Analysis of Endophyte Diversity of Rheum palmatum from Different Production Areas in Gansu Province of China and the Association with Secondary Metabolite

Abstract

Investigations of the differences in the metabolites of medicinal plants have typically focused on the effects of external environmental factors. However, little is known about the relationship between endophytes diversity and host metabolites. We used high-throughput sequencing methods to compare the endophyte diversity of Rheum palmatum from eight different production areas in Gansu Province of China and to analyze the association between those areas and five secondary metabolites (aloe-emodin, rhein, emodin, chrysophanol, and physcion). The results show that the diversity and OTUs (Operational taxonomic units) abundance of endophytic fungi and bacteria of R. palmatum differed according to production area. Spearman analysis showed that the five secondary metabolites of R. palmatum were positively correlated with the diversity and abundance of endophytic fungi. Comparing both space and environmental differences to determine influences on community structure, VPA analysis revealed that geographic factors explained more difference in community composition of fungal and bacterial endophytes than climate factors. PICRUSt and FUNGuild predictive analysis indicated that metabolites were the primary components of endophytic bacteria in all samples, while the function of endophytic fungi was composed of dominant trophic modes (saprotroph and pathotroph), and relative abundances were different. Our results help elucidate the correlation of plant–microbe interactions and offer pivotal information to reveal the role of endophytes in the production of R. palmatum and its important secondary metabolite.

Analysis of endophyte diversity of two Gentiana plants species and the association with secondary metabolite

“…It indicated that the content of loganic acid was correlated with endophytic fungi, the content of gentiopicroside, swertiamarine and sweroside were correlated with endophytic bacteria in the G. officinalis and G. siphonantha . However, Chen et al [ 3 ] and Cui et al [ 28 ] reported that that metabolites content of Rheum palmatum and Cynomorium songaricum were only correlated with endophytic fungi. Endophyte play a important role on the accumulation of secondary metabolite in medicinal plants [ 3 ], while endophyte may have different effects on the accumulation of secondary metabolites in different plants.…”

“…To sterilize the surface of the plant parts, the root samples were successively immersed in 70% ethanol for 5 min, 2.5% sodium hypochlorite for 1–2 min, and 70% ethanol for 1 min, and then rinsed five times with sterile Millipore water. The last portion of the washing water was inoculated in PDA (potato dextrose agar) at 28 °C for 10 d and NA (nutrient agar) at 37 °C for 3 d to validate sterilization efficiency [ 3 ]. All samples were stored at − 80 °C until DNA extraction.…”

“…The DNA extracts were analyzed for their concentration using NanoDrop spectrophotometer (Termo Fisher Scientifc, Model 2000, MA, USA) and stored at − 20 °C for further PCR amplifcation. The PCR assays were performed in 20 μL mixture containing 4 μL of 5× Fast-Pfu buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL of FastPfu Polymerase, 10 ng of templateDNA and ddH 2 O. Bacterial 16S gene was amplified with primers 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) [ 3 ]. Amplification was performed under the following conditions: initial denaturation at 95 °C for 3 min, 30 cycles at 95 °C for 30 s, 52 °C for 30 s, and 72 °C for 45 s, and a final extension at 72 °C for 5 min.…”

“…Amplification was performed under the following conditions: initial denaturation at 95 °C for 3 min, 30 cycles at 95 °C for 30 s, 52 °C for 30 s, and 72 °C for 45 s, and a final extension at 72 °C for 5 min. The fungal ITS genes were amplifed using the primers ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS2R (5′-GCTGCGTTCTTCATCGATGC-3′) [ 3 ]. The PCR reactions were conducted using the following program: 3 min of denaturation at 95 °C, 35 cycles of 30 s at 95 °C, 30 s for annealing at 55 °C, and 45 s for elongation at 72 °C, and a final extension at 72 °C for 10 min.…”

“…Endophytes existed the internal tissues of plant, but it can not cause any disease symptoms, which played a vital role in plant growth, development, tolerance of abiotic or biotic stress and accumulation of host secondary metabolites [ 1 , 2 ]. Chen et al [ 3 ] found that the five secondary metabolites content of Rheum palmatum were positively correlated with the fungal endophyte. Song et al [ 4 ] reported that Bacillus altitudinis was isolated from Panax ginseng, which can improve ginsenoside accumulation.…”