Effect And Mechanism Of Endophytic Fungi On Grape Anthocyanins

In this study,grape callus(Vitis vinifera L.cultivar:Gamay)and endophytic fungi were chose to construct two type of co-culture systems:endophytic fungi-grape cells contact co-culture and dual co-culture systems.The effects of endophytic fungi on anthocyanins of grape and its possible mechanism were discussed by analyzing the growth of grape cells and their anthocyanins content,composition and the expression of key genes after co-culture with different endophytic fungal strains or strain extracts.The main results are as follows:1.Grape callus contact co-culture(CC)with most endophytic fungi strains,significantly inhibited the growth and total anthocyanins(TA)content of grape cells,while nearly 50%of endophytic fungi strains were dual co-culture with grape callus increased the total anthocyanins content in grape cells.Among them,fungal strains XHYN-2(Alternaria alternata)and R2-21(Epicoccum sp.)significantly(P≤0.05)increased the total anthocyanins content of grape cells.Our results illustrated that dual co-culture respectively with fungal strains XHYN-2,R2-21 and B2-17(Alternaria sp.)the components and relative contents of anthocyanins in grape cells were specifically changed.It indicates that endophytic fungi have potential application value in regulating grape color and stability.The results of fluorescence quantitative PCR indicated that the expression of anthocyanin biosynthesis structural genes VvCHS2,VvF3’5′H,VvF3′H1,VvUFGT were down-regulated with varying degrees and regulatory gene VvMybAl were up-regulated in grape cells after co-cultured with fungi.It indicated that the endophytic fungi have an impact on anthocyanin synthesis of grape cells and it was mainly achieved by controlling the expression of upstream transcription factors.2.Using the Multiplex Research to analyze the anthocyanin index(ANTH-RG)of grape cells that dual co-cultured with three fungal strains,the results showed that the three fungal strains had certain promoting impacts on the ANTH-RG of grape cells.During the whole culture period,the ANTH-RG of grape cells increased in the first stage,peaked on the 3rd day then decreased.By analyzing the anthocyanin index of grape berries after dual co-culture with fungal strains XHYN-2,R2-21 and B2-17,the results showed that the fungal strains can improve the ANTH-RG of grape berries throughout the whole culture period.It indicated that the endophytic fungi can also regulate the synthesis of anthocyanins in grape berries in addition to the grape callus.3.With adding different concentrations of endophytic fungal water extracts to the culture medium,the total anthocyanins content of grape cells was affected with varying degrees and showed obvious dose effect.With the increase of the concentration of fungal strain XHYN-2 water extracts,the total anthocyanins content of grape cells was also increased.The total anthocyanins content significantly promoted on the 6th day with XHYN-2 water extract of 4 mg/L and reached a very significant(P≤0.01)level.With the increase of endophytic fungal R2-21 water extracts concentration,the promotion effect on total anthocyanins content of co-culture grape cells decreased gradually.And on the 9th day of with low concentration R2-21 water extracts(2 mg/L and 1 mg/L)for co-culture,it showed a significant effect on the total anthocyanins content in grape cells on the 9th day.