| Parasitic nematodes in domestic animals are the pathogens of a large class of diseases,among which Haemonchus contortus is one of the most common parasitic diseases that seriously affect the healthy development of cattle and sheep breeding industry.Its treatment mainly depends on drugs,such as ivermectin and other broad-spectrum insect repellents.These drugs not only caused environmental pollution and chemical residues,but also lead to drug resistance of nematodes.In order to avoide the spread of drug resistance,it is urgent to find new methods to control parasitic nematodes.China has been committed to the implementation of green and healthy production mode and sustainable development strategy,and biological control has gradually entered people's attention.Among them,as an important candidate strain for biological control of animal parasitic nematodes,D.flagrans mainly includes two stages: hyphae and spores(chlamydospores and conidia).Usually,hyphae can produce predatory structure to kill nematodes.The insect resistance mechanism is only limited to the mechanical damage of nematodes caused by the penetration of mycelium through the horn skin of nematodes.Whether there are other insect resistance mechanisms is unknown.The previous research of our research team found that D.flagrans chlamydospores had dormancy during the culture process.At this time,the spores could not produce hyphae,and then lost their insect resistance activity.However,the conditions for the formation and release of dormancy have not been reported.In addition,fungi are an important source of many small molecular compounds to be used.It is not known whether the secondary metabolites produced by the fungus in the later stage of its growth have the same effect against H.contortus as the mycelium.Clarifying the dormancy characteristics and releasing dormancy conditions of chlamydospores of D.flagrans is the prerequisite for studying the anti-nematode effect of D.flagrans.In conclusion,clarifying the dormancy characteristics of chlamydospores of D.flagrans and the conditions for releasing dormancy are the preconditions for studying the anti-nematode effect by D.flagrans metabolites.Therefore,the dormancy characteristics of D.flagrans chlamydospores and the mechanism of anti-H.contortus were studied,as follows:1.Dormancy characteristics of chlamydospores of D.flagrans(1)Morphological structure of chlamydospores at different physiological stages:using MTT staining and laser confocal observation,it was found that the cell wall of dormant spores was significantly thicker than that of non-dormant spores,and the nucleus was relatively complete.The cytoplasmic vesicles of non-dormant spores were small granules,while the cytoplasm of dormant spores fused to form several large masses.The results showed that cell wall thickness and nuclear integrity were the main basis for distinguishing dormant and non-dormant spores.(2)Effects of physical and chemical factors on chlamydospores: using physical factors such as different temperature,p H,light,hypoxia,hypertonic environment and drying,as well as the effects of 6 carbon sources and 3 amino acids on chlamydospores germination rate.The results showed that the optimal germination temperature of spores was 30 ? and p H was 6-8.Visible light irradiation could inhibit spore germination,but the spore germination rate did not decrease significantly after ultraviolet irradiation.Hypoxia could significantly reduce the spore germination rate.Among various drying methods,vacuum freeze-drying decreased the spore germination rate the fastest.In addition,0.03% arginine,1% fructose and 0.3% lysine could significantly promote spore germination(p<0.05),and 1% trehalose and glycerol could significantly inhibit spore germination(p<0.05).The results suggest that temperature,p H,hypoxia and vacuum freeze-drying are the most important factors affecting the germination rate of chlamydospores of D.flagrans.(3)Determination of related substances in chlamydospores at different physiological stages: it was found by gas chromatography-mass spectrometry(GC-MS)that non dormant spores contained one saturated fatty acid and one unsaturated fatty acid,dormant spores contained one saturated fatty acid and two unsaturated fatty acids,and non-dormant spores contained two saturated fatty acids and seven unsaturated fatty acids.The content of trehalose in non-dormant spores was significantly higher than that in dormant spores,and the content of glycerol was significantly lower than that in dormant spores(p<0.05).The content of c AMP in resting spores was significantly higher than that in non resting spores(p<0.05).The protein content of dormant spores was higher than that of non-dormant spores.These results indicating that the types of fatty acids,trehalose,glycerol and c AMP were important factors affecting the dormancy of chlamydospores.(4)Study on dormancy release conditions of dormant spores: the spores were dried by vacuum freeze-drying method.The results showed that the longer the culture time,the lower the spore germination rate,the higher the culture humidity and germination rate,and the spores entered structural dormancy in the dehydrated environment.Dormant spores can be released from dormancy after storage at 4 ?,-20 ? and 28 ?(RH90 ? 95%),which reveals that culture time and freeze-drying are the physical and chemical basis of chlamydospore dormancy.The storage temperature and humidity are important factors for chlamydospore dormancy release,which can provide a key basis for the study of spore dormancy release.2.Mechanism of anti-H.contortus by D.flagrans(1)Effects of different treatments on the of affecting chlamydospores on H.contortus: chlamydospores treated in different ways were co-incubated with the third stage larvae of H.contortus.The results showed that 70 ?,p H 1-3,5%?saturated sodium chloride solution and ultraviolet irradiation were the main reasons for reducing the killing rate of chlamydospores,and-20 ? treatment was an important factor for maintaining the killing rate of chlamydospores.(2)Anti-nematode mechanism of D.flagrans: the semi terpene epoxy cyclohexene compounds NF1702-1 and NF1702-2 in the crude ethyl acetate extract of D.flagrans were incubated with the third stage larvae of H.contortus respectively,and the anti-nematode mechanism of H.contortus was studied.At the same time,D.flagrans fermentation broth and mycelium were used to compare the model nematode(Caenorhabditis elegans).JC-1 method and mitosox method were used to detect the mitochondrial membrane potential and reactive oxygen species content of H.contortus and C.elegans respectively.It was found that the mitochondrial membrane potential of the two nematodes decreased significantly,while the content of reactive oxygen species and the expression of apoptosis related genes increased significantly.Western blot was used to detect the phosphorylation of major molecules in MAPK signaling pathway.The results showed that the phosphorylation levels of p38 and JNK in H.contortus and C.elegans were significantly increased(p<0.05).In addition,the phosphorylation level of ERK in C.elegans was also significantly increased.It is suggested that the crude ethyl acetate extract and active monomer of D.flagrans may participate in apoptosis pathway and activate MAPK signal pathway when playing the role of anti H.contortus.Compared with the control group,the expression level of ced-3 in the third stage larvae of H.contortus could be significantly reduced by RNA interference(p<0.05).In conclusion,this study preliminarily clarified the relevant characteristics of D.flagrans chlamydospore dormancy and dormancy release and clarified the effects of different treatments on chlamydospore anti-H.contortus.On this basis,it was explored that D.flagrans Ethyl acetate crude extract and active monomers have a role in killing H.contortus and may activate cell apoptosis pathway and MAPK signal pathway,it provides an important basis for in-depth study of fungi against parasitic nematodes. |
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