First report of Rhabditis (Rhabditella) axei with the invasive palm borer Paysandisia archon.

Rhabditis (Rhabditella) axei is a free-living, pseudoparasitic, necromenic, and parasitic nematode, depending on the host. This species feeds mainly on bacteria present in decaying organic matter, soil, and other substrates; however, in its parasitic form, it can colonize some species of snails. Moreover, the presence of R. axei has also been detected in birds and mammals, including humans. In 2021-2023, during monitoring of the palm borer Paysandisia archon in Central Italy, R. axei emerged from dead larvae of this alien invasive moth and was extracted from palm fibres of Trachycarpus fortunei in three independent sites. The nematode was identified by morphological and morphometric analyses. Molecular analyses using SSU and LSU gene fragments were used to confirm the identification and to perform Bayesian reconstruction of the phylogeny. Each sampling site showed a unique haplotype. Concerning the pathogenicity of this nematode against insects, the test performed on Galleria mellonella larvae did not show any entomopathogenic effect. This is the first time that R. axei was found associated with P. archon, and this recurrent association was discussed.

Effectiveness of field-exposed attract-and-kill devices against the adults of Popillia japonica (Coleoptera: Scarabaeidae): a study on duration, form and storage.

BACKGROUND: The Japanese beetle Popillia japonica Newman is an insect pest native to Japan that has spread into North America, the Azores and, recently, into continental Europe. Here, we present a study assessing the effectiveness of a long-lasting insecticide-treated net (LLIN), assembled in semiochemical-baited attract-and-kill devices (A&Ks) as a low environmental impact means to control P. japonica in the field. We compared the attractiveness of three different forms of A&Ks that were left outdoors throughout the summer, and the residence time of P. japonica landing on them. Moreover, we performed a preliminary study testing the effectiveness of new LLINs after storage. Collected data also allowed us to investigate the beetles' diel flight patterns in relation to meteorological conditions. RESULTS: Killing effectiveness of the field-exposed A&Ks declined steadily over the flight season (from 100% to 37.5%) associated with a decrease in residues of α-cypermethrin, the active ingredient in the LLINs. The different A&K forms (pyramidal, octahedral and ellipsoidal) attracted similar numbers of beetles. Individual beetles' residence time ranged from 75 to 95 s and differed slightly between A&K forms. Effectiveness of LLINs decreased by ≈30% after 1 year storage. Based on numbers landing on A&Ks, the beetles' flight activity peaked about 14:30 h and was inversely correlated with relative humidity. CONCLUSION: This study indicates that semiochemical-baited A&Ks are effective for controlling P. japonica in the field. Because of active ingredient decay, the LLINs should be replaced after 30-40 days of field exposure to ensure that the A&Ks remain fully functional. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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.

Popillia japonica - Italian outbreak management.

Popillia japonica, a priority pest for the EU, was first detected in Northern Italy in 2014. Since its discovery, the outbreak extended over an area of more than 16,000 square kilometers in Northern Italy and Southern Switzerland. In this review, we summarize the state-of-the-art of research conducted in Italy on both the spreading capacity and control measures of P. japonica. Chemical, physical, and biological control measures deployed since its detection are presented, by highlighting their strengths and weaknesses. An in-depth study of the ecosystems invaded by P. japonica disclosed the presence and pathogenicity of natural strains of entomopathogenic fungi and nematodes, some of which have shown to be particularly aggressive towards the larvae of this pest under laboratory conditions. The Plant Health authorities of the Lombardy and Piedmont regions, with the support of several research institutions, played a crucial role in the initial eradication attempt and subsequently in containing the spread of P. japonica. Control measures were performed in the infested area to suppress adult populations of P. japonica by installing several traps (e.g., for mass trapping, for auto-dissemination of the fungus Metarhizium anisopliae, and "attract & kill"). For larval control, the infested fields were treated with commercial strains of the entomopathogenic fungus M. anisopliae and nematode Heterorhabditis bacteriophora. Future studies will aim at integrating phenological and spread models developed with the most effective control measures, within an ecologically sustainable approach.

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.

Assessing immunocompetence in red palm weevil adult and immature stages in response to bacterial challenge and entomopathogenic nematode infection.

Parasites and pathogens can follow different patterns of infection depending on the host developmental stage or sex. In fact, immune function is energetically costly for hosts and trade-offs exist between immune defenses and life history traits as growth, development and reproduction and organisms should thus optimize immune defense through their life cycle according to their developmental stage. Identifying the most susceptible target and the most virulent pathogen is particularly important in the case of insect pests, in order to develop effective control strategies targeting the most vulnerable individuals with the most effective control agent. Here, we carried out laboratory tests to identify the most susceptible target of infection by infecting different stages of the red palm weevil Rhynchophorus ferrugineus (larvae, pupae, male, and female adults) with both a generic pathogen, antibiotic-resistant Gram-negative bacteria Escherichia coli XL1-Blue, and two specific strains of entomopathogenic nematodes (EPNs), Steinernema carpocapsae ItS-CAO1 and Heterorhabditis bacteriophora ItH-LU1. By evaluating bacterial clearance, host mortality and parasite progeny release, we demonstrate that larvae are more resistant than adults to bacterial challenge and they release less EPNs progeny after infection despite a higher mortality compared to adults. Considering the two EPN strains, S. carpocapsae was more virulent than H. bacteriophora both in terms of host mortality and more abundant progeny released by hosts after death. The outcomes attained with unspecific and specific pathogens provide useful information for a more efficient and sustainable management of this invasive pest.

How ecosystems change following invasion by Robinia pseudoacacia: Insights from soil chemical properties and soil microbial, nematode, microarthropod and plant communities.

Biological invasions are a global threat to biodiversity. Since the spread of invasive alien plants may have many impacts, an integrated approach, assessing effects across various ecosystem components, is needed for a correct understanding of the invasion process and its consequences. The nitrogen-fixing tree Robinia pseudoacacia (black locust) is a major invasive species worldwide and is used in forestry production. While its effects on plant communities and soils are well known, there have been few studies on soil fauna and microbes. We investigated the impacts of the tree on several ecosystem components, using a multi-trophic approach to combine evidence of soil chemical properties and soil microbial, nematode, microarthropod and plant communities. We sampled soil and vegetation in managed forests, comparing those dominated by black locust with native deciduous oak stands. We found qualitative and quantitative changes in all components analysed, such as the well-known soil nitrification and acidification in stands invaded by black locust. Bacterial richness was the only component favoured by the invasion. On the contrary, abundance and richness of microarthropods, richness of nematodes, and richness and diversity of plant communities decreased significantly in invaded stands. The invasion process caused a compositional shift in all studied biotic communities and in relationships between the different ecosystem components. We obtained clear insights into the effects of invasion of managed native forests by black locust. Our data confirms that the alien species transforms several ecosystem components, modifying the plant-soil community and affecting biodiversity at different levels. Correct management of this aggressive invader in temperate forests is urgently required.

Hexamermis popilliae n. sp. (Nematoda: Mermithidae) parasitizing the Japanese beetle Popillia japonica Newman (Coleoptera: Scarabaeidae) in Italy.

A new species of mermithid nematode, Hexamermis popilliae n. sp. (Nematoda: Mermithidae) is described from the Japanese beetle Popillia japonica Newman in Italy, an area of new introduction for this invasive pest. The combination of the following characters separates H. popilliae from other members of the genus Hexamermis Steiner, 1924: adult head obtuse; amphidial pouches slightly posterior to lateral head papillae in female but adjacent to lateral head papillae in males; amphidial openings large, well developed; amphidial pouches elliptical in females and oblong in males; cuticular vulvar cone well developed, vulvar lips greatly reduced or lacking, vagina curved at tip where meeting uteri, without reverse bend (not S-shaped), spicules slightly curved, with a slight bend in the basal portion, approximately equal to body width at cloaca. This is the first record of a species of Hexamermis parasitizing the Japanese beetle Popillia japonica. The only previous mention of mermithid nematodes from P. japonica was an undescribed species of Psammomermis in North America. Hexamermis popilliae will be evaluated as a potential biological control agent in an integrated control program of the Japanese beetle in Italy.

Occurrence of Panagrellus (Rhabditida: Panagrolaimidae) Nematodes in a Morphologically Aberrant Adult Specimen of Rhynchophorus ferrugineus (Coleoptera: Dryophthoridae).

An aberrant specimen of Rhynchophorus ferrugineus (Coleoptera: Dryophthoridae) also known as red palm weevil (RPW), the most economically important insect pest of palms in the world, was found among a batch of conspecifics reared for research purposes. A morphological analysis of this weevil revealed the presence of nematodes associated with a structured cuticle defect of the thorax. These nematodes were not able to be cultured, but were characterized by molecular analysis using 28S and 18S ribosomal DNA and shown to belong to the family Panagrolaimidae (Rhabditida), within a clade of Panagrellus. While most nematodes in the insect were juveniles, a single male adult was partially characterized by light microscopy. Morphometrics showed similarities to a species described from Germany. Excluding the entomopathogenic nematodes (EPN), only five other genera of entomophilic or saprophytic rhabditid nematodes are associated with this weevil. This is the first report of panagrolaimid nematodes associated with this invasive pest. Possible mechanisms of nematode-insect association are discussed.

Oscheius tipulae in Italy: Evidence of an Alien Isolate in the Integral Natural Reserve of Montecristo Island (Tuscany).

Montecristo Island is an integral natural reserve of the Tuscan Archipelago National Park (Central Italy), characterized by a peculiar assemblage of flora and fauna, with several endemic taxa, and also with a high number of alien species. During a soil survey, we found an alien Oscheius tipulae Lam & Webster, 1971 isolate, phylogenetically close to others from South America. In this article, we examined the possible pathways of introduction of this nematode. Because of the high number of alien plants in this protected area and the low desiccation survival ability of O. tipulae, we hypothesized that the presence of this alien nematode isolate may be related to the soil of introduced plants, although historical association with plant-associated invertebrates is also possible. Further studies with more populations and marker molecules are necessary to investigate the distribution of O. tipulae and the possible impact on this natural reserve.

Oscheius onirici sp. n. (Nematoda: Rhabditidae): a new entomopathogenic nematode from an Italian cave.

Oscheius onirici sp. n. (Nematoda: Rhabditidae) was isolated from a karst cave soil of Central Italy. Molecular and morphological analyses were performed. Total DNA was extracted from individual nematodes and the mitochondrial COI, the ITS containing region, the D2-D3 expansion domains of the 28S rRNA gene and the 18S rRNA gene were amplified and sequenced. BLAST search at NCBI by using all molecular markers revealed that this taxon is similar to Oscheius species. Phylogenetic trees of ITS, 28S and 18S rDNA revealed that O. onirici sp. n. belongs to Dolichura-group. Oscheius onirici sp. n. is characterized by small body size and stoma rhabditoid type. Female reproductive system is amphidelphic. Males are rare with peloderan bursa, spicules slender and small, nine pairs of papillae of different lengths, arranged in a 1+1+1/3+3 pattern. Entomopathogenicity bioassay revealed that this nematode is capable of infecting larvae of Galleria mellonella and Tenebrio molitor.