Pea Protein Alginate Encapsulated Bacillus subtilis B26, a Plant Biostimulant, Provides Controlled Release and Increased Storage Survival

Abstract

Plant growth promoting bacteria (PGPB) represent a wide variety of soil and endophytic bacteria that have the ability to promote growth or to protect against stress of its host plant. Encapsulation of PGPB, using vegetable proteins instead of animal or petroleum-derived polymers, is a new technology to crop production and protection. Pea protein isolates (PPI) alginate capsules were synthesized and used for the protection and delivery of Bacillus subtilis B26 as plant inoculum for agricultural applications. The capsules provided a protective site to B26 strain allowing good survival, between 45 to 50%, after 112 days of incubation at different temperatures. The loaded microcapsules sustained a large population of bacteria, up to 8.3 LogCFUg -1 of soil after 3 weeks post application. The soil concentration stabilized to 7.3 Log CFUg -1 after 8 weeks post application. Capsules loaded with B26, once incorporated in the soil, successfully colonized test plants, and cell numbers inside plant tissues were sustained at 3.5 and 3.3 Log 7.3 CFUg -1 in the root and the shoot respectively. These results indicate that PPIalginate technology represents a good choice for commercial application of B. subtilis B26 in agriculture.Citation: Gagné-Bourque F, Meng Xu, Dumont MJ, Jabaji S (2015) Materials and Methods Maintenance and preparation of B. subtilis B26 inoculumThe B.subtilis strain B26, previously isolated from switchgrass and fully characterized [15], was maintained on Luria Broth (LB) (1.0% Tryptone, 0.5% Yeast Extract, 1.0% NaCl) with glycerol (25% final volume) and stored at-80°C. B. subtilis B26 was revived on LBA (1.5% Agar) plates. The inoculum was prepared by placing a single colony of B. subtilis B26 in 250 mL of LB and incubated for 18 h at 37°C until an OD600 of 0.7, representing colony forming units (CFU) of 3.38 × 10 8 / mL was reached on a shaker at 250 rpm to the mid-log phase, pelleted by centrifugation, washed and suspended in sterile distilled water. Preparation of alginate capsules and microencapsulationPea protein isolates (PPI) (Propulse N™) containing 81.73% protein, <10.3% sugars, <0.7% starch, <3.4% moisture, <0.5% fat, <4.0% ash was obtained from Nutri-Pea Ltd. (Portage la Prairie, MB, Canada). Alginic acid sodium salt with low viscosity (viscosity of 1% aq. solution: <300cps) was purchased from MP biomedicals (Solon, OH, USA). Calcium chloride (CaCl 2 ) dehydrate, hydrochloric acid (HCl) and dimethyl sulfoxide (DMSO) were purchased from Fisher Scientific (Fair Lawn, NJ). Sodium hydroxide was purchased from EMD (Damstadt, Germany). Tryptone, Yeast Extract, NaCl, and Agar were purchased from Difco (Franklin Lakes, NJ, USA). Ammonium sulphate, Potassium phosphate dibasic trihydrate, monopotassium phosphate, trisodium citrate and magnesium sulfate heptahydrate were purchased from Sigma-Aldrich (Co., St. Louis, MO). Glycerol was purchased from ThermoFisher Scientific (Waltham, MA). The qPCR SYBRII master mix and the ROX were purchased from Agilent Technologies (Morrisville, NC, USA) while B...

Evaluation of eliciting activity of peptidil prolyl cys/trans isomerase from <em>Pseudonomas fluorescens</em> encapsulated in sodium alginate regarding plant resistance to viral and fungal pahogens

“…As we have already mentioned, a possibility to use ALG for production of encapsulated microbial biopreparations for soil fertilization has been reported in a series of studies [29],[30]. One of the recently published studies was devoted to the development of ALG microcapsules containing plant growth stimulator based on a Bacillus subtilis strain [44]; according to authors, such encapsulation of bacterial cells significantly improved their survival in soil and provided a possibility of commercial production of such preparations. In our case, a wide range of action of PPIase and its high heat resistance facilitating the isolation and purification of this protein [9] represent additional advantages for the industrial production of PPIase-based biopesticides.…”

Microencapsulation of Bacillus Strains for Improving Wheat (Triticum turgidum Subsp. durum) Growth and Development

“…In addition, among different strains, there is a range of variability in responses to growth or stability [ 35 ]. In our study, bacteria were encapsulated with an optimal yield of 133.9% to 338.0%, the numbers are greater as compared to other encapsulation methods, i.e., L. casei encapsulated inside alginate-pectin microbeads with yields less than 79% [ 40 ], and a 75% encapsulation yield of B. subtilis B26 in microbeads of calcium alginate and pea protein [ 41 ]. In our study, the increase in the bacterial population during encapsulation is attributed to the procedure itself, because before encapsulating the bacterial suspension + sodium alginate, it is incubated for a period of 1 h and 24 h after the encapsulation for the drying of the microbeads (32 °C), considering that the bacteria in alginate medium have an average doubling time of ~1.92 h, sufficient time for bacterial reproduction.…”

Current Techniques to Study Beneficial Plant-Microbe Interactions

“…Regarding the material used for PGPB encapsulation among biopolymers alginate, xanthan gum, starch, pea proteins, humic acids, bentonite, and skim milk have been widely used [ 242 , 243 , 244 , 245 ]. Synthetic polymers are less suitable for bacterial encapsulation due to their low biocompatibility related to the use of organic solvents during the procedure [ 225 ].…”