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.

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.

Oleogels have been widely studied as delivery matrices for lipophilic compounds. Despite their potential, oleogels are underexplored for the formulation and release of semiochemicals for insect behavior manipulation strategies. An ethylcellulose‐candelilla wax‐oleogel as a suitable carrier for the slow release of volatile semiochemicals, employing the sesquiterpene β‐caryophyllene as a model substance is presented. A current drawback of ethylcellulose oleogels is the high temperature needed during the oleogel preparation procedure, leading to excessive volatilization of the active compounds. Reducing the gelling temperature from 104.8 to 66.9 °C and, thus, the volatile addition temperature by addition of the softener octyldodecanol results in 95.28% entrapment efficiency. Experimental results demonstrate release modifiability by tailoring the matrix crystalline state due to candelilla wax addition. Furthermore, a method is established for analyzing the self‐adhesive properties of the gel on plant leaves. In addition, the gel's self‐adhesive properties employed on apple leaves are synergistically affected by oil leakage and gel stiffness. Olfactometer experiments showed that the formulation is able to enhance the attractiveness of aphid‐infested plants for parasitic wasps. The present study provides a novel matrix‐type semiochemical formulation based on non‐toxic, biobased and biodegradable materials and will contribute to the establishment of oleogels as delivery systems for semiochemicals.

Pandora sp. nov. inedit. (ARSEF13372) is a recently isolated entomophthoralean fungus with high potential for psyllid pest control. The use of entomopathogenic fungi as biocontrol agents in large-scale field application requires biomass production in a sufficient quantity and quality. This work is the first attempt to explore the biomass production of the novel entomophthoralean Pandora species in a liquid medium. This study aims at establishing a submerged fermentation process with potential for mass production of the fungus. Three different complex nutrient sources were evaluated for maximising biomass in a liquid shaking culture. Pandora sp. nov. grew best in media containing skimmed milk, but even faster in a mixture of skimmed milk, yeast extract and a low-cost protein hydrolysate from animal by-products. Subsequently, the produced biomass can be encapsulated in biopolymer beads or granules in order to transfer the fungus into an easily applicable form. Therefore, growth as finely dispersed mycelium was promoted by increased media osmolality through the addition of sodium chloride. After the medium had been transferred to a stirred tank bioreactor with a working volume of 8 L, a maximum biomass dry weight of 21 g·L⁻¹ was reached after 48 h. These promising results can pave the way for large-scale fermentation and formulation processes of this novel Pandora species for biological psyllid pest control.

Batkoa is a genus of entomophthoralean fungi often associated with insect epizootics, particularly in phytophagous hemipterans. Encapsulation has become a promising strategy for improving the shelf life and sporulation of these fungi post-application. This study aims to (i) compare the virulence of the submerged propagules and primary conidia of Batkoa sp. ESALQ1199 against Dalbulus maidis; (ii) formulate submerged propagules in calcium alginate beads with co-formulants; (iii) assess the colony-forming units and sporulation of encapsulated beads dried with different kaolin concentrations (0%, 2%, 4%, 8% and 10%); (iv) determine the shelf life of dried bead formulations containing 10% kaolin, comparing washed and unwashed beads treated with a 4% sucrose solution; and (v) assess the sporulation capacity of beads with 10% kaolin, washed and unwashed with 4% sucrose solution, over time under humid conditions. Our results demonstrated that primary conidia and submerged propagules effectively killed 82.4% and 57.8% of adult corn leafhoppers, respectively. Co-formulants maintained viability above 80% in dried propagules, while control samples dropped to 45%, indicating the sensitivity of submerged propagules to the drying process. Encapsulated Batkoa sp. retained the same concentration of viable propagules per bead and the number of conidia produced (sporulation) for 30 days at 28 °C. The sporulation of fresh beads increased during the incubation period, plateauing after 27 days. This suggests that Batkoa sp. beads can produce primary conidia under humid field conditions, serving as a potential inoculum source for new infections.