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 vacuumsealed 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.

Los hongos entomopatógenos Metarhizium anisopliae (Ascomycota: Clavicipitaceae) y Beauveria bassiana (Ascomycota: Cordycipitaceae) son una alternativa para el control de plagas de gramíneas (e.g. Rhammatocerus schistocercoides (Orthoptera: Acrididae) y de soya (e.g. Cerotoma tingomariana; Coleoptera: Chrysomelidae). Con el fin de utilizar estos microorganismos como principio activo de micoinsecticidas, es necesario contar con métodos de control de calidad microbiológico, fisicoquímico y de actividad biológica que sean repetibles y reproducibles en el tiempo. Por tal razón, y dada la dificultad en la recolecta de individuos en el campo y en el establecimiento de crías masivas de R. schistocercoides y de C. tingomariana, se establecieron métodos para la evaluación rutinaria de la actividad biológica mediante el uso de hospederos alternativos. Para M. anisopliae, dirigido al control de R. schistocercoides, se evaluaron las especies Diatraea saccharalis (Lepidoptera: Crambidae) y Spodoptera frugiperda (Lepidoptera: Noctuidae) mientras que para B. bassiana, dirigido al control del insecto C. tingomariana, se evaluaron D. saccharalis y Chloridea virescens (Lepidoptera: Noctuidae). Se seleccionaron a D. saccharalis y C. virescens como insectos alternativos para evaluar la eficacia de M. anisopliae y B. bassiana, respectivamente con eficacia cercana al 80 %, en los dos casos. Los métodos con los insectos alternativos seleccionados fueron estandarizados y se demostró que son repetibles y reproducibles bajo las condiciones evaluadas. Los hongos entomopatógenos Metarhizium anisopliae (Ascomycota: Clavicipitaceae) y Beauveria bassiana (Ascomycota: Cordycipitaceae) son una alternativa para el control de plagas de gramíneas (e.g. Rhammatocerus schistocercoides (Orthoptera: Acrididae) y de soya (e.g. Cerotoma tingomariana; Coleoptera: Chrysomelidae). Con el fin de utilizar estos microorganismos como principio activo de micoinsecticidas, es necesario contar con métodos de control de calidad microbiológico, fisicoquímico y de actividad biológica que sean repetibles y reproducibles en el tiempo. Por tal razón, y dada la dificultad en la recolecta de individuos en el campo y en el establecimiento de crías masivas de R. schistocercoides y de C. tingomariana, se establecieron métodos para la evaluación rutinaria de la actividad biológica mediante el uso de hospederos alternativos. Para M. anisopliae, dirigido al control de R. schistocercoides, se evaluaron las especies Diatraea saccharalis (Lepidoptera: Crambidae) y Spodoptera frugiperda (Lepidoptera: Noctuidae) mientras que para B. bassiana, dirigido al control del insecto C. tingomariana, se evaluaron D. saccharalis y Chloridea virescens (Lepidoptera: Noctuidae). Se seleccionaron a D. saccharalis y C. virescens como insectos alternativos para evaluar la eficacia de M. anisopliae y B. bassiana, respectivamente con eficacia cercana al 80 %, en los dos casos. Los métodos con los insectos alternativos seleccionados fueron estandarizados y se demostró que son repetibles y reproducibles bajo las condiciones evaluadas.

Biological control (BCAs) such as Trichoderma spp. minimize fungicides inputs in agriculture, and increase both the plant nutrition and the efficacy against fungal diseases. Novel bioproducts in 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 compare two hydrogel capsules prototypes based on alginate (P1) and amidated pectin (P2), containing Trichoderma koningiopsis Th003 conidia. Capsules were prepared by the ionic gelation method and calcium gluconate as crosslinker was used. The capsules were characterized by various properties such as conidia releasing at different pH values, drying conidia survival, storage stability, and biocontrol activity against sheath blight (R. solani) in rice. P2 prototype provided higher survival to Th003 up to 98% in fluid bed drying, faster conidia release at pH 5.8, storage stability > 6 months at 18°C, and a disease reduction up to 67%. Amidated pectin provides advantages on drying survival and conidia release at pH 5.8. Both biopolymers facilitate the antagonistic activity against R. solani, and therefore can be incorporated in hydrogel capsules for novel bioinoculants development. This research highlights the knowledge regarding the influence of biopolymer in hydrogel capsules on survival and shelf-life of Trichoderma spp conidia.

Utilizing the interactions of microorganisms with plants offers a favorable path to increase crop production and replace the use of synthetic fertilizers. Different bacteria and fungi have been used as biofertilizers to improve agricultural production, yield, and sustainability. Beneficial microorganisms can act as free-living organisms, symbiotes, and endophytes. Soil bacteria called plant growth-promoting bacteria (PGPB) and fungi called arbuscular mycorrhizae fungi (AMF) stimulate the growth and health of plants by direct and indirect mechanisms including nitrogen fixation, phosphorus solubilization, phytohormone production, enzyme production, antibiotic synthesis, and induced systemic resistance. To use these microorganisms as a biofertilizer, it is necessary to assess their efficacy under laboratory and greenhouse conditions. Few reports detail the methods used to develop a test under different environmental conditions, and without these details it is difficult to develop suitable methodologies to evaluate microorganism-plant relationships. We describe four protocols that go from sample preparation to testing in vitro the efficacy of different biofertilizers. Each protocol can be used to test a different biofertilizer microorganism, focusing on bacteria such as Rhizobium sp., Azotobacter sp., Azospirillum sp., Bacillus sp. as well as AMF such as Glomus sp. These protocols can be used in several stages of biofertilizer development, including microorganism selection, microorganism characterization, and in vitro evaluation of efficacy for the registration process.

The search for commercially viable entomopathogenic fungi for use in integrated pest management programs involves several steps. Fungal species must first be obtained from diseased insects or the environment and identified. Then, they must be evaluated under laboratory conditions to identify the most promising candidates. Because of that, bioassays must be repeatable and reliable to determine accurate pathogenicity or virulence. Variability in results may be caused by the variation in the components of an assay. However, the availability of a standardized bioassay is limited. Few reports detail the methods used to develop bioassays for a specific purpose and, without these details, it is difficult to develop bioassay methodologies suitable to evaluate the fungus-host relationship. We described a protocol based on the immersion method to evaluate entomopathogenic fungi (larval and adult stages), that can be reproduced to reduce variability. This protocol can be used in several stages of biopesticide development: selection of the biological control agent, characterization of the microorganism, formulation compatibility, and in vitro evaluation of efficacy.