Abstract
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.
Highlights
The formulation of living biocontrol agents as capsules improves their applicability, shelf life and storability and prolongs the sporulation duration after field application by serving as a "microfermenter" formulation [9]
Compared to manual counting of discharged conidia which is a subjective and time-wasting procedure, automatized image analysis improves the comparability of results
The aim of this study was the development of a method for faster and more precise high-throughput quantification of fungal sporulation by computer-assisted image analysis
Summary
The formulation of living biocontrol agents as capsules improves their applicability, shelf life and storability and prolongs the sporulation duration after field application by serving as a "microfermenter" formulation [9]. When considering entomopathogenic fungi for use in biological control, one of the most important issues is a reliable method for quantification of virulent conidia formed by encapsulated fungal cells. Fungi of the order of Entomophthorales bear an exceptional high potential for biological insect pest control because of their narrow host range and fast speed-to-kill [8] and the genus Pandora contains several species with potential for biological control. The conidia of Pandora spp. stick, by aid of mucoid substances and a partly detached cell wall, to surfaces like the host insect cuticle [4,7]. The role of this mucous layer is not yet completely clear, it makes classical collection and counting of the conidia using common surfactants like polysorbates, e.g. Tween 80, difficult or even impossible
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