Plant powder teabags: a novel and practical approach to resolve culturability and diversity of rhizobacteria

Abstract

We have developed teabags packed with dehydrated plant powders, without any supplements, for preparation of plant infusions necessary to develop media for culturing rhizobacteria. These bacteria are efficiently cultivated on such plant teabag culture media, with better progressive in situ recoverability compared to standard chemically synthetic culture media. Combining various plant-based culture media and incubation conditions enabled us to resolve unique denaturing gradient gel electrophoresis (DGGE) bands that were not resolved by tested standard culture media. Based on polymerase chain reaction PCR-DGGE of 16S rDNA fingerprints and sequencing, the plant teabag culture media supported higher diversity and significant increases in the richness of endo-rhizobacteria, namely Gammaproteobacteria (Enterobacteriaceae) and predominantly Alphaproteobacteria (Rhizobiaceae). This culminated in greater retrieval of the rhizobacteria taxa associated with the plant roots. We conclude that the plant teabag culture medium by itself, without any nutritional supplements, is sufficient and efficient for recovering and mirroring the complex and diverse communities of rhizobacteria. Our message to fellow microbial ecologists is: simply dehydrate your plant canopy, teabag it and soak it to prepare your culture media, with no need for any additional supplementary nutrients.

Plant-fed versus chemicals-fed rhizobacteria of Lucerne: Plant-only teabags culture media not only increase culturability of rhizobacteria but also recover a previously uncultured Lysobacter sp., Novosphingobium sp. and Pedobacter sp.

“…The PCR reaction was performed according to Sarhan et al . [11] in a total volume of 25 μl using SYBR ® green master mix (Bio-Rad) containing 10–30 ng genomic DNA and 8.25 pmol of each primer [19]; the universal forward 519f () and reverse 907r (). The cycling program and both standard and melting curves construction were done according to Sarhan et al .…”

“…The cycling program and both standard and melting curves construction were done according to Sarhan et al . [11]. Bacterial cell numbers were indirectly calculated assuming an average 16S rDNA copy number of 3.6 per bacterial cell [20,21].…”

“…[11]. Fresh vegetative parts, including leaves and stems, of Lucerne were oven dried at 65°C for 18 hrs.…”

“…The PCR thermal cycling program was adjusted according to Sarhan et al . [11]. A QIAquick PCR Purification Kit (Qiagen Inc., Hilden, Germany) was used to purify the PCR product, according to the manufacturers’ instructions.…”

“…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 [11].…”