Technologies for the Selection, Culture and Metabolic Profiling of Unique Rhizosphere Microorganisms for Natural Product Discovery

Abstract

Small molecule discovery has benefitted from the development of technologies that have aided in the culture and identification of soil microorganisms and the subsequent analysis of their respective metabolomes. We report herein on the use of both culture dependent and independent approaches for evaluation of soil microbial diversity in the rhizosphere of canola, a crop known to support a diverse microbiome, including plant growth promoting rhizobacteria. Initial screening of rhizosphere soils showed that microbial diversity, particularly bacterial, was greatest at crop maturity; therefore organismal recovery was attempted with soil collected at canola harvest. Two standard media (Mueller Hinton and gellan gum) were evaluated following inoculation with soil aqueous suspensions and compared with a novel “rhizochip” prototype buried in a living canola crop rhizosphere for microbial culture in situ. Following successful recovery and identification of 375 rhizosphere microbiota of interest from all culture methods, isolates were identified by Sanger sequencing and/or characterization using morphological and biochemical traits. Three bacterial isolates of interest were randomly selected as case studies for intensive metabolic profiling. After successful culture in liquid media and solvent extraction, individual extracts were subjected to evaluation by UHPLC-DAD-QToF-MS, resulting in the rapid characterization of metabolites of interest from cultures of two isolates. After evaluation of key molecular features, unique or unusual bacterial metabolites were annotated and are reported herein.

Comparative Analysis of the Cultured and Total Bacterial Community in the Wheat Rhizosphere Microbiome Using Culture-Dependent and Culture-Independent Approaches

“…Similarly for the Jensen medium experiments, where agar was replaced with Phytagel and the PS protocol was used (SJM Phytagel), 61 to 273% and 10 to 127% more CFU counts compared to the JM agar medium (PT protocol) and SJM agar medium (PS protocol), respectively, were measured. Our findings are in agreement with previous publications supporting the use of gellan gum as a solidifying agent because it resulted in higher CFU counts and greater culturability of bacteria isolated from soil ( 32 , 35 ) or seawater ( 18 ) than agar. The variation in culturability observed between gellan gum substrates and agar-based substrates may be due to the possibility that the two solidifying agents are composed of different sugars, each supporting the growth of specific phylotypes of microorganisms ( 18 , 27 ).…”

“…Many factors influence the culturability of microbes, such as the culture medium (e.g., medium composition and solidifying agents), growth conditions, and other cultivation techniques ( 21 , 32 , 33 ). Any modifications of these approaches may promote the growth of taxa that did not grow under the original conditions ( 34 ).…”

Reaching unreachables: Obstacles and successes of microbial cultivation and their reasons

“…Acuna ( Acuna et al, 2020 ) used microwell chambers, devices similar to the iChip in design, to culture rhizobacterial populations. Rhizosphere microorganisms were also cultured in situ using the Rhizochip, an acrylic device with holes, in which microorganisms are randomly and not evenly inoculated, and placed into a plant rhizosphere ( Gurusinghe et al, 2019 ). All these examples show that when the unreachables stay in their environments, we are more likely to reach them in cultures.…”

Current State and Future Directions of Genetics and Genomics of Endophytic Fungi for Bioprospecting Efforts

“…This setup facilitates the cultivation of novel species without the need to optimize artificial growth conditions and allows for capturing broader biological diversity. The use of this technology has not been reported for plant-associated fungi, but a so-called “rhizochip” was developed to study bacteria in the rhizosphere of the canola plant (Gurusinghe et al, 2019 ). For the study of epiphytes, living on the surface of a plant, we could imagine that techniques to attach a FIND to a plant could be developed.…”