Formalin-casein enhances water absorbency of calcium alginate beads and activity of encapsulated Metarhizium brunneum and Saccharomyces cerevisiae.

The control of root-feeding wireworms has become more challenging as synthetic soil insecticides have been progressively phased out due to environmental risk concerns. Innovative microbial control alternatives such as the so-called attract-and-kill strategy depend on the rapid and successful development of dried encapsulated microorganisms, which is initiated by rehydration. Casein is a functional additive that is already used in food or pharmaceutical industry due to its water binding capacity. Cross-linked forms such as formalin-casein (FC), exhibit altered network structures. To determine whether FC influences the rehydration of alginate beads in order to increase the efficacy of an attract-and-kill formulation for wireworm pest control, we incorporated either casein or FC in different alginate/starch formulations. We investigated the porous properties of alginate/starch beads and subsequently evaluated the activities of the encapsulated entomopathogenic fungus Metarhizium brunneum and the CO2 producing yeast Saccharomyces cerevisiae. Adding caseins altered the porous structure of beads. FC decreased the bead density from (1.0197 ± 0.0008) g/mL to (1.0144 ± 0.0008) g/mL and the pore diameter by 31%. In contrast to casein, FC enhanced the water absorbency of alginate/starch beads by 40%. Furthermore, incorporating FC quadrupled the spore density on beads containing M. brunneum and S. cerevisiae, and simultaneous venting increased the spore density even by a factor of 18. Moreover, FC increased the total CO2 produced by M. brunneum and S. cerevisiae by 29%. Thus, our findings suggest that rehydration is enhanced by larger capillaries, resulting in an increased water absorption capacity. Our data further suggest that gas exchange is improved by FC. Therefore, our results indicate that FC enhances the fungal activity of both fungi M. brunneum and S. cerevisiae, presumably leading to an enhanced attract-and-kill efficacy for pest control.

Unraveling the interaction of co-encapsulated Saccharomyces cerevisiae and Metarhizium brunneum in calcium alginate-based attract-and-kill beads.

BACKGROUND: Attract-and-kill (AK) beads are biological, microbial insecticides developed as an alternative to synthetic soil insecticides. For wireworm control, beads are based on calcium alginate/starch co-encapsulating the carbon dioxide (CO2) producing yeast Saccharomyces cerevisiae H205 as the attract component, and the entomopathogenic fungus Metarhizium brunneum CB15-III as the kill component. However, the physicochemical processes inside beads during co-cultivation are still unclear. Here we reveal for the first time the spatiotemporal conditions of oxygen and pH inside AK beads measured with microelectrodes and describe the impact of S. cerevisiae on CO2 and conidia formation. RESULTS: Measurements revealed a steep oxygen gradient already 2 days after co-encapsulation, with an internal hypoxic zone. Encapsulating either S. cerevisiae or M. brunneum already decreased the average pH from 5.5 to 4.7 and 4.6, respectively. However, on day 3, co-cultivation lead to temporal strong acidification of beads down to pH 3.6 which followed the maximum CO2 productivity and coincided with the maximum conidiation rate. Decreasing the yeast load decreased the total CO2 productivity to half, and the conidial production by 93%, while specific productivities normalized to 1% yeast load increased eight-fold and three-fold, respectively, with day 3 being an exception. CONCLUSION: Our findings indicate a general beneficial interaction between M. brunneum and S. cerevisiae, but also suggest competition for resources. These findings will contribute to develop innovative co-formulations with maximum efficiency to save application rates and costs. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Polyvinyl alcohol coating releasing fungal blastospores improves kill effect of attract-and-kill beads.

Polyvinyl alcohol (PVA) is a biodegradable, water-soluble polymer with excellent film forming properties, commonly studied or used as tablet coating, food packaging or controlled release fertilizers. Attract-and-kill (AK) beads are sustainable, microbial alternatives to synthetic soil insecticides, whose onset of lethal effect largely depend on how fast the encapsulated entomopathogenic fungus forms virulent conidia. Therefore, the objective of this study was to develop a water-soluble coating accelerating the kill effect of AK beads by immediately releasing virulent Metarhizium brunneum CB15-III blastospores. We assessed three PVA types (PVA 4-88, 8-88, 10-98) which differed in their degree of hydrolysis or molecular weight for their ability to release viable blastospores from thin films after drying at 60-40 °C, and examined how polyethylene glycol and soy-lecithin impact the blastospore survival. Finally, we evaluated the effectiveness of coated AK beads in a bioassay against Tenebrio molitor larvae. The blastospore release rate quadrupled within the first 5 min with decreasing molecular weight and degree of hydrolysis, with PVA 4-88 releasing 79 ± 19% blastospores. Polyethylene glycol and soy-lecithin significantly increased the blastospore survival to 18-28% for all three PVA types. Coated beads exhibited a uniform, 22.4 ± 7.3 µm thin coating layer, with embedded blastospores, as confirmed by scanning electron microscopy. The blastospore coating increased the mortality rate of T. molitor larvae over uncoated AK beads, decreasing the median lethal time from 10 to 6 days. Consequently, the blastospore coating accelerated the kill effect of regular AK beads. These findings pave the way to enhanced pest control efficacy from coated systems such as beads or seeds.