Mycelial Culture And Genetic Background Analysis Of The Lactarius Deterrimus Complex

The Lactarius deterrimus complex is composed of several closely related species that have high host specificity with spruces,distributed in the north temperate zone.The species of this complex are all edible,but,the symbiotic nature limits the growth of mycelia on artificial medium.Isolating and culturing the mycelia and evaluating the effects of different culture conditions on the growth rate of mycelia will be of great importance for the follow-up conservation of natural resources.In this study,385 samples of L.deterrimus complex were collected from northwestern Yunnan and western Sichuan in the Hengduan Mountains in 2019 and 2020,and 59 strains were successfully isolated from them.The mycelia were cultured on four media: 1.PDA medium;2.PDA medium with addition of smashed sporocarp of L.deterrimus;3.a mixed medium of PDB(Commercial potato dextrose water)and MMN(1:1 v:v);4.a mixed medium of PDA and MMN(2:1 v:v).The mycelia grew the fastest on the PDA medium with addition of smashed sporocarp of L.deterrimus.The reason might be that the micronutrients in the sporocarps nourished the growth of the mycelia,or the filtrate of the sporocarps functioned as a buffer in the medium.Effects of four kinds of hormones(Salicylic acid,Strigolactone,Methyl Jasmonate and Adenosine)on the growth of mycelia were tested.The results show that three hormones(Salicylic acid,Methyl Jasmonate and Adenosine),regardless of concentrations,had no obvious promoting effects on the growth of mycelia.Strigolactone,which was found to be not able to obviously promote mycelia branching of arbuscular mycorrhizal fungi in previous studies,2.5ng/ml of that had obvious stimulating effect on the growth of mycelia at the three concentrations tested.Significant differences of growth rate were observed among different strains.Genetic background of the mycelia was analyzed using fungal DNA barcode ITS region and microsatellite molecular markers(Simple Sequence Repeat,SSR).ITS sequences of all the strains were obtained and based on this the strains can be assigned into four known species:L.deterrimus,L.fennoscandicus,L.hengduanensis,and L.pseudohatsudake and potential species L.sp03.ITS sequences of these stains are classified into 27 genotypes,12 of which are heterozygous.The possible cross combinations are L.sp03 × L.sp04,L.pseudohatsudake × L.hengduanensis,L.hengduanensis × L.sp03,and L.deterrimus × L.fennoscandicus.Analysis of the ITS sequences of these strains showed that the mycelia with faster growth were mainly heterozygotes between species and homozygotes,while the mycelia with slow growth were heterozygotes within speciesEight pairs of SSR primers were used to amplify 23 strains.All the loci contain both homozygotes and heterozygotes.Data analysis of the eight loci showed that the number of alleles ranged from 2 to 13,showing significant variation among individuals.Correlation analysis between the growth rate and SSR data showed that the strains with the fastest growth rate are homozygotes at most loci.During the research,a new species of Lactarius,L.mirus,was found.The morphology was studied and ITS-LSU sequences were amplified and obtained successfully.This is the first report of species with yellowing latex in L.subg.Plinthogalus in China.