Formulating bacterial endophyte: Pre-conditioning of cells and the encapsulation in amidated pectin beads.

Despite the benefits of bacterial endophytes, recent studies on the mostly Gram-negative bacteria lack of regard for formulation strategies. The encapsulation into biopolymeric materials such as amidated pectins hydrogels is a suitable alternative. Here, this research aimed at supporting the capability of the plant growth-promoting bacteria Kosakonia radicincitans DSM16656T to endophytically colonize plant seedlings. In this approach, the pre-conditioned cells through osmoadaptation and hydroxyectoine accumulation were used. In general, pre-osmoadapted and hydroxyectoine-supplemented bacteria cells formulated in amidated pectin dried beads increased the endophytic activity by 10-fold. Moreover, plant promotion in radish plants enhanced by 18.9% and 20.7% for a dry matter of tuber and leaves. Confocal microscopy studies with GFP-tagged bacteria revealed that bacterial aggregates formed during the activation of beads play an essential role in early colonization stages. This research encourages the integration of fermentation and formulation strategies in a bioprocess engineering approach for exploiting endophytic bacteria.

Anhydrobiotic engineering for the endophyte bacterium Kosakonia radicincitans by osmoadaptation and providing exogenously hydroxyectoine.

This study presents an anhydrobiotic engineering approach aiming at conferring a high degree of desiccation tolerance to the Gram-negative endophyte Kosakonia radicincitans. In particular, pre-conditioning of bacteria under high salinities provides a remarkable positive influence on drying survival. The endophytic bacteria accumulate exogenous hydroxyectoine > 500 µmol g-1 dry weight cells exerted by osmotic stress at 4% NaCl. Microfermentation research demonstrated that hydroxyectoine provides positive effects on reducing the lag phase duration and alleviates the dissolved oxygen consumption under high salinity conditions. Beyond the amassing of hydroxyectoine, this work provides evidence supporting the notion that hydroxyectoine can produce significant changes in the endogenous bacterial metabolome during the exponential growth phase at high-osmolarity. Metabolome changes include alterations on tricarboxylic acid cycle, novo-synthesis of specific intracellular metabolites such as mannitol, myo-inositol and trehalose, and fold changes on amino acids such as L-leucine, L-asparagine, L-serine, L-methionine and L-proline. The significant fold change of L-aspartate suggests a potential acidic proteome at high-osmolarity environments, extending the knowledge of salt-stressed bacterial endophytes. Thus, these findings place the metabolic salt stress response and the hydroxyectoine accumulation by K. radicincitans into a physiological context, paving the way into the interaction between cellular phenotype associated with salt stress tolerance and drying survival capacity of Gram-negative endophytes.

Salt stress and hydroxyectoine enhance phosphate solubilisation and plant colonisation capacity of Kosakonia radicincitans.

Gram-negative bacterial endophytes have attracted research interest caused by their advantageous over epiphytic bacteria in plant nutrition and protection. However, research on these typically Gram-negative endophytes has deficiencies concerning the role of cultivation and pre-formulation strategies on further plant colonisation capabilities. Besides, the influence of cultivation conditions and osmotic stress within bacterial endophytes on their phosphate solubilising ability has not yet been addressed. By pre-conditioning cells with an osmoadaptation and a hydroxyectoine accumulation approach, this research aimed at enhancing the capability of the plant growth promoting bacterium Kosakonia radicincitans strain DSM 16656T to both solubilise phosphate and colonise plant seedlings. The results showed that halotolerant bacterial phenotypes increased the root-colonising capability by approximately 3-fold and presented growth-promoting effects in radish plants. Interestingly, findings also demonstrated that salt stress in the culture media along with the accumulation of hydroxyectoine led to an increase in the in vitro phosphate-solubilising ability by affecting the production of acid phosphatases, from 1.24 to 3.34 U mg-1 for non-salt stressed cells and hydroxyectoine-added cells respectively. Thus, this approach provides a useful knowledge upon which the salt stress and compatible solutes in bacteria endophytes can confer phenotypic adaptations to support the eco-physiological performance concerning phosphate-solubilising abilities and endosphere establishment.

Endogenous arabitol and mannitol improve shelf life of encapsulated Metarhizium brunneum.

Successful commercialization of microbial biocontrol agents, such as Metarhizium spp., is often constrained by poor drying survival and shelf life. Here, we hypothesized that culture age would influence endogenous arabitol, erythritol, mannitol and trehalose contents in M. brunneum mycelium and that elevated levels of these compounds would improve drying survival and shelf life of encapsulated mycelium coupled with enhanced fungal virulence against T. molitor larvae. We found that culture age significantly influenced endogenous arabitol and mannitol contents in mycelium with highest concentrations of 0.6 ± 0.2 and 2.1 ± 0.2 µg/mg after 72 h, respectively. Drying survival of encapsulated mycelium was independent of culture age and polyol content with 41.1 ± 4.4 to 55.0 ± 6.2%. Best shelf life was determined for biomass harvested after 72 h at all investigated storage temperatures with maximum values of 59.5 ± 3.3% at 5 °C followed by 54.5 ± 1.6% at 18 °C and 19.4 ± 1.3% at 25 °C after 6 months. Finally, high fungal virulence against T. molitor larvae of 83.3 ± 7.6 to 98.0 ± 1.8% was maintained during storage of encapsulated mycelium for 12 months with larval mortalities being independent of culture age and polyol content. In conclusion, our findings indicate beneficial effects of endogenous polyols in improving shelf life of encapsulated mycelium and this may spur the successful development of microbial biocontrol agents in the future.

Cellulase enhances endophytism of encapsulated Metarhizium brunneum in potato plants.

The recent discovery that entomopathogenic fungi can grow endophytically in plant tissues has spurred research into novel plant protection measures. However, current applications of fungi aiming at endophytism mostly lack targeted formulation strategies resulting in low efficacy. Here, we aimed at enhancing Metarhizium brunneum CB15 endophytism in potato plants by (i) improvement of fungal growth from beads and (ii) cellulase formation or addition to encapsulated mycelium. We found that beads supplemented with cellulose alone or in addition with inactivated baker's yeast exhibited cellulase activity and increased mycelial growth by 12.6 % and 13.6 %, respectively. Higher enzymatic activity achieved by cellulase co-encapsulation promoted a shift from mycelial growth to spore formation with maximum numbers of 2.5 × 108 ± 6.1 × 107 per bead. This correlated with improved endophytism in potato plants by 61.2 % compared to non-supplemented beads. Our study provides first evidence that customized formulations of fungal entomopathogens with enzymes can improve endophytism and this may increase efficacy in plant protection strategies against herbivorous pests.