Isolation of rhizobacteria from salt tolerant plant species and evaluation of their plant growth-promotion

Abstract

Bacteria from rhizosphere samples of Hordeum secalinum and Plantago winteri from a natural salt meadow in Hessen, Germany, a natural undisturbed ecosystem, were isolated with emphasis on diazotrophs (NFB&LG agar), phosphate- and phytate-mobilising bacteria (CP&IHP agar), ACC (1-aminocyclopropane-1-carboxylate, a precursor of ethylene) deaminase-active bacteria (DF agar) as well as IAA (indole-3-acetic-acid)-producing bacteria (LBT agar) as source of potential halotolerant potential growth-promoting rhizospheric bacteria. Twenty-two isolates, out of 100, mostly belonging to diazotrophic, ACC deaminase producer and P-mobilising bacteria, were selected and their potential plant growth-promoting activities determined in pure culture by evaluating their abilities to grow on specific media, measuring specific metabolic functions. The effect of these 22 isolates on barley plants (Hordeum vulgare L.) was evaluated in non-sterile soil under salt stress in greenhouse conditions. Results surprisingly showed that the best performing isolates in pure culture were not those ones displaying the best plant growth-promoting activity in plant growth assays. The partial 16S rRNA gene sequence of the bacteria showed that they belong to the Proteobacteria, Actinobacteria, and Firmicutes. Among the isolates two new species belonging to genus Rheinheimera, R. hassiensis E48T and R. muenzenbergensis E49T, a new species of genus Cellvibrio, C. diazotrophicus E50T, and new genus and species Hartmannibacter diazotrophicus E19T were proposed and accepted. Strain E19T is able to grow on agar containing not water-soluble phosphate sources (Ca3(PO4)2, AlPO4 FePO4 and Phytate), DF media agar supplemented with ACC, nitrogen free media, reduce acetylene, and it has phosphatase and ACC-deaminase activities. In addition, reduction of ethylene emission measured on barley plantlets under salt stress showed positive stress relieving effect of E19T due to its ACC deaminase activity.Plant growth-promoting activity of the new taxonomically proposed strains were first tested under gnotobiotic conditions in a newly designed liquid plant growth system, and in Mitscherlich pots using subsoil under salt stress with unsuccessful results under such experimental conditions. However, experiments using non-sterile soil under salt stress allowed to evidence the plant growth-promotion capability of H. diazotrophicus E19T on barley plants (Hordeum vulgare L.) by significantly increasing root and shoot dry weights, water content in the root system, root-to-shoot ratio and decreasing root Na+ concentration and root surface sodium uptake. The capability of strain E19T to colonize barley roots under salt stress conditions was revealed with a specifically designed fluorescence in situ hybridization (FISH) probe.Draft genome sequence of H. diazotrophicus E19T is used at the moment in order to identify the presence of genes contributing directly or indirectly to enable PGPR effects on plants aiming to better understand the mode of interaction of the bacterium and plants, and to furthermore study the phylogenetic relationship with closest genome sequenced related organisms.