Encapsulation of the psyllid-pathogenic fungus Pandora sp. nov. inedit. and experimental infection of target insects.

BACKGROUND: Pandora sp. nov. inedit. (Entomophthorales: Entomophthoraceae) (ARSEF 13372) is a recently isolated entomophthoralean fungus with potential for psyllid pest control. This study aimed to develop a formulation based on biocompatible hydrogel beads in order to transfer the fungus into an easily applicable form and to test the effects on psyllids. RESULTS: After encapsulation in calcium alginate beads, Pandora sp. nov. grew from the beads and discharged conidia over 12 days under optimal humidity conditions at 18 °C. Conidial number was increased 2.95-fold by the addition of skimmed milk as nutritional formulation adjuvant to the beads. The virulence of the encapsulated fungus was assessed with the two target psyllid species; the summer apple psyllid, Cacopsylla picta and the pear psyllid, Cacopsylla pyri. Beads containing skimmed milk as nutritional adjuvant led to the highest mortalities (48.3% on C. picta and 75.0% on C. pyri). In a second bioassay, survival time of C. pyri exposed to beads containing different concentration (10%, 20% or 40%) of Pandora sp. nov. was tested. The survival time of C. pyri was significantly reduced when exposed to beads containing 10% or 20% Pandora sp. nov. The median survival time was reached after 5-6 days past inoculation and the cumulative mortality for C. pyri treated with Pandora sp. nov. beads showed up to 89% mortality. CONCLUSION: The promising results of this study will ease the way for large-scale field application of a novel Pandora species in biological psyllid pest control.

Image analysis-based quantification of fungal sporulation by automatic conidia counting and gray value correlation.

The present work describes a new computer-assisted image analysis method for the rapid, simple, objective and reproducible quantification of actively discharged fungal spores which can serve as a manual for laboratories working in this context. The method can be used with conventional laboratory equipment by using bright field microscopes, standard scanners and the open-source software ImageJ. Compared to other conidia quantification methods by computer-assisted image analysis, the presented method bears a higher potential to be applied for large-scale sample quantities. The key to make quantification faster is the calculation of the linear relationship between the gray value and the automatically counted number of conidia that has only to be performed once in the beginning of analysis. Afterwards, the gray value is used as single parameter for quantification. The fast, easy and objective determination of sporulation capacity enables facilitated quality control of fungal formulations designed for biological pest control.•Rapid, simple, objective and reproducible quantification of fungal sporulation suitable for large-scale sample quantities.•Requires conventional laboratory equipment and open-source software without technical or computational expertise.•The number of automatically counted conidia can be correlated with the gray value and after initial calculation of a linear fit, the gray value can be applied as single quantification parameter.

Chitin increases drying survival of encapsulated Metarhizium pemphigi blastospores for Ixodes ricinus control.

Ticks, like Ixodes ricinus, have negative impacts on human and animal health in Germany and worldwide, with almost no specific scientifically proven biological control agent commercially available. Biological control agents containing entomopathogenic fungi present many advantages over chemical acaricides but usually high doses of aerial conidia (1013-1014 conidia/ha) are required to control arthropod pests in the field. A suitable formulation containing nutrients not only makes sensitive blastospores applicable but also functions as a microfermenter to multiply the biomass and thus significantly reduce the required application dosage. For this approach, Metarhizium pemphigi X1c blastospores were encapsulated in calcium alginate beads with granular corn starch or chitin powder as nutrients to ensure formation of aerial conidia on the surface and were then dried. The highest concentration was obtained with moist beads containing chitin (4.68 ±â€¯0.71∙107 conidia∙bead-1). The highest drying survival was also obtained with chitin as the additive (14.7 ±â€¯2.18%). Newly formed aerial conidia of all formulations showed high virulence and caused 100% mortality of I. ricinus nymphs. Altogether, this study paves the way for a lower dose and cost-effective application of blastospores for the control of above ground arthropod pests.

Development of an attract-and-kill co-formulation containing Saccharomyces cerevisiae and neem extract attractive towards wireworms.

BACKGROUND: Wireworms (Coleoptera: Elateridae) are major insect pests of worldwide relevance. Owing to the progressive phasing-out of chemical insecticides, there is great demand for innovative control options. This study reports on the development of an attract-and-kill co-formulation based on Ca-alginate beads, which release CO2 and contain neem extract as a bioinsecticidal compound. The objectives of this study were to discover: (1) whether neem extract can be immobilized efficiently, (2) whether CO2 -releasing Saccharomyces cerevisiae and neem extract are suitable for co-encapsulation, and (3) whether co-encapsulated neem extract affects the attractiveness of CO2 -releasing beads towards wireworms. RESULTS: Neem extract was co-encapsulated together with S. cerevisiae, starch and amyloglucosidase with a high encapsulation efficiency of 98.6% (based on measurement of azadirachtin A as the main active ingredient). Even at enhanced concentrations, neem extract allowed growth of S. cerevisiae, and beads containing neem extract exhibited CO2 -emission comparable with beads without neem extract. When applied to the soil, the beads established a CO2 gradient of >15 cm. The co-formulation containing neem extract showed no repellent effects and was attractive for wireworms within the first 24 h after exposure. CONCLUSION: Co-encapsulation of S. cerevisiae and neem extract is a promising approach for the development of attract-and-kill formulations for the control of wireworms. This study offers new options for the application of neem extracts in soil. © 2017 Society of Chemical Industry.

Co-encapsulation of amyloglucosidase with starch and Saccharomyces cerevisiae as basis for a long-lasting CO2 release.

CO2 is known as a major attractant for many arthropod pests which can be exploited for pest control within novel attract-and-kill strategies. This study reports on the development of a slow-release system for CO2 based on calcium alginate beads containing granular corn starch, amyloglucosidase and Saccharomyces cerevisiae. Our aim was to evaluate the conditions which influence the CO2 release and to clarify the biochemical reactions taking place within the beads. The amyloglucosidase was immobilized with a high encapsulation efficiency of 87% in Ca-alginate beads supplemented with corn starch and S. cerevisiae biomass. The CO2 release from the beads was shown to be significantly affected by the concentration of amyloglucosidase and corn starch within the beads as well as by the incubation temperature. Beads prepared with 0.1 amyloglucosidase units/g matrix solution led to a long-lasting CO2 emission at temperatures between 6 and 25 °C. Starch degradation data correlated well with the CO2 release from beads during incubation and scanning electron microscopy micrographs visualized the degradation of corn starch granules by the co-encapsulated amyloglucosidase. By implementing MALDI-ToF mass spectrometry imaging for the analysis of Ca-alginate beads, we verified that the encapsulated amyloglucosidase converts starch into glucose which is immediately consumed by S. cerevisiae cells. When applied into the soil, the beads increased the CO2 concentration in soil significantly. Finally, we demonstrated that dried beads showed a CO2 production in soil comparable to the moist beads. The long-lasting CO2-releasing beads will pave the way towards novel attract-and-kill strategies in pest control.

Calcium gluconate as cross-linker improves survival and shelf life of encapsulated and dried Metarhizium brunneum and Saccharomyces cerevisiae for the application as biological control agents.

Calcium chloride (CC) is the most common cross-linker for the encapsulation of biocontrol microorganisms in alginate beads. The aim of this study was to evaluate if calcium gluconate (CG) can replace CC as cross-linker and at the same time improve viability after drying and rehydration, hygroscopic properties, shelf life and nutrient supply. Hence, the biocontrol fungi Metarhizium brunneum and Saccharomyces cerevisiae were encapsulated in Ca-alginate beads supplemented with starch. Beads were dried and maximum survival was found in beads cross-linked with CG. Beads prepared with CG showed lower hygroscopic properties, but a higher shelf life for encapsulated fungi. Moreover, we demonstrated that gluconate has a nutritive effect on encapsulated fungi, leading to increased mycelium growth of M. brunneum and to enhanced CO2 release from beads containing Saccharomyces cerevisiae. The application of CG as cross-linker will pave the way towards increasing drying survival and shelf life of various, especially drying-sensitive microbes.