Article First Report of Entomopathogenic Fungi Occurrence in Forest Soils in Croatia Marta Kovaˇ c 1, * , Cezary Tkaczuk 2 and Milan Pernek 1   Citation: Kovaˇ c, M.; Tkaczuk, C.; Pernek, M. First Report of Entomopathogenic Fungi Occurrence in Forest Soils in Croatia. Forests 2021, 12, 1690. https://doi.org/10.3390/ f12121690 Academic Editors: Lassaâd Belbahri, Justyna A. Nowakowska and Tomasz Oszako Received: 25 October 2021 Accepted: 30 November 2021 Published: 2 December 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 Croatian Forest Research Institute, Cvjetno naselje 41, 10450 Jastrebarsko, Croatia; milanp@sumins.hr 2 Institute of Agriculture and Horticulture, Siedlce University of Natural Sciences and Humanities, 08-110 Siedlce, Poland; cezary.tkaczuk@uph.edu.pl * Correspondence: martam@sumins.hr Abstract: Entomopathogenic fungi (EPF) in Croatian forests are known only from observations of insect cadavers that show obvious signs of disease. To date, their presence in soils has not been investigated. The aim of this study was to investigate their occurrence, diversity, and distribution, and to assess their density in tested soils. Soil samples were collected during 2018, 2019, and 2020 at different localities throughout the country, and analyzed by using a method of isolation of fungi on selective culture media. To assess the density of EPF in tested soils, colonies of individual fungal species were counted and recorded; the results were expressed as the number of colony-forming units (CFU) per gram of dry soil. After morphological and molecular analysis, five entomopathogenic fungal genera were identified: Beauveria spp., Metarhizium spp., Purpureocillium spp., Lecanicillium spp., and Paecilomyces spp. Results also showed that the range of a total EPF colony density in the soil varies from 4 × 10 3 to 27.4 × 10 3 CFU g −1 . The most common were EPF of the genus Beauveria, which were recorded at four of five locations, and at 16 of 25 sampling points, but the highest average number (density) of colonies belonged to the genus Metarhizium. Since this type of research was never conducted in Croatia previously, this is the first evidence that insect pathogenic fungi are present in soils of different natural forest habitats. Such research can be useful in selecting and utilizing entomopathogens that are suitable for biological pest control in certain target areas. Keywords: Beauveria; Metarhizium; Purpureocillium; Lecanicillium; Paecilomyces; entomopathogens; selective culture media; colony-forming units; biological pest control; natural forests 1. Introduction Entomopathogenic fungi (EPF) have a wide distribution and live in almost all terres- trial ecosystems in the world. Their highest diversity has been recorded in tropical forests, but they can also be found in extreme habitats such as the Arctic [1,2] and Antarctica [3,4]. They are an important factor in the regulation of insect pests that can cause great levels of mortality, and they are capable of breaking down pest populations during outbreaks in for- est habitats [5–8]. Among these fungi, Beauveria spp., Metarhizium spp., Isaria (=Cordyceps) spp., and Lecanicillium spp. are common genera found in agricultural and forest soils and have the greatest potential for biological control [9–12]. Many hypocrealean EPF are adapted to the soil, where they spend most of their life cy- cles, a feature that allows them to infect and utilize not only typical soil-inhabiting pests but also a large group of insect pests that use soil as a site for hibernation, pupation, or periodic diapause [12–15]. It is estimated that soil plays habitat to more than 90% of all insect pests at some point in their development cycle [16]. Entomopathogenic fungi can exist in the soil as saprophytes, colonizing fragments of insect cadavers and organic matter [17]. Soil is a stable structure that mitigates the fluctuation of their populations and protects them from harmful abiotic influences such as temperature, solar radiation, or drying [18,19]. Many species can be recovered from conventional isolation sites by using specific methods such as isolation on selective culture media [20] or insect-baiting methods [21]. Since a wide Forests 2021, 12, 1690. https://doi.org/10.3390/f12121690 https://www.mdpi.com/journal/forests