The spread of organic farming and the development of resistance to anthelmintics by parasites, especially in small ruminants, have necessitated the search for alternative methods of nematode control. Biological control using nematophagous fungi is one option; however, few studies have been conducted with native strains. The present study was divided into two phases. In the first phase, we aimed to isolate, identify, and assess the in vitro predatory activity of nematophagous fungi that had been isolated on Trichostrongylidae third-instar larvae. In the second phase, the isolate with superior predatory activity in vitro was molecularly characterized, and its morphological plasticity was observed using scanning electron microscopy (SEM) on Haemonchus third-instar larvae. Of the 56 soil samples from different regions of Paraná State, Brazil, 57 fungal strains were recovered, of which four exhibited predatory activity. Two pure isolates were obtained: the CED and LIN strains. After demonstrating 96.35 % predatory activity for the CED strain, this strain was selected and characterized using molecular criteria by sequencing the rDNA internal transcribed spacer and was identified as Arthrobotrys conoides (GenBank ID: JN191309). Morphological patterns in this strain during the interaction between the fungus and the nematode were revealed by SEM, in which two extensions of the infection bulb that was used to pierce the nematode's cuticle were clearly visible.
[Show abstract][Hide abstract] ABSTRACT: Proteases form nematophagous fungi are most important extracellular hydrolytic enzymes paying a central role in cuticle degradation. In the present study, protease from two nematode-trapping fungi, A. conoides GenBank accession no. JX979095 and D. flagrans JX979096 were studied at biochemical and molecular level. Crude protease of A. conoides showed maximum activity at pH 7 and temperature 50°C while protese of D. flagrans was functioning best at pH 8 and temperature 55°C. 726 and 716 bp serine protease gene fragment from each fungus was amplified and sequences were submitted to GenBank under the accession No. KC769585 and KC862257. Phylogenetic relationships showed homology with serine proteases of other nematode-trapping fungi. Moreover protease activity in culture broth of fungi inoculated with nematodes was increased. Biochemical assay demonstrated 2.14 and 6.4 fold increase in protease activity in induced culture of A. conoides and D. flagrans respectively. Real Time-PCR assay confirmed 4.27 and 23.8 fold increase in serine protease gene activity in A. conoides and D. flagrans respectively. Thus like other nematode trapping fungi, serine protease of these two fungi plays important role in virulence against nematodes.
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