Choosing source of microorganisms and processing technology for next generation beet bioinoculant

Abstract

The increase of human population and associated increasing demand for agricultural products lead to soil over-exploitation. Biofertilizers based on lyophilized plant material containing living plant growth-promoting microorganisms (PGPM) could be an alternative to conventional fertilizers that fits into sustainable agricultural technologies ideas. We aimed to: (1) assess the diversity of endophytic bacteria in sugar and sea beet roots and (2) determine the influence of osmoprotectants (trehalose and ectoine) addition during lyophilization on bacterial density, viability and salt tolerance. Microbiome diversity was assessed based on 16S rRNA amplicons sequencing, bacterial density and salt tolerance was evaluated in cultures, while bacterial viability was calculated by using fluorescence microscopy and flow cytometry. Here we show that plant genotype shapes its endophytic microbiome diversity and determines rhizosphere soil properties. Sea beet endophytic microbiome, consisting of genera characteristic for extreme environments, is more diverse and salt resistant than its crop relative. Supplementing osmoprotectants during root tissue lyophilization exerts a positive effect on bacterial community salt stress tolerance, viability and density. Trehalose improves the above-mentioned parameters more effectively than ectoine, moreover its use is economically advantageous, thus it may be used to formulate improved biofertilizers.

Rhizosphere bacterial colonization of beet occurs in discrete phases regardless of bioinoculation with the wild sea beet root community

“…Collected plants were immediately refrigerated in styrofoam boxes filled with cooling pads frozen at -80ºC. Upon transfer to the laboratory, roots were separated from above-ground parts and the former were surface sterilized, homogenized with Warring blender, mixed with trehalose (1 mg/g) and lyophilized as previously described [5].…”

“…Plates were incubated for 7 days at 26ºC. Salt tolerance was estimated according to previously published methodology [5] using Biolog Micro Station plate reader (Biolog, CA).…”

“…Upon arrival at the laboratory, the roots were separated from the aboveground parts, and the roots were subjected to surface sterilization. Subsequently, they were homogenized using a Warring blender combined with trehalose (at a concentration of 1 mg/g) and then lyophilized following a previously described method [10]. The obtained fine powder inoculant was stored at -80°C.…”

“…For the estimation of salt tolerance, a previously published methodology [10] was employed. A Biolog Micro Station plate reader from Biolog, CA, was utilized for this purpose.…”

“…For example, bioinoculation with synthetic microbial community was shown to restore growth under stress conditions in Arabidopsis thaliana [4]. We found previously that beet genotype influences both endophytic and rhizosphere bacterial communities, moreover, lyophilized beet roots can be a source of viable microorganisms for inoculation [5]. As to the problem of devising formulation, various strategies based on liquids, slurries or solids are used, reviewed e.g.…”