Isolating Effective Fungi To Facilitate Seed Germination Of Paphiopedilum Spicerianum And Application In Conservation Practice

Paphiopedilum spicerianum is a species that included in the Plant Species with Extremely Small Population Project(PSESP)in China,which has high ornamental value and scientific research value.P.spicerianum is one of the star species in Orchidaceae plant’s protection.In China,only one remnant population was found on the steep slope of the river at the entrance of an immigrant village in Simao District,Pu’er City,Yunnan Province.The main strategy for the effective protection of P.spicerianum is establish a self-sustaining population in the wild.On the basis of P.spicerianum conservation practice in the past,this study summarized the datas of the asymbiotic germination plants’ s growth and reproduction status,which had been reintroduced or assisted conolized in the wild.By the technology of ex-situ symbiotic germination,we isolated the effective symbiotic fungi of P.spicerianum.Using the isolated effective symbiotic germination fungi to carry out the germination promotion experiment of P.spicerianum and produce symbiotic seedlings.In order to achieve effective protection for P.spicerianum,we using the isolated fungi made fungal seeds bags and try to apply it in the assisted conolization.1.The living status of asymbiotic seedlings of P.spicerianum after reintroduced and conolized in the fieldThis study analyzed and evaluated the growth status of asymbiotic germination seedlings of P.spicerianum after reintroduced and conolized in the field.It was found that the survival rate of the plants was high in the first two years.However,the survival rate of the plant decreased rapidly between the second year and the third year.P.spicerianum asymbiotic germination seedlings can bloom after a certain period of growth in the reintroduced and assisted conolization sites.The survival individuals could obtain reproductive fitness successfully in the reintroduced sites,but it is still need to further exploring whether the survival individuals can obtain fruits in the assisted conolization sites.2.Isolating the endophytic fungi of P.spicerianum(1)Put the seeds bags in the field for in-situ symbiotic germination,after 6 months of continuous observation,no symbiotic protocorms or seedlings obtained.In ex-situ symbiotic germination,seeds germinated to protocorms and seedlings on the substrate which taken in the rainy season.However,seeds cannot germinate on the substrate which collected in the dry season,may be attributed to the inappropriate sampling time.(2)Two endophytic fungi GYBQ01 and GYBQ02 were successfully isolated from the symbiotic protocorms of P.spicerianum.The strains obtained by molecular identification are all fungi of the family Tulasnellaceae,and their sequences have been uploaded to GeneBank with the numbers MN733451 and MN793984,respectively.MN733451 is most similar to the fungus with accession number JX514385.1 in Tulasnella,the maximum similarity is 99.84 %;MN793984 is most similar to the fungus with accession number JX545225.1,the maximum similarity is 99.64 %.3.Testing the fungal capacity and specificity in promoting seed germination(1)Through the inoculation germination experiment of P.spicerianum seeds sown on oat medium(OMA),the results showed that the two isolated fungi can effectively promote the germination of P.spicerianum seeds developed to seedlings,but no protocorm was formed in the blank control group.The seed germination rate and protocorm formation rate after the seeds were incubated with GYBQ01 strain for 60 days were 78.93 ± 1.02 % and 22.86 ± 2.91 %,respectively.The seed germination rate and protocorm formation rate after the seeds were incubated with GYBQ02 strain for 60 days were 78.93 ± 1.02 % and 35.83 ± 2.96 %,respectively.A large number of seedlings were formed after the seeds were cultured for 90 d after GYBQ01 and GYBQ02 strains were cultivated,and the seedling formation rates were 30.83 ± 2.56 % and 43.45 ± 3.16 %,respectively.In comparison,the seedling formation rate of P.spicerianum after being cultured with GYBQ02 strain for 120 days was higher,reaching 57.62 ± 3.58 %,while the seedling formation rate of GYBQ01 strain was 38.45 ± 2.93 %.(2)The effective symbiotic germination fungus Tulasnella sp.2 of Arundina graminifolia was co-cultivated with P.spicerianum seeds.After 120 days,P.spicerianum seeds did not form protocorm.In the inoculation germination experiment of Paphiopedilum hirsutissimum,after 60 days,the treatment with GYBQ01 strain can form the protocorm,but it can not be successfully developed to seedling and other inoculation treatments did not form protocorm.From these results,it can be seen that the isolated fungi have a high specificity for the seed germination promotion,and the growth of the P.spicerianum seeds to the seedling stage rely on the specific fungi.4.Preliminary study on effective symbiotic germination fungus of P.spicerianum for direct application in conservation practiceThe isolated effective germination-promoting fungi are used to make fungal seed bags,which are placed in the sterilized original substrate and assisted transplantation site respectively.After 120 days of cultivation and observation,the results showed that the seed bags on the sterilized substrate in the laboratory can germinate to seedlings and grow very well.Theoretically,the fungal seed bags can be used in the protection practice of P.spicerianum directly.5.Using isolated effective symbiotic germination-promoting fungi cultivating symbiotic seedlings of P.spicerianumObtained the seedlings that have leaves and roots,after the seeds sown on oatmeal medium(OMA)inculated the effective symbiotic fungi cultivating for 120 days.Then the seedlings transplanted on the original substrate of P.spicerianum for further cultivation in the laboratory,and obtained many symbiotic plants of P.spicerianum,which could be used for wild reintroduction.