Isolation And Identification Of Nematode-trapping Fungi And Screening Of High-performance Strains

Nematode parasites in the animal gastrointestinal cause long-term, nutrition consume mechanical injury, reducing the efficiency of feed utilization, causing slow growth in livestock and lead to decline of animal products in quantity and quality, which causes highest economic losses and seriously harm for the development of animal husbandry. At present, the control of nematode disease mainly depends on chemical drugs, which not only lead to drug residues, make the parasites body resistance, but also cause serious environmental pollution. As nematodes predators, nematode-trapping fungi were a kind of effective and important nematode natural control factors. However, studies on nematode-trapping fungi in domestic are lagging behind compared to foreign countries. It is an urgent need to study the biological characteristics of nematode-trapping fungi for the development of environment-friendly method to control the animal gastrointestinal nematode disease.In order to develop the biological reagents to prevent animal gastrointestinal nematode disease, nematode-trapping fungi in soil samples collected from the Northern Tianshan Mountain slope pasture were isolated, and then identified using morphological and molecular biological techniques. The predacious activity and the ability of pass through sheep digestive tract were determinated. High efficient strains which can efficiently kill livestock gastrointestinal nematodes were screened. The main contents and results of this study are as follows:1. Isolation of Arthrobotrys oligospora in soils samples from the northern Tianshan mountain slope pasture. Nematode-trapping fungi in soil samples were isolated from the Northern Tianshan Mountain slope pasture using Haemonchus contortus (H. contortus) L3as bait nematode by microbiological and parasitological methods, and then identified by morphological and molecular techniques and elementary predacious activity were assayed. As the result,26nematode-trapping fungi strains were successfully isolated.9in26isolates had the characteristics of typical solitary, separated conidiophores erect, oval conidia and networks, which were consistent with morphological characteristics of Arthrobotrys oligospora(A. oligospora).5.8S rDNA-ITS2genes of isolates were then amplified by PCR for the sequences comparison with known strains. There was the highest identities (99.69%.) among isolates and Arthrobotrys oligospora isolate128Hongkong isolate, which confirmed that these isolates were Arthrobotrys oligospora strains.2. Observation of predation process of Arthrobotrys oligospora and assay of predacious activity against Haemonchus contortus L3. The interaction of nematode-trapping fungus A. oligospora against H. contortus larvae (L3) using the inverted microscope were observed. Predacious activity of9strains of A. oligospora in different conditions such as nutrient concentration (O.Og/L,0.2g/L,0.4g/L,0.6g/L,0.8g/L,1.0g/L,2.0g/L), ambient temperature (5℃,10℃,15℃,20℃,25℃,30℃,35℃) and the larval concentrations (500,1000,2000,3000,4000,5000), were assayed, respectively. The results show that:①The predation structures are present after6h of H. contortus L3joined; the simple three-dimensional network are formation after8h; predatory structure continues to develop, become mature three-dimensional network after12h;②When the nutrient concentration, ambient temperature and larval concentrations respectively were0.4g/L,20℃and3000of each panel, the predacious rate of strains against H.contortus reached a maximum level, respectively85.25%,89.90%and93.23%. Show that the predacious activity of nine A, oligospora strains were increased firstly and then decreased with the increase of the nutrient concentration and the ambient temperature, but then sustained in a high level with the increase of the larval concentrations.3. Predation of A. oligospora against nematode in feces and the ability of passage through of sheep digestive tract:Using the method of adding quantitative nematode and recovery the residual nematode after the predatory of A. oligospora, the predacious activity of9strains against H.contortus L3in feces before and after passage through the sheep gastrointestinal tract were determinated. The results showed that:①The A-XJ1isolate displayed the highest predacious activity (97.6%) followed by B-XJ2, L-XJ12, D-XJ4, and M-XJ13strains (ranging from96.9%to90.8%) before passage through the sheep gastrointestinal tract, while the predatory activity of C-XJ3, H-XJ8, J-XJ10, K-XJ114strains were93.4%,91.8%,93.8%and93.2%, respectively;②Isolates B-XJ2, C-XJ3, D-XJ4, H-XJ8, J-XJ10, L-XJ12could pass through the sheep gastrointestinal tract and had significant reductions in the number of Haemonchus contortus larvae. The highest is the isolate B-XJ2was78.7%, followed by D-XJ476.9%, and L-XJ1276.1%) isolate. The least was J-XJ10isolate with the predatory activity of72.4%, while the predation activity of C-XJ3and H-XJ8strains were74.5%and73.8%, respectively.In conclusion,9strains of nematode-trapping fungi were successfully isolates and identified as A. oligospora, These isolates displayed high nematode activities under laboratory conditions, and some strains remained high nematode-trapping ability after through the sheep digestive tract. The study showed that these isolates had the potentiality for the development the biological agent to control livestock gastrointestinal nematode disease.