Date Published: July 7, 2017
Publisher: Public Library of Science
Author(s): Nabil A. Hegazi, Mohamed S. Sarhan, Mohamed Fayez, Sascha Patz, Brian R. Murphy, Silke Ruppel, Dawn L Arnold.
In an effort to axenically culture the previously uncultivable populations of the rhizobacteria of Lucerne (Medicago sativa L.), we propose plant-only teabags culture media to mimic the nutritional matrix available in the rhizosphere. Here, we show that culture media prepared from Lucerne powder teabags substantially increased the cultivability of Lucerne rhizobacteria compared with a standard nutrient agar, where we found that the cultivable populations significantly increased by up to 60% of the total bacterial numbers as estimated by Quantitative Real-time Polymerase Chain Reaction (qRT-PCR). Cluster analysis of 16S rDNA Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) of cultivable Colony-Forming Units (CFUs) revealed a more distinct composition and separation of bacterial populations recovered on the plant-only teabags culture media than those developed on a standard nutrient agar. Further, the new plant medium gave preference to the micro-symbiont Sinorhizobium meliloti, and succeeded in isolating a number of not-yet-cultured bacteria, most closely matched to Novosphingobium sp., Lysobacter sp. and Pedobacter sp. The present study may encourage other researchers to consider moving from the well-established standard culture media to the challenging new plant-only culture media. Such a move may reveal previously hidden members of rhizobacteria, and help to further explore their potential environmental impacts.
It has been long established that more than 90% of environmental microorganisms are not amenable to cultivation on/in standard laboratory media . Bacterial databases are continually updated with new bacterial taxa, most of which are not yet cultured. Microbial ecologists struggle to recover and maintain these unculturables as viable entities grown as pure cultures. The last decade has witnessed the development of culture media and conditions for culturing new bacterial members from various environments [2–8]. In 2012, we proposed the use of plant-only culture media as a new approach for culturing rhizobacteria that might resist conventional cultivation. Crude plant juices, saps and powders, without any amendments, supported good growth and recoverability of rhizobacteria, comparable with the standard culture media [9–11]. For ease of application and practicability, tea bags packed with plant powders were successfully introduced to obtain the plant infusions necessary to prepare culture media .
Throughout the recent history of microbiological research, different types of culture media have been designed to meet the nutritional requirements of microorganisms cultured in vitro. While these media have been mostly successful for the general cultivation of a broad range of microorganisms, they have been less successful when it comes to meeting the requirements for cultivating endophytes. We have addressed this issue by using a novel approach which focuses on the use of plant based culture media for the recovery and cultivation of bacterial endophytes. The proposed plant-only culture medium is of a complex nature that mimics the nutritional matrix present in the plant root and/or rhizosphere. Such plant-only-based culture media contain relatively well-balanced amounts of nutrients [9–11], being of natural origin, concentration and diversity. We found that a culture medium with concentrations as low as 0.25 g plant powder per liter supported the cultivability of a large number of Lucerne rhizobacteria, to the extent that no statistical significant differences were observed between the tested concentrations of plan-only teabags culture medium (0.25, 0.5, 1.0, 4.0 g l-1). The population densities of rhizobacteria, in terms of total CFUs, were higher on plant-only teabags media compared with the rich Standard I nutrient agar; recovering up to 62–71% and 2–16% of the total bacterial numbers (as measured by qPCR), respectively. This result corresponds with a number of other techniques that have been proposed to develop culture media with low nutrient concentrations in order to facilitate culturing the unculturables [30–32]. Further, the community composition of rhizobacteria recovered on plant-only teabags culture media was substantially different, being clustered apart from those fed on Standard I nutrient agar. In fact, the use of plant-only teabags culture media resulted in the development of more diverse populations of Lucerne rhizobacteria and extended the range of cultivability among its microbiome. This enabled us to easily recover pure isolates of globally distributed rhizobacteria, predominantly Sinorhizobium meliloti. It appeared that the plant-only teabags culture media gave preference to culturability and recovery of the specific micro-symbiont of Lucerne, namely S. meliloti. Moreover, they were able to reveal fastidious not-yet-cultured communities of Lucerne rhizobacteria (Table 2). Both of our two isolates LP1.2 and LP3.2 represent a high similarity to the uncultured Bacterium (AM697055) detected in the dust of indoor environments in Finland , and the uncultured Novosphingobium sp. (HM438566) which have been detected in a PCR-amplified 16S rDNA sequence from Mexican soil . Two more isolates were originally isolated on plant-only teabags culture media and were subsequently able to regenerate on Standard I nutrient agar. The isolate LP3.11 was most closely (99% similarity) related to an uncultured bacterium (HF586982.1), detected in a batch sample of activated sludge (Germany), and Lysobacter pocheonensis (accession no. EU273938) isolated from the soil of ginger and ginseng fields in South Korea and China. The second isolate LP2.2 was found to be most closely related (99% similarity) to an uncultured Pedobacter- JN697525 , and the nearest cultivated type strain is Pedobacter steynii isolated from a spring of the Westerhofer Bach, Westerhof, Germany . So far and to the best of our knowledge, those 3 genera (Novosphingobium, Lysobacter, and Pedobacter) brought into cultivation via plant-only teabags culture media were not reported in literature as culturable endophytes of Lucerne.