Studies Of Biochemical Mechanism Of Trapping And Killing Nematodes By Arthrobotrys Oligospora

The parasites, especially the nematodes, cause great damage to livestock. Dur- ing the past 50 years, nematicides have been used extensively to control nematodes diseases in animals, but their useage has become increasingly restricted duce to anth- elmintic resistance, public health and environmental concerns. To get rid of these pro- blems, nematode-trapping fungi have attracted much attention. But it is necessary to ascertain its biochemical mechanism with the need for control of parasites, so the study was carried out. The results were as follows:(1)Caenorhabditis elegans could induce Arthrobotrys oligospora to produce great quantities of traps, and were trapped two hours later. Besides, it seemed that traps touched the nematodes were more muscular and were covered with liquid materia; There were also many electron-dense microbodies in the edge of cells of traps under TEM.The touch points of fungus with nemaotdes were most on heads and tails, which showed that there were more specific sites in these parts; There were also many introcessions in the touch points and appresorium formed later, then it dilated into infection bulbs after hyphae penetrated the body walls of nematodes. Soon they developped into common vegetative hyphae and contents of nematodes were digested during the stage, then the hyphae filled the nematodes and the nematodes were digested completely.(2)Among the 10 carbon sources and 8 nitrogen compounds tested, the optimal carbon and nitrogen sources for mycelial growth in solid culture were sucrose and peptone, but it was sweet potato filtrate and ammonium sulfate in liquid culture. A C/N ratio of 5:1 at pH 6.8 gave the maximum growth in solid culture and 10:1 at pH 6.8 gave the maximum biomass in liquid culture.The initial pH value had a significant effect on mycelial growth, with the optimal rangs being 7.0-8.5 for growth in solid culture and 5.5-6.0 for growth in liquid culture.(3)The effects of amino acids on traps forming was tested on the CMA.The results showed that amino acids could induce the traps formation and these traps could kill larvae of nematodes effectively. Different amino acids had different effects on traps forming. Among all of the amino acids tested, L-Phenylalanine, L-Methionine and DL-valine of 0.05g/L solutions had the best effects to induce traps formation, and L-Threonine, L-Isoleucine, L-Tryptophanea, L-Leucine and L-valine had better effects than the others. The traps forming in initial stage and in intermediate stage was better than in the final stage, anyway it was best in the initial stage. Seen from the above, Amino acids mainly induced young hyhape of A.oligospora to form traps.(4)The biochemistry composition of A. oligospora and the third larvae of nematode were measured by conventional methods. The contents of crude proteins and amino acids in the hyphae with traps were much higher than in vegetative hyphae and conidia, but were lower than the third stage larvae; The SDS-PAGE result showed that the protein contents in the hyphae with traps and in common hyphae were different. Besides, the contents of total sugar in vegetative hyphae and the hyphae with traps were 30.69% and 24.77% respectively. All these showed that biochemistry composition changed when the vegetative hyphae transformed into the traps.(5)In this experiment,the extraction methods of surface polymers from the hyphae with traps and the effects of extraction buffers, enzymes and protease inhibitors on growth and trapping of fungus were studied. The results showed that surface polymers were very important in the process of adhension and it mainly contained proteins or compouds with proteins. Besides, we detected protease activity and phosphotase activity in surface polymers for the first time and it was much higher in hyphae with traps than in vegetative ones, which gave indirect evidences that protease and phosphotase might play important roles in trapping process.(6)The composition of the culture and the inductors can signinficantly influence metabolic process of A.oligospora, and the composition of ferments induced by C.elegans and by amino acids were different. we also found that the ferments with high protease activity had low phosphotase activity. Besides, a protrease with molecular mass of 38KD was purified from the LMZ culture. it had a serine residue in its active site, suggesting it belonged to subtilase family of serine proteases. The pH optimum was found to be pH6-8.5 and the optimal temperature was 50℃. The purification of the extracellar protease provided important data for the trapping and killing mechanism of nematode-trapping fungus.(7)A 1108bp fragment of the protease gene from A. oligospora CHIM has been sequenced, which contained 6 kinds of restriction sites(BstXI, DraII, EcoRI, SacI, SalI and XbaI) in the fragment. The homology in protease genes from A.oligospora of Inner Mongolia compared with the strains from Sweden or from Yunnan were 80.3% and 84.1% respectively. The similarity between animo acids deduced from gene and a sequence reported in GenBank were 100%. Based on Blastn analyses, the fragment that we had amplified were partial gene of serine protease. All the results showed again that the nematode-trapping fungus—A. oligospora were different in strains from different nations and regions.