Genetic variability of Metarhizium isolates from the Ticino Valley Natural Park (Northern Italy) as a possible microbiological resource for the management of Popillia japonica.

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.

Impact of Super-High Density Olive Orchard Management System on Soil Free-Living and Plant-Parasitic Nematodes in Central and South Italy.

The soil nematode community plays an important role in ecosystem services. The objective of this study was to assess the effect of Super-high density (SHD) olive orchards on the nematode community in five sites with different soils, climates, and cultivars. At each site, the SHD management system was compared to the adjacent olive orchard traditional (TRAD) system, in which the same soil management and phytosanitary measures were applied. Soil management was assessed by total organic carbon content (TOC), while the soil nematode community was evaluated using the nematode taxa abundances and soil nematode indicators. TOC was significantly decreased in the SHD olive orchard system compared to TRAD in the sites characterized by conventional tillage and mineral fertilization. The two-way ANOSIM analysis on nematode abundance showed no difference between the two olive management methods, instead showing only a significant difference per site mainly due to variabilities in plant-parasitic nematode assemblage. However, a negative impact of SHD management was evident in environments stressed by summer droughts and conventional tillage: the ratio of obligate plant-parasites to bacterivores and fungivores (Pp/(B+F)) was significantly higher in SHD than in the TRAD olive orchard system, and the prey-to-predator θ mass ratio showed the lowest values in the sites under organic fertilization or green manure. The canonical correspondence analysis showed that the free-living nematodes were only slightly affected by SHD olive orchards; instead, the presence of plant-parasitic nematodes families such as Telotylenchidae, Paratylenchidae, Meloidogynidae, and Criconematidae was favored, in comparison to Longidoridae, Heteroderidae, and Pratylenchidae.

Evaluation of Indigenous Entomopathogenic Nematodes as Potential Biocontrol Agents against Popillia japonica (Coleoptera: Scarabaeidae) in Northern Italy.

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.

First report of Bursaphelenchus fungivorus (Nematoda: Aphelenchida) in Italy and an overview of nematodes associated with Crocus sativus L.

Saffron is one of the most precious spices in the world. It is derived from the dried stigmas of the Crocus sativus L. flowers. This plant is triploid sterile and propagated by corms. As a subterranean organ, the corm can come into contact with different nematodes. In this contribution Bursaphelenchus fungivorus was reported for the first time in Italy. It was found associated with C. sativus corms and characterized based on morphological and morphometrical characteristics for this species. The identification was confirmed using molecular analyses. Moreover, a review of nematodes associated with C. sativus worldwide is also provided.Saffron is one of the most precious spices in the world. It is derived from the dried stigmas of the Crocus sativus L. flowers. This plant is triploid sterile and propagated by corms. As a subterranean organ, the corm can come into contact with different nematodes. In this contribution Bursaphelenchus fungivorus was reported for the first time in Italy. It was found associated with C. sativus corms and characterized based on morphological and morphometrical characteristics for this species. The identification was confirmed using molecular analyses. Moreover, a review of nematodes associated with C. sativus worldwide is also provided.