Abstract
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.
Highlights
It has been long established that more than 90% of environmental microorganisms are not amenable to cultivation on/in standard laboratory media [1]
Relating Colony-Forming Units (CFUs) numbers to the total bacterial numbers measured by Quantitative real-time PCR (qPCR) revealed higher culturability efficiencies on plantonly teabags culture media (62–71%) compared to the Standard I nutrient agar (2–16%), with the extension of incubation period up to 15 days (Table 1)
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)
Summary
It has been long established that more than 90% of environmental microorganisms are not amenable to cultivation on/in standard laboratory media [1]. The last decade has witnessed the development of culture media and conditions for culturing new bacterial members from various environments [2,3,4,5,6,7,8]. In 2012, we proposed the use of plant-only culture media as a new approach for culturing rhizobacteria that might resist conventional cultivation. Saps and powders, without any amendments, supported good growth and recoverability of rhizobacteria, comparable with the standard culture media [9,10,11]. 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 [11]
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