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Simple Summary: The Japanese beetle Popillia japonica is considered one of the most harmful organisms in the world for crops and the urban landscape. Popillia japonica spends most of its life cycle in the soil as a larva. At this stage, this beetle is more susceptible to biological control agents like entomopathogenic nematodes, which are obligate parasites of main soil-inhabiting insects, killing their host in just a few days. In 2014, P. japonica was detected in Northern Italy between the Piedmont and Lombardy regions. This research aims to investigate the natural occurrence of indigenous and locally adapted entomopathogenic nematodes along the Piedmont part of the Ticino river and test the most performing of them via bioassays. Natural isolates were recovered from 39 out of the 155 soil samples collected. The virulence of all entomopathogenic nematodes assessed by laboratory and semi-field assays highlighted that two isolates resulted in more efficiency in controlling P. japonica grubs. This result is very encouraging, and the use of these natural biocontrol agents against this pest is a fundamental component of eco-friendly management. The natural presence of entomopathogenic nematodes (EPNs) has been investigated in the Piedmont region (Northern Italy) in areas infested by the Japanese beetle Popillia japonica. Thirty-nine out of 155 soil samples (25.2%) were positive for EPNs. Most of the samples contained only steinermatids (92.3%), 5.1% contained heterorhabditids, and one sample (2.6%) contained both genera. All the recovered isolates were identified at species level both morphologically and molecularly. Steinernema carpocapsae was the most abundant and it was mainly distributed in open habitats, such as perennial meadows, uncultivated soils, and cropland, characterized by sandy loam soil texture and acidic pH. Steinernema feltiae has been found associated mainly with closed habitats such as coniferous and deciduous woodland, characterized by sandy loam-texture and extremely acidic soil. The three isolates of Heterorhabditis bacteriophora were collected only in open habitats (perennial meadows and uncultivated fields) characterized by strongly acidic soils with sandy loam texture. The virulence of all EPN natural strains was evaluated by laboratory assays against P. japonica third-instar larvae collected during two different periods of the year (spring, autumn). The results showed that larval mortality was higher for pre-wintering larvae than post-wintering ones. The five more promising EPN isolates were tested in the semi-field assay in which H. bacteriophora natural strains have been shown to be more efficient in controlling P. japonica grubs. All of these results are finally discussed considering the use of these natural EPNs as biological control agents against P. japonica, within an eco-friendly perspective of management. [ABSTRACT FROM AUTHOR]

[Display omitted] • The natural occurrence of entomopathogenic fungi (EPF) was investigated in Piedmont. • Eighty-three out of 155 soil samples were positive for EPF presence. • Most of the isolates (94%) belonged to the genus Metarhizium. • M. robertsii 17/T02 showed high efficacy against P. japonica larvae in the lab tests. The natural occurrence of entomopathogenic fungi (EPF) was investigated along the Ticino River (Ticino River Natural Park, Novara Province, Piedmont, Italy), at the center of the area of the first settlement of the invasive alien pest Popillia japonica. Using Zimmermann's " Galleria bait method", EPF were successfully isolated from 83 out of 155 soil samples from different habitats (perennial, cultivated, or uncultivated meadows, woodlands, and riverbanks). Sequencing of the 5' end of the Translation Elongation Factor 1 alfa (5'-TEF) region allowed the assignment of 94% of the isolates to Metarhizium spp., while 8% and 7% were assigned to Beauveria spp. and Paecilomyces spp., respectively. Four Metarhizium species were identified: Metarhizium robertsii was the most common one (61.5% of the isolates), followed by M. brunneum (24.4%), M. lepidiotae (9%), and M. guizhouense (5.1%). Microsatellite marker analysis of the Metarhizium isolates revealed the presence of 27 different genotypes, i.e., 10 genotypes among M. robertsii , 8 among M. brunneum , 5 among M. lepidiotae, and 4 among M. guizhouense. Metarhizium brunneum appeared to be associated with woodlands and more acid soils, while the other species showed no clear association with a particular habitat. Laboratory virulence tests against P. japonica 3rd instar larvae allowed the identification of one M. robertsii isolate that showed efficacy as high as 80.3%. The importance of this kind of study in the frame of eco-friendly microbiological control is discussed. [ABSTRACT FROM AUTHOR]