Diversity of Endophytic Microbes in Taxus yunnanensis and Their Potential for Plant Growth Promotion and Taxane Accumulation

Abstract

Taxus spp. are ancient tree species that have survived from the Quaternary glacier period, and their metabolites, such as taxol, have been used as anticancer drugs globally. Plant-endophytic microbial interaction plays a crucial role in exerting a profound impact on host growth and secondary metabolite synthesis. In this study, high-throughput sequencing was employed to explore endophytic microbial diversity in the roots, stems, and leaves of the Taxus yunnanensis (T. yunnanensis). The analysis revealed some dominant genera of endophytic bacteria, such as Pseudomonas, Neorhizobium, Acidovorax, and Flavobacterium, with Cladosporium, Phyllosticta, Fusarium, and Codinaeopsis as prominent endophytic fungi genera. We isolated 108 endophytic bacteria and 27 endophytic fungi from roots, stems, and leaves. In vitro assays were utilized to screen for endophytic bacteria with growth-promoting capabilities, including IAA production, cellulase, siderophore production, protease and ACC deaminase activity, inorganic phosphate solubilization, and nitrogen fixation. Three promising strains, Kocuria sp. TRI2-1, Micromonospora sp. TSI4-1, and Sphingomonas sp. MG-2, were selected based on their superior growth-promotion characteristics. These strains exhibited preferable plant growth promotion when applied to Arabidopsis thaliana growth. Fermentation broths of these three strains were also found to significantly promote the accumulation of taxanes in T. yunnanensis stem cells, among which strain TSI4-1 demonstrated outstanding increase potentials, with an effective induction of taxol, baccatin III, and 10-DAB contents. After six days of treatment, the contents of these metabolites were 3.28 times, 2.23 times, and 2.17 times the initial amounts, reaching 8720, 331, and 371 ng/g of dry weight of stem cells, respectively. These findings present new insight into the industrialization of taxol production through Taxus stem cell fermentation, thereby promoting the conservation of wild Taxus resources by maximizing their potential economic benefits.

Keywords: Taxus yunnanensis; endophytes; growth-promotion; taxanes.

Figure 1

The phylogenetic tree of endophytic bacteria at the genus level. The representative sequences of the top 100 genera were obtained through multiple sequence alignment, and the phylogenetic tree was constructed based on the horizontal species of each genus. The colors of the branches and fan rings correspond to their respective clades. The stacked histograms outside the fan rings convey the abundance distribution information for each genus across various samples.

Figure 2

LEfSe analysis of endophytic bacteria in Taxus yunnanensis: (a) TR vs. TS; (b) TL vs. TR. In the evolutionary branch diagram, the circles radiating from inside to outside represent the classification level from phylum to genus (or species). Each small circle at different classification levels represents a classification at this level, and the diameter of the small circle is proportional to the relative abundance. Coloring principle: The species with no significant differences were uniformly colored as yellow, and the biomarkers of the different species followed the group for coloring. The red node represents the microbial group that plays an important role in the red group, and the green node represents the microbial group that plays an important role in the green group. If a group is missing in the figure, it indicates that there are no significant differences in this group. The species names represented by the English letters in the figure are displayed in the right legend. TR, root. TL, leaf. TS, stem.

Figure 3

LEfSe analysis of endophytic fungi in Taxus yunnanensis: (a) TR vs. TS; (b) TL vs. TR. Each small circle at different classification levels represents a classification at this level, and the diameter of the small circle is proportional to the relative abundance. Coloring principle: The species with no significant differences were uniformly colored as yellow, and the biomarkers of the different species followed the group for coloring. The red node represents the microbial group that plays an important role in the red group, and the green node represents the microbial group that plays an important role in the green group. TR, root. TL, leaf. TS, stem.

Figure 4

Cluster heat map of PICRUSt functional annotation of endophytes in Taxus yunnanensis: (a) endophytic bacteria; (b) endophytic fungi.

Figure 5

The results of IAA production, phosphorus solubilization ability, nitrogen fixation ability, and ACC deaminase production ability of endophytic bacteria in Taxus yunnanensis: (a) IAA qualitative experiment, partial results; (b) quantitative experiment of 18 strains with strong IAA production capacity; (c) phosphate dissolving capacity qualitative experiment, partial results; (d) quantitative experiment of 14 strains with strong phosphate dissolving capacity; (e) potential of endophytic bacteria producing ACC deaminase of strains TRI4-1 and MG-2; (f) nitrogen fixation potential of strains TSI2-4 and TSI2-6.

Figure 6

Qualitative experiments of endophytes producing siderophore, cellulase, and protease: (a) endophytes producing siderophore plate experiment; (b) the ratio of transparent circle diameter (D) to colony diameter (d) in iron carrier plate experiment; (c) endophytes producing cellulase plate experiment; (d) the ratio of transparent circle diameter to colony diameter in cellulase-producing plate experiment; (e) endophyte protease plate experiment; (f) the ratio of transparent circle diameter to colony diameter in protease-producing plate experiment.

Figure 7

Phylogenetic tree of Sphingomonas sp. MG-2, Kocuria sp. TRI2-1, and Micromonospora sp. TSI4-1. The phylogenetic tree was drawn using the full-length sequence of 16S rRNA gene. MEGA X was used to construct phylogenetic trees based on the alignment of all sequences using the ClusterW method and subsequent trimming of the aligned sequences back and forth. The maximum likelihood method was applied to construct the trees, with 1000 bootstrap replications selected and default values used for other parameters.

Figure 8

Effects of strains MG-2, TRI2-1, or TSI4-1 on the growth of Arabidopsis: (a–e) the growth and related index statistics of Arabidopsis treated with MG-2, TRI2-1, or TSI4-1 for 7 days; (f–j) the growth and related index statistics of Arabidopsis treated with MG-2, TRI2-1, or TSI4-1 for 14 days; (k–m) the growth of Arabidopsis and related growth index statistics after different root irrigation treatments of MG-2, TRI2-1, or TSI4-1. CK refers to the control group treated with sterile water, MY refers to the experimental group treated with strain MG-2, RY refers to the experimental group treated with strain TRI2-1, and SY refers to the experimental group treated with strain TSI4-1.

Figure 9

The content of paclitaxel and its precursors in Taxus yunnanensis stem cells after different treatments of MG-2, TRI2-1, or TSI4-1: (a–c) the content of paclitaxel and its precursor in stem cells after treatment with Sphingomonas sp. strain MG-2; (d–f) the content of paclitaxel and its precursor in stem cells after treatment with Micromonospora sp. strain TSI4-1; (g–i) the content of paclitaxel and its precursor in stem cells after treatment with Kocuria sp. strain TRI2-1. MY, the experimental group treated by the filtrate of the bacterial liquid of the strain MG-2; MT, the experimental group treated by the inactivated thallus of strain MG-2; RY, the experimental group treated with the filtrate of strain TRI2-1; RT, the experimental group treated by the inactivated thallus of strain TRI2-1; SY, the experimental group treated by the bacterial liquid filtrate of strain TSI4-1; ST, the experimental group treated by the inactivated thallus of strain TSI4-1. ** Represent statistically significant differences in comparison.