Study On Life Cycle Of Ophiocordyceps Xuefengensis

Ophiocordyceps xuefengensis,a new species associated with larv ae of Phassus nodus(Hepialidae),was found that has the closest genetic relatives with Ophiocordyceps sinensis.Currently,the micros copic characteristics and chemical composition of this fungus have been studied;however,the studies about its artificial cultivation and life cycle have not yet been reported.This is the first time to stud y the life cycle of Ophiocordyceps xuefengensis systematically,the main contents and conclusions are as follows:1.Artificial cultivation in different developmental stages of Ophiocordyceps xuefengensis.Modified the cultivation conditions of Ophiocordyceps xuefengensis,and then observed the different stages of development and collected the spores to make the aennination experiments.The results showed that the mycelium were white,villiform in the initial stage,and later was pale rust.The mycelia were kinked when they were matured,and then gradually formed primordium.The primordium was gradually increasing which developed into asexual stromas.The top of asexual stromas was white,which the middle and lower part was pale rust.Continue to cultivate,asexual stromas gradually increased which the top still was white,then the middle and lower part become deep rust.The spore germinated and developed into mycelium.2.Morpho-anatomy features in different developmental stages of Ophiocordyceps xuefengensis.The temporary slide in different developmental stages of Ophiocordyceps xuefengensis staining with Congo red showed that: a.the mycelium with many septa were multiple.b."H" type fusion phenomenon between the mycelium.c.solitary conidiophore were unbranched or branched.d.asexual stromas can be divided into cortex andmedullary layer.TEM Photomicrograph of the asexual stroma showed that the surface of asexual stroma covered densely conidia,which size ranged(3.2~5.0)um×(2.1~3.3)um.Each conidiogenous cell produced one conidia which was elliptical,smooth.The cortex and medulla of the stroma is composed of mycelial cell,which the small cortical cells were arranged closely,and an obvious gap could be seen between medullary cells.Cultured mature mycelium,mycelium and spores on asextual stroma were stained by fluorochrome(DAPI)and observed under fluorescence microscope.The results were as follows: the mycelia were various in size at different growth stages,which the membrane was clearly visible.Most of the cultured mature mycelia and the mycelia of asextual stroma are amononuclear and binuclear,while most of conidia had only one nucleus.3.Study on life cycle of Ophiocordyceps xuefengensis using SNPs molecular markersIn this study,30 pairs of primers were designed based on the genomic library of Ophiocordyceps xuefengensis.The same primers were used to amplify in different strains or samples.A total of 36 SNPs were found in Ophiocordyceps xuefengensis,representing an SNP frequency of 0.129%.The sequences and chromatographs indicated that the mycelium and asexual stromas was a homokaryons,while the ascus part was a heterokaryon.4.Detection of mating type genes MAT1-1-1 and MAT1-2-1in Ophiocordyceps xuefengensisThe specific primers were designed based on the conserved sequences of MAT1-1-1 and MAT1-2-1 in Ophiocordyceps sinensis.Through the detection of MAT1-1-1 and MAT1-2-1 in different Ophiocordyceps xuefengensis samples,then discovered the dead insects body?stroma(not having ascocarp),and isolates of Ophiocordyceps xuefengensis containing MAT1-1-1 or MAT1-2-1,but included two mating type gene in mature site of stroma(with ascocarps),the results showed that Ophiocordyceps xuefengensis is heterothallic.Based on our observations and data,we proposed the following life cycle of Ophiocordyceps xuefengensis.In the wild,the parasite spores germination and mycelial growth in larvae of Phassus nodus after infecting spores.When the mycelium grow to a certain stage,sclerotium were developed from the aggregation of hyphae.Under certain conditions,Ophiocordyceps xuefengensis could form asexual stroma with single mating type gene or homokaryon,which could forcibly ejected their ascospores.When the asexual stroma matured,the sexual reproduction of Ophiocordyceps xuefengensis beginning with complementary mating on the surface of mature asexual stroma.