Hydrogel capsules as new delivery system for Trichoderma koningiopsis Th003 to control Rhizoctonia solani in rice (Oryza sativa).

The incorporation of biological control agents (BCAs) such as Trichoderma spp. in agricultural systems favors the transition towards sustainable practices of plant nutrition and diseases control. Novel bioproducts for crop management are called to guarantee sustainable antagonism activity of BCAs and increase the acceptance of the farmers. The encapsulation in polymeric matrices play a prominent role for providing an effective carrier/protector and long-lasting bioproduct. This research aimed to study the influence of biopolymer in hydrogel capsules on survival and shelf-life of T. koningiopsis. Thus, two hydrogel capsules prototypes based on alginate (P1) and amidated pectin (P2), containing conidia of T. koningiopsis Th003 were formulated. Capsules were prepared by the ionic gelation method and calcium gluconate as crosslinker. Conidia releasing under different pH values of the medium, survival of conidia in drying capsules, storage stability, and biocontrol activity against rice sheath blight (Rhizoctonia solani) were studied. P2 prototype provided up to 98% survival to Th003 in fluid bed drying, faster conidia releasing at pH 5.8, storage stability greater than 6 months at 18 °C, and up to 67% of disease reduction. However, both biopolymers facilitate the antagonistic activity against R. solani, and therefore can be incorporated in novel hydrogel capsules-based biopreparations. This work incites to develop novel biopesticides-based formulations with potential to improve the delivery process in the target site and the protection of the active ingredient from the environmental factors.

Hydrogel capsules as new approach for increasing drying survival of plant biostimulant gram-negative consortium.

Several plant growth-promoting bacteria (PGPB) are gram-negative, and their cell viability is affected during the bio-inoculant production. Hence, formulation-drying processes provide challenges that limit the adoption of these beneficial microorganisms in sustainable agricultural production. Among delivery system strategies for gram-negative PGPB, the encapsulating cells in biopolymeric materials are emerging as a promising alternative. This research aims to evaluate the effect of additives and crosslinking agents on the survival of the consortium of Herbaspirillum frisingense AP21, Azospirillum brasilense D7, and Rhizobium leguminosarum T88 in hydrogel capsules. Three crosslinkers and diverse potential drying protectors were tested. Calcium gluconate provides notable consortium survival advantages regarding colony-forming units (CFUs) (losses of up to 4 log CFU) compared to calcium lactate and calcium chloride (up to 6 log CFU). Additives such as skimmed milk, whey protein, and Gelita® EC improve the recovery of viable cells after the drying process, demonstrating an increase in cell survival of the three bacteria by up to 4 log CFU. The combination of these substances into a capsule prototype extends the storage stability of bacterial consortium up to 3 months at 18 ± 2 °C. This study expands the knowledge for formulating gram-negative PGPB consortium, regarding the crosslinker and drying protector relationship on encapsulation processes with drying survival and further storage stability performance. KEY POINTS: • Hydrogel immobilization formulation approach for PGPB consortium • Enhancing drying survival of gram-negative PGPB consortium • Increasing storage stability of PGPB consortium at 18 °C.

Trichoderma koningiopsis Survival on Coated Seeds and Effect on Plant Growth Promotion in Rice (Oryza sativa).

Seed coating is an alternative delivery system for beneficial plant microorganisms into the soil. Although seed coats are widely used for the application of agrochemicals, the incorporation of beneficial microorganisms has not been explored deeply and their survival on seeds while in storage is unknown. The study aimed to evaluate the effect of the coating process on microbial survival and on plant growth promotion. Two coating formulations were designed, and assessed by two coating processes: rotating drum and fluidized bed. The rotating drum process resulted in more uniform coatings than in the fluidized bed process. In addition, with this coating technique, lower viability losses over time were observed. The rotatory drum prototype containing a biopolymer and a clay mineral derivate (P90) showed the best behavior at the three temperatures evaluated, with superior viabilities compared to the other prototypes and the lowest loss of viability after 12 months. The formulation of this coating prototype may preserve the viability of Trichoderma koningiopsis Th003 up to 15 months at 8 °C, 9 months at 18 °C, and 3 months at 28 °C, which are very promising shelf-life results. Regarding the effect of seed coating on plant growth, prototypes showed higher yields > 16% than the control, comparable to the conventional use of Tricotec® WG, which may reduce the number of applications and water consumption for dissolution of the inoculant. The results demonstrated that the formulation composition, as well as the coating process may impact the microbial survival on seeds.

High-Throughput Assessments for Storage Stability, In Vitro Release, and Particle Size of Encapsulated Biocontrol Fungi in Hydrogel Beads.

Use of biocontrol fungi (BF) such as Trichoderma spp. minimizes fungicide input and increases both plant nutrition and protection from disease. Thus, the introduction of BF by novel inoculants in crop management is an excellent strategy to promote sustainable antagonism activity. Within these strategies, encapsulation in polymeric matrices such as hydrogel beads will play a prominent role in providing an effective carrier/protector and long-lasting bioproduct. These studies have used biomaterials with tunable physicochemical features, providing differential morphologies, compaction, and disintegration, among other parameters. Aiming at developing bioproducts within polymeric hydrogel beads, viability of encapsulated conidia, storage stability, release of active ingredient, and particle size are essential. However, there are no reports that detail standardized and comprehensive methods to evaluate the characterization of these bioproducts. We describe step-by-step protocols that go from sample preparation to testing the viability and storage stability using vacuum-sealed aluminum foil bags. We also describe a high-throughput in vitro method for quantifying released fungal conidia of BF at different pH values. Finally, the particle size of beads is established by bright-field microscopy. These protocols could be transferable to other biological actives, accessible to researchers in the microbiology and bioengineering communities. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Viability of encapsulated conidia Basic Protocol 2: Study of stability of prototypes under storage Basic Protocol 3: Release profile of encapsulated conidia at different pH conditions Basic Protocol 4: Determination of capsule size.

Seed coating as a delivery system for the endophyte Trichoderma koningiopsis Th003 in rice (Oryza sativa).

Seed coating is a technique to cover seeds with external agents to upgrade their performance, handling, and plant establishment. Plant beneficial microbes (PBMs), such as plant growth-promoting bacteria, mycorrhizal fungi, and other fungi (e.g., Trichoderma spp.), decrease agrochemical inputs, enhance tolerance to biotic-abiotic stresses, and increase essential plant nutrition. The demand for pre-treated seeds as delivery systems for biological agents is advancing. Here, a seed coating formulation containing Trichoderma koningiopsis is presented. The physicochemical and biological characterization of the seed coating prototypes included drying protector screening, the effect of the inoculum concentration on survival, the assessment of microbial release profiles in soil extract, and plant tissue colonization capability under semi-controlled conditions. Gelatine and pectin, two of the tested drying protectors, maintained fungus germination after 60 days at 18 °C with significantly higher values of up to 38% compared with the control. The initial concentration of 106 colony-forming units (CFU) per seed undergoes a positive effect on survival over time. Regarding plant tissue colonization, the fungus establishes endophytically in rice. In conclusion, seed coating is a promising alternative for the formulation of beneficial microbial agents such as Trichoderma sp., maintaining cell survival and further promoting the establishment in rice systems.Key points• Enhancing drying survival of T. koningiopsis formulates• Seed coating formulation approach for T. koningiopsis in rice• Colonization capacity of formulated T. koningiopsis in rice tissue.

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.