Diversity, chemical constituents and biological activities of endophytic fungi from Alisma orientale (Sam.) Juzep

Abstract

The dried tuber of Alisma orientale (Sam.) Juzep. (AOJ) is a traditional Chinese medicine with high medicinal value. The endophytic fungi of medicinal plants are a treasure house of natural compounds. However, there is a lack of research on the diversity and biological activity of endophytic fungi of AOJ. In this study, high-throughput sequencing technology was used to study the diversity of endophytic fungi in the roots and stems of AOJ, and endophytic fungi with a high output of phenols and flavonoids were screened by chromogenic reaction, and the antioxidant and antibacterial activities and chemical constituents of crude extracts of their fermentation broth were studied. A total of 3,426 amplicon sequence variants (ASVs) belonging to 9 phyla, 27 classes, 64 orders, 152 families, and 277 genera were identified from AOJ. There were significant differences in the endophytic fungal communities of AOJ roots and stems, as well as in the endophytic fungal communities of triangular AOJ and circular AOJ. In addition, 31 strains of endophytic fungi were isolated from AOJ, of which 6 strains had good antioxidant and antibacterial activities. The crude extract of YG-2 had the strongest free radical scavenging ability and bacteriostatic ability, and its IC_{50 DPPH}, IC_{50 ABTS}, and IC_50⋅OH values were 0.009 ± 0.000 mg/mL, 0.023 ± 0.002 mg/mL, and 0.081 ± 0.006 mg/mL, respectively. The results of LC-MS showed that the main component of the crude extract of YG-2 was caffeic acid (10.12 μmol/g). Overall, the results of this study preliminarily elucidated the diversity and community composition of endophytic fungi of AOJ, indicating that AOJ endophytic fungi have abundant secondary metabolites and good antioxidant and antibacterial activities. This study provides an important reference for further research, development and utilization of AOJ endophytic fungi and a theoretical basis for the further development of the endophytic fungus YG-2 (Chaetomium globosum) as a source of antioxidants.

Keywords: Alisma orientale (Sam.) Juzep.; antioxidant; bacteriostatic activity; chemical constituents; diversity; endophytic fungi; high-throughput sequencing.

FIGURE 1

Two kinds of Alisma orientale (Sam.) Juzep.

FIGURE 2

High throughput sequencing. (A) ASV rarefaction curves. (B) Venn diagram showing the number of shared and specific ASVs for each group.

FIGURE 3

Composition of endophytic fungi in root and stem of AOJ. (A) Multiple sample principal coordinate analysis (PCoA) of the ASV level. (B) Multiple sample Non-metric multidimensional scaling (NMDS) of the ASV level. (C) Relative abundance of phyla in each sample. (D) Relative abundance of genus in each sample.

FIGURE 4

Phylogenetic tree of endophytic fungi in Alisma orientale (Sam.) Juzep.

FIGURE 5

Total Phenol and Total Flavonoid Content of YG-2, SJ-11, YJ-4, YJ-9, YJ-10, and SJ-2 extracts. a–e and A–E: significant differences between different groups (p < 0.05).

FIGURE 6

Antioxidant activity in vitro assays of YG-2, SJ-11, YJ-4, YJ-9, YJ-10, and SJ-2 extracts. (A) DPPH clearance assay; (B) ABTS clearance assay; (C) ⋅OH clearance assay; (D) superoxide anion clearance assay.