Effects of bacterial wilt on community composition and diversity of culturable endophytic fungi in Alpinia galanga

Abstract Hongdoukou plant ( Alpinia galanga Willd.) is a perennial herbaceous plant that usually has a stable microflora living in the inter-root and stem and leaf tissues, which assists the host in normal growth and development. The bacterial wilt disease that had been investigated in planting bases of A. galanga , was a new soil-borne disease infected by pathogenic bacteria ( Ralstonia solanacearum (Smith) Yabuuchi et al.), which disrupts the A. galanga -microbe-soil microecological balance. For this reason, it is important to study the changes of the community composition and diversity of endophytic fungi in healthy and diseased A. galanga (HDK_J and HDK_B), and to mine the active endophytic fungal resources in order to lay the foundation for exploring the functional microbial communities for artificial synthesis. All experimental materials were from the healthy and diseased plant clusters of A. galanga in the traditional planting areas in Guangdong Province. The endophytic fungi were isolated from the stems and leaves of A. galanga by separated plant tissue blocks cultured in PDA medium. The colonization rate (CR) was calculated, and the isolated strains were amplified by PCR with the ITS1/ITS4 genes as the fungal molecular markers, to identify the endophytic fungi species, and to analyze the diversity of endophytic fungi in the healthy and diseased clusters of HDK_J and HDK_B, and the indexes included: relative segregation frequency (SF), relative abundance (RA) plotted by ggplot2 under R language, Shannon-Wiener diversity index ( H′ ), Simpson index ( D ), Margalef richness index ( R ) and Pielou uniformity index ( J ), and finally, a phylogenetic tree of the endophytic fungi of healthy and diseased A. galanga was constructed by MEGA. A total of 685 endophytic fungal strains were isolated from the stems and leaves of healthy (HDK_J) and diseased (HDK_B) A. galanga , of which 27 endophytic fungal species were identified in healthy A. galanga . and 8 in diseased A. galanga , which belonged to 3 Phyla, 6 Classes and 13 Families. At the genus level, the RA of HDK_B endophytic fungi were only 38.93% of HDK_J, with significant difference ( P <0.05), indicating that the composition and abundance of endophytic fungi in HDK_B were lower than that in HDK_J. Meanwhile, the endophytic fungi species isolated from the stems and leaves of the HDK_J and HDK_B were the same, but the RA of leaf organs was higher than that of stems, with the RA in the leaves in HDK_J higher than that of stems by 124.23%, and the RA of leaves in HDK_B higher than that of stems by 78.23%. The RA of HDK_J leaves was 78.08% higher than that of stems. all the diversity indexes of HDK_J were higher than that of HDK_B, with significant differences ( P <0.05), and the Shannon-Wiener index ( H′ ), Simpson’s index ( D ), and Margalef’s index ( R ) behaved as follows: HDK_J leaves (1.16, 0.87, 5.12) > HDK_J stems (0.82, 0.72, 4.22) > HDK_B leaves (0.57, 0.24, 2.97) > HDK_B stems (0.33, 0.14, 2.25), whereas Pielou’s index ( J ) indicated that the endophytic fungal species were more uniformly distributed in HDK_J leaves, and there was not much difference in the homogeneity of the leaves of the HDK_J stems and the HDK_B stems. The mapping phylogenetic trees showed there were 4 major development branches of the species on endophytic fungi in HDK_J, and especially, there were many long development branches under the Ascomycota. The genetic relationships among different species were completely revealed in phylogenetic trees. The average genetic similarity among genera was 52.80%. Conversely, there only were1major branch (Ascomycota), and few sub-branches under the Ascomycota in HDK_B. The average genetic similarity among genera was 84.59%, and the difference was small. The bacterial wilt significantly affected the composition and RA of endophytic fungi in A. galanga . The diversity indices showed decreasing trend in A. galanga after infected by R. solanacearum . The dominant species were changed. The parts of sensitive endophytic fungi were disappeared. This result will be helpful for the study on the relationship between the artificial minimal microbial community and the role of the host as well as the study on synthetic microbiomics.