosporia, a widespread fungal parasite of potato cyst nematodes (PCN), Globodera spp., and root-knot nematodes (RKN), Meloidogyne spp., has been studied as a biological control agent. Three Portuguese isolates (Pc1, Pc2, Pc3) obtained from PCN eggs and two non-native isolates (Vc10, Pc280) were characterised using ERIC-PCR and screened by in vitro assays for their ability to produce chlamydospores, parasitise eggs of Globodera rostochiensis and Meloidogyne chitwoodi and colonise the rhizosphere of barley. The effects of temperature on growth, sporulation, parasitism and enzymatic activity were also evaluated. Isolates Pc1 and Pc3, despite their different geographical origins, had identical molecular profiles. Pc2 produced the higher numbers of chlamydospores in solid medium (1.15 × 107 chlamydospores g−1), whereas Pc3 produced the least (3 × 105 chlamydospores g−1). These isolates extensively colonised the rhizosphere of barley (>90% root fragments) and the proportion of parasitised eggs, detected on agar plates, was low (
Arthrobotrys musiformis is a nematophagous fungus with potential for the biological control of Haemonchus contortus larvae. This study aimed to identify and demonstrate the proteolytic activity of extracellular products from A musiformis cultured in a liquid medium against H contortus infective larvae. A musiformis was cultured on a solid medium and further grown in a liquid medium, which was then processed through ion exchange and hydrophobic interaction chromatography. The proteolytic activity of the purified fraction was assayed with either gelatin or bovine serum albumin as substrate. Optimum proteolytic activity was observed at pH 8 and a temperature of 37°C. Results obtained with specific inhibitors suggest the enzyme belongs to the serine-dependent protease family. The purified fraction concentrate from A musiformis was tested against H contortus infective larvae. A time-dependent effect was observed with 77 per cent immobility after 48 hours incubation, with alteration of the sheath. It is concluded that A
musiformis is a potential candidate for biological control because of its resistant structures and also because of its excretion of extracellular products such as proteases. The present study contributes to the identification of one of the in vitro mechanisms of action of Amusiformis, namely the extracellular production of proteases against H contortus infective larvae. More investigations should be undertaken into how these products could be used to decrease the nematode population in sheep flocks under field conditions, thereby improving animal health while simultaneously diminishing the human and environmental impact of chemical-based drugs.
For the first time, the specific activities of chitinases, esterases, lipases and a serine protease (VCP1) produced by different isolates of the nematophagous fungus Pochonia chlamydosporia were quantified and compared. The isolates were grown for different time periods in a minimal liquid medium or media supplemented with 1 % chitin, 0.2 % gelatin or 2 % olive oil. Enzyme-specific activities were quantified in filtered culture supernatants using chromogenic p-nitrophenyl substrates (for chitinases, lipases and esterases) and a p-nitroanilide substrate (to measure the activity of the proteinase VCP1). Additionally, information on parasitic growth (nematode egg parasitism) and saprotrophic growth (plant rhizosphere colonisation) was collected. Results showed that the production of extracellular enzymes was influenced by the type of medium (p < 0.05) in which P. chlamydosporia was grown. Enzyme activity differed with time (p < 0.05), and significant differences were found between isolates (p < 0.001) and the amounts of enzymes produced (p < 0.001). However, no significant relationships were found between enzyme activities and parasitic or saprotrophic growth using Kendall's coefficient of concordance or Spearman rank correlation coefficient. The results provided new information about enzyme production in P. chlamydosporia and suggested that the mechanisms which regulate the trophic switch in this fungus are complex and dependent on several factors.
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