Isolation And Purification Of Two Endophytic Fungi In Zizania Latifolia And Optimization Of Culture Conditions

Zizania latifolia is a perennial herb of Zanthoxylum which is the second largest aquatic vegetable in China.It is not only nutritious,but also has health and medicinal value.The formation of its swollen stem is closely related to the infection of Ustilago esculent.There are three kinds of phenotypes,including thenormal,thegray and the males,and these three phenotypes are closely related to the growth of Ustilago esculent.When the Ustilago esculent successfully infected Zizania latifolia plants,the stem tissue is enlarged to form a fleshy stem,"normal Zizania latifolia".If the plant can resist the infection of Ustilago esculent,it will then flowering and bearing,and the stems will not swollen,which is called "the male".If the latent period of Ustilago esculent is shortened,and form a dark brown plutonium spores,and lead to the stems filling with the black spots,which are called "the gray plants".The study shows that the incidence rate of the male and the graywill be around 30%,if it is not selected for two years,and which seriously affects the economic benefits of farmers.This topic took Zizania latifolia as the research object,and studied the species and their training conditions of endophytic fungi of Zizania latifolia.The main results are as follows:1.Two kinds of endophytic fungi were isolated and purified by the combination of three-point purification and monospore isolation.Take the identification of endophytic fungi by a series of molecular biological methods,including DNA extraction and the fungal primers designation,fragment PCR amplification,bacterial fragment recovery,T vector attachment and the corresponding plasmid extraction and so on.The sequencing results were 627 bp and 618 bp respectively.The results showed that the two kinds of endophytic fungi were Mucor and Bjerkandera adusta.2.On the basis of the purification and identification of endophytic fungi,the pure colony of Mucor was obtained,and its culture conditions were systematically selected,including the culture mediums,inoculation quantity,p H value,rotational speed,temperature and time.The results showed that Mucor grew well in the modified Martin medium with the inoculation amount of 3/50 ml,p H 6.5,rotational speed of 130 r/min and temperature of 28 ?,and reached the best level at 108 h.3.The purified colony of Bjerkandera adusta was obtained by the same methods,and the culture conditions such as culture medium,inoculation quantity,p H value and time was also determined.The results showed that Bjerkandera adusta grew well in PDA medium with the inoculation quantity of 3/50 ml,p H 6.5,rotational speed of 130 r/min and temperature of 28 ?,and it reached the best level at 96h.