Marina Vemmer

Development of a CO2-releasing coformulation based on starch, Saccharomyces cerevisiae and Beauveria bassiana attractive towards western corn rootworm larvae

BACKGROUND CO2 is known as an attractant for many soil-dwelling pests. To implement an attract-and-kill strategy for soil pest control, CO2 -emitting formulations need to be developed. The aim of the present work was to develop a slow-release bead system in order to bridge the gap between application and hatching of western corn rootworm larvae. RESULTS We compared different Ca-alginate beads containing Saccharomyces cerevisiae for their potential to release CO2 over a period of several weeks. The addition of starch improved CO2 release, resulting in significantly higher CO2 concentrations in soil for at least 4 weeks. The missing amylase activity was compensated for either by microorganisms present in the soil or by coencapsulation of Beauveria bassiana. Formulations containing S. cerevisiae, starch and B. bassiana were attractive for western corn rootworm larvae within the first 4 h following exposure; however, when considering the whole testing period, the maize root systems remained more attractive for the larvae. CONCLUSION Coencapsulation of S. cerevisiae, starch and B. bassiana is a promising approach for the development of attractive formulations for soil applications. For biological control strategies, the attractiveness needs to be increased by phagostimuli to extend contact between larvae and the entomopathogenic fungus growing out of these formulations.

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

Abstract 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.

The role of carbon dioxide as an orientation cue for western corn rootworm larvae within the maize root system: implications for an attract‐and‐kill approach

BACKGROUND Western corn rootworm larvae use CO2 to locate maize roots. However, the importance of CO2 as a specific orientation cue close to maize roots has not been investigated unequivocally. This study aimed at elucidating the effect of CO2 -emitting capsules in combination with a soil insecticide (Tefluthrin = attract and kill) within the root system. We hypothesized that the capsules would result in aggregation of the larvae at the soil insecticide, thus increasing its efficacy. A nondestructive observation device was used to study larval distribution and behaviour. RESULTS Spatial analysis of distance indices (SADIE) revealed aggregation of the larvae around the capsules in an attract-and-kill treatment after 4 h, which was not found with the conventional treatment without the capsules. However, larval mortality did not differ between treatments. CONCLUSION CO2 is a weak attractant for western corn rootworm larvae within the root system. Consequently, an attract-and-kill strategy based on a CO2 product will not contribute to better control compared with conventional Tefluthrin applications. Host-specific compounds, combined with a CO2 source, should be used to target more larvae, making attract and kill a feasible management option against this pest.

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.

A bioencapsulation and drying method increases shelf life and efficacy of Metarhizium brunneum conidia

Abstract This study reports the development of encapsulated and dried entomopathogenic fungus Metarhiuzm brunneum with reduced conidia content, increased conidiation, a high drying survival and enhanced shelf life. Dried beads prepared with the fillers corn starch, potato starch, carboxymethylcellulose or autoclaved baker’s yeast, showed enhanced survival with increasing filler content. The maximum survival of 82% was found for beads with 20% corn starch at <0.1 water activity. While increasing starch content inhibits the conidiation, autoclaved baker’s yeast and a combination with starch enhanced the conidiation to 1.0 × 108 conidia/bead. Beads with conidia content reduced to 0.01% multiplied conidia in a “microfermentation” by the factor 1000. A bioassay confirmed that conidia formed from rehydrated beads were virulent against Tenebrior molitor larvae. After six months of storage, encapsulated conidia showed improved shelf life compared to non-formulated conidia. This “microfermenter” will pave the way for encapsulated fungi to be used as cost-effective biocontrol agents.

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

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.

Increased neem extract content enhances drying survival of co-encapsulated Saccharomyces cerevisiae and decreases relative release of azadirachtin

ABSTRACT Plant extracts from the neem tree can be applied as a bioinsecticidal compound in ‘attract-and-kill’ co-formulations to control soil-dwelling insect pests. Since insects are attracted to the bioinsecticide, the attract-and-kill approach benefits from a reduced dose of insecticide needed. Here, we demonstrate that a powdery neem seed kernel extract in combination with corn starch significantly enhances the drying survival of co-encapsulated CO2-releasing Saccharomyces cerevisiae. Additionally, an increased content of the neem extract was shown to slow down the relative release of azadirachtin A. Both findings are of high relevance for the development of formulations in the field of biological pest control.

Technical scale production of encapsulated Saccharomyces cerevisiae and Metarhizium brunneum attractive to wireworms

ABSTRACT Wireworms (Coleoptera: Elateridae) have recently become an increasing problem as agricultural insect pests due to the phasing out of effective control options. Entomopathogenic fungi such as Metarhizium brunneum have proven to be a promising microbial antagonist for wireworm control. Here, we tested whether the efficacy of M. brunneum can be increased through a combination with CO2, emitted by Saccharomyces cerevisiae, as an attractant (=attract-and-kill). We aimed at a technical scale production of a formulated biological control agent offering a practical and economically feasible application for wireworm control. Therefore, a novel technical formulation process for encapsulated S. cerevisiae (Attract beads) and M. brunneum (Kill beads) was investigated. For the bead production by jet cutting, the parameters nozzle diameter, pump speed, cutting device speed and collecting distance were evaluated. In order to dry the beads in a short time while maintaining a high cell viability, different drying temperatures during fluidised-bed drying were tested and the best results were obtained with an inlet air temperature profile between 50°C and 40°C. CO2 production of the beads in the soil was highest for co-applied Attract and Kill beads. The potential of beads to modify wireworm behaviour (Agriotes sputator) was tested in a rhizotron experiment. The Attract-and-Kill treatment (co-applied beads) significantly attracted wireworms, whereas Attract beads and Kill beads alone showed a weak, but non-significant attraction. Wireworm mortality could not be enhanced due to a low rate of mycosis from M. brunneum infection.

Entwicklung von Verkapselungsmethoden für Pflanzenextrakte im Projekt DevelOPAR

Die Daten der Vergleichsbetriebe stellen den Status quo im Pflanzenschutz in Deutschland dar. Der Behandlungsindex in Mohre und Frischkohl betrug im Durchschnitt der Jahre (2007-2014) bei 6,2 bzw. 9,2. Mohren wurden vorrangig mit Herbiziden und Frischkohl mit Insektiziden behandelt. Unter dem Aspekt des Pflanzenschutzes und zur Reduktion der Behandlungsintensitat mit Insektiziden kann Abbildung 1: Behandlungsindex der Herbizide (Herbizid-BI) in Abhangigkeit der Vorfrucht in den Vergleichsbetrieben fur Mohre, 2007-2014, in Deutschland, ungleiche Buchstaben symbolisieren signifikante Unterschiede (p<0,05), nBlattfrucht=58, nGemuse=28, nHalmfruchte=115 208 72. ALVA-Tagung, Seminarhotel Wesenufer, 2017 der Fruhkohlanbau empfohlen werden. Im Kohl- und Mohrenanbau konnen Halmvorfruchte Schadlingsbefall vorbeugen. Die Mittelaufwandmengen wurden vorrangig bei Herbizidanwendungen reduziert In beiden Kulturen wurden Abweichungen vom notwendigen Mas verzeichnet. Die Pflanzenschutzberatung sowie die regelmasige Durchfuhrung von Bonituren konnen Pflanzenschutzmittelanwendungen dem notwendigen Mas naher bringen und die Umsetzung des IPS in der Praxis verbessern. Fur die Ausschopfung nichtchemischer Masnahmen sind Praxisdemonstrationen und der intensive Austausch zwischen Wissenschaft und Praxis notig, genauso wie Forschungsforderung zur Entwicklung neuer und Optimierung bestehender integrierter Verfahren und neuer Wirkstoffe. Vorbildhaft wird der IPS von den Demonstrationsbetrieben u. a. in den Kulturen Mohre und Weiskohl, seit 2014 umgesetzt. Die Betriebe werden individuell und intensiv betraten, um Pflanzenschutzmittelanwendungen auf das notwendige Mas zu beschranken. Hierfur werden Bonituren unter Nutzung von Schadschwellen, Warndiensthinweisen und Monitoringverfahren durchgefuhrt.