Isolation,Biological Characteristics,in Vitro And In Vivo Studies Of The Duddingtonia Flagrans Of The Nematophagous Fungus

As a candidate for biocontrol of parasitic nematode in domestic animals,nematophagous fungus Duddingtonia flagrans has been widely concerned at home and abroad.Thus,the purpose of this study is to isolate D.flagrans in China,and to study the biological and genetical characters,observe the ultrastructure of the isolates and the interaction with the third stage larvae(L3)of trichostrongylides,and assess the in vitro predatory capacity and predatory L3 capacity of all isolates of nematophagous fungi after passing through the gastrointestinal tract of sheep.The total frequency of D.flagrans detected in 1532 different types of samples was 1.70%(26/1532).Among all samples,this fungus was detected in the feces of cattle and sheep,the dung composted in barns,pasture soil but not detected in the soil around barns.After purification,total of 13 pure cultures of D.flagrans were obtained.Morphological studies showed that the isolates could produce the conidiospores never recorded in literature,such as long bar,meniscus and "V" shapes at two or more septum and found that conidiophores could be multiplied occasionally.The sequences of 18 S rRNA,28 S rRNA and ITS1-5.8S rRNA-ITS2 of D.flagrans showed that the isolates shared 98%–100% identity with D.flagrans sequence in GenBank.Phylogenetic evolution tree analysis showed that seven isolates and other isolates were on one branch.The tested isolates could grow in the temperature range from 11–35 ?,with the optimal growth temperature at 30 ?.The fungus did not grow in the pH interval from 1 to 3 and from 13 to 14 and grew in the pH interval from 4 to 12,and best growth achieved between pH 7 and 8.Eleven fungal isolates were cultured on 2% WBA,2% CMA,WA and PDA of culture mediums,andthis result showed that the radial growth of all isolates on 2% CMA and 2% WA displayed faster than that of other culture mediums(2% WBA and PDA)(p <0.05),except the isolate NBS063 and SFG170.The ultrastructure of conidia and chlamydospore of different developmental stages for D.flagrans,and the interaction between the fungus and the L3 of trichstrongylides were obtained by scanning electron microscopy.At 6–8 h after the addition of nematodes,the L3 was captured.At 6 h post-capture,the L3 was penetrated by hyphae.At 24 h post-capture,the L3 began to be digested and at 48 h post-capture,L3 s were completely digested and intercalary chlamydospores were formed and emerged in the body of L3.In in vitro test,the reduction percentage of L3 of Trichostrongylus colubriformis ranged from 57.21 to 99.83%,and that of Haemonchus contortus ranged from 62.12 to 99.88%.The spores of representative isolate were excreted from feces of sheep 12–96 h after oral administration,while the time of fungal excretion in feces after its administration on a full stomach has a 24 h lag compared with that on an empty stomach.Three isolates of D.flagrans were oral administrated to the Rex rabbits.The spores of the two isolates were excreted from feces of rabbits 4–24 h after oral administration,and one isolate was disappeared in the feces at 18 h.Ten isolates of D.flagrans were assessed to pass through the digestive tract after oral administration of sheep,while the reduction percentage of L3 ranged from 55.15 to 98.82%.