Characterisation, metabolite production and biological activities of endophytes from Artemisia afra and Lessertia frutescens

Abstract

Medicinal plants remain a valuable source for natural drug bioprospecting owing to their multitarget spectrum. However, their use as raw materials for novel drug synthesis has been greatly limited by unsustainable harvesting leading to decimation of their wild populations coupled with inherent low concentrations of constituent secondary metabolites. Thus, adding value to the medicinal plants research dynamics calls for adequate attention. Medicinal plant cultivation is considered as a gateway to high-quality products and commercialization. Consequently, stimulating metabolite production in medicinal plants by using endophytes as plant growth promoters has been demonstrated to be one of the most effective methods for increasing metabolite syntheses. Thus, this study aimed to isolate endophytes from Artemisia afra Jacq. ex Willd. var. afra and Lessertia frutescens (L.) Goldblatt & J.C. Manning subsp. Frutescens and determine their biological activities such as their ability to promote plant growth and enhance plant metabolite production. In order to achieve this, four objectives were formulated. Objective 1 was to evaluate metabolite variation of Artemisia afra (A. Afra) and Lessertia frutescens (L. frutescens) and determine if endophytes have an impact on the production of metabolites in these two plants. Objective 2 was to isolate, identify and characterise bacterial endophytes from A. afra and L. frutescens using both culture-based and DNA barcoding approaches. Objective 3 was to determine plant growth-promoting attributes of the endophytes isolated from A. afra and L. frutescens. Objective 4 was to examine if endophytes can accelerate the growth of A. afra and L. frutescens and enhance their metabolite production in a pot trial. Using nuclear magnetic resonance (NMR) and 16S rDNA gene sequencing, metabolite profiles and endophytic bacterial communities of Artemisia afra sourced from two different environments where two cultivation methods (seed cultivation and vegetative cutting cultivation method) were used. The NMR analysis revealed that more metabolites were recovered in A. afra samples collected from Pretoria where vegetative cutting cultivation method was used compared to samples collected from Klerksdorp where seed cultivation was used. Additionally, plant parts and cultivation method also had an effect in bacterial endophytes' abundance and community structure. The most dominant phyla were Proteobacteria, Actinobacteria, and Firmicutes. Redundancy analysis (RDA) demonstrated a positive correlation between bacterial endophytes and metabolites from A. afra leaves and stems, indicating a close relationship between metabolites and endophytes. On the other hand, the metabolite profiles and endophytic bacterial communities of L. frutescens plant organs were determined and there was variability in chemical profiles as well as the endophytic microbial communities across different plant organs in L. frutescens. On leaves, metabolites including n-acetylglucosamine positively correlated with bacterial endophytes such as Arthrobacter, Xanthomonas, Pseudomonas and Kineococcus. Bacterial endophytes have plant growth promoting attributes that presents significant potential for sustainable agriculture. In-vitro capabilities of bacterial endophytes isolated from L. frutescens for plant growth promotion and their effect on initial seedling growth was evaluated. Using a culture-dependent approach, plant samples of L. frutescens were screened for bacterial endophytes. The isolated bacterial endophytes were subsequently evaluated for their plant growth-promoting attributes along with their ability to produce hydrolytic enzymes. The obtained results revealed that many of the bacterial endophytes had potential to promote plant growth. Specifically, 86% of the endophytes possessed nitrogen-fixing, phosphate solubilizing, and Indole Acetic acid (IAA)-producing abilities, while approximately 71% were able to exhibit siderophore-producing capabilities. The endophytes exhibited significant production of essential hydrolytic enzymes, including amylase (86%), gelatinase (86%), protease (29%), lipase (43%), and D-nase (57%). The two best isolates were chosen and identified as Bacillus licheniformis and Bacillus velezensis and their plant growth-promoting properties such as their ability to enhance plant height and their ability to be used as bio-agent were further confirmed in the pot trial study as they enhanced the growth of L. frutescens seedlings compared to the control. On the other hand, endophytes from Artemisia afra sourced from two different environments under different cultivation methods were also assessed for plant growth promotion and enzyme activities. Data obtained in this study revealed that 64% of the endophytes fixed biological N, 79% solubilised insoluble tricalcium phosphate, 86% produced IAA, and 79% produced siderophores. The endophytes also demonstrated a substantial ability to produce essential extracellular enzymes (protease (57%), gelatinase (64%), d-nase (43%), amylase (57%) and lipase (36%). The notable role of endophytes in promoting host plant growth stems from their ability to enhance nutrient acquisition, induce stress tolerance, and stimulate the biosynthesis of bioactive compounds. To evaluate the effect of Bacillus endophytes on A. afra and L. frutescens growth and metabolite production, a pot trial experiment was conducted, and NMR analysis was used to assess the metabolites from pure bacterial endophytes and these medicinal plants leaves after harvesting. In a pot trial experiment, isolated endophytic Bacillus species promoted A. afra and L. frutescens growths as evidenced in a significant increase in plant height, wet and dry total, and shoot and root biomass. These medicinal plants' nutrient composition was likewise impacted by the endophytes, with a greater impact seen on macronutrients. Exploring interactions between plants and microbes presents significant potential for sustainable agriculture, providing innovative approaches to enhance crop yield while minimizing dependence on chemical interventions. These Bacillus endophytes also increased plant metabolite production, as more diverse metabolites were obtained from inoculated plants compared to the controls. Pure Bacillus endophytes L. frutescens and A. afra produced more diverse metabolites than inoculated. Endophyte inoculation presents a promising avenue for enhancing the productivity and health of medicinal plants while contributing to sustainable agricultural practices. Ongoing research and innovation in this field are essential for unlocking the full potential of endophytes in both agriculture and medicine, offering a pathway toward more resilient and effective cultivation methods.