Attraction of entomopathogenic nematodes to black truffle and its volatile organic compounds: A new approach for truffle beetle biocontrol.

The European truffle beetle, Leiodes cinnamomeus, is the most important pest in black truffle (Tuber melanosporum) plantations. Entomopathogenic nematodes (EPNs) are a promising biological control agents against L. cinnamomeus. EPNs may employ multiple sensory cues while seeking for hosts, such as volatile organic compounds (VOCs) and CO2 gradients. We report for the first time the attraction of EPNs to truffle fruitbodies, and identified some VOCs potentially playing a key role in this interaction. We conducted olfactometer assays to investigate the attraction behavior of Steinernema feltiae and Steinernema carpocapsae towards both T. melanosporum fruitbodies and larvae of L. cinnamomeus. Subsequently, a chemotaxis assay using agar plates was performed to determine which of the 14 of the main VOCs emitted by the fruitbodies attracted S. feltiae at low (0.1 %) and high (mg/100 g truffle) concentrations. Both EPN species were attracted to mature fruitbodies of T. melanosporum, which may enhance the likelihood of encountering L. cinnamomeus during field applications. L. cinnamomeus larvae in the presence of truffles did not significantly affect the behavior of EPNs 24 h after application, underscoring the importance of the chemical compounds emitted by truffles themselves. Chemotaxis assays showed that four long-chain alcohol compounds emitted by T. melanosporum fruitbodies attracted S. feltiae, especially at low concentration, providing a first hint in the chemical ecology of a little-studied ecological system of great economical value. Further studies should be conducted to gain a finer understanding of the tritrophic interactions between T. melanosporum, EPNs, and L. cinnamomeus, as this knowledge may have practical implications for the efficacy of EPNs in the biological control of this pest.

Natural occurrence of entomopathogenic nematodes (Steinernema and Heterorhabditis) and Pristionchus nematodes in black truffle soils from Spain.

The European truffle beetle Leiodes cinnamomeus is the most important pest in black truffle (Tuber melanosporum) plantations. Current control methods against it are inefficient, so entomopathogenic nematodes (EPNs) could play an important role in their population regulation due to their efficacy against many soil-dwelling insect pests. A survey of EPNs and Pristionchus nematodes was conducted in truffle soils of Spain, considering environmental and physical-chemical soil factors. A total of 164 soil samples were collected from forests, productive plantations and null-low productive plantations, representing three distinct black truffle-growing habitat types. EPNs were isolated from seven soil samples (4.3%); four nematodes were identified as Steinernema feltiae and three as Heterorhabditis bacteriophora. Both species were sampled in three types of soil texture (loam, sandy loam or sandy clay loam), characterized by alkaline pH (7.5 to 8.5) and high organic matter (2.1-11.04%). The presence of these EPNs was influenced by habitat type and organic matter content. Pristionchus nematodes were isolated from truffle soil, around truffle fruit bodies and under the elytra of L. cinnamomeus, with Pristionchus maupasi being the most commonly identified species. No significant associations were found between environmental and soil factors and the occurrence of Pristionchus nematodes. These nematodes were found in alkaline soils (pH 7.75 to 8.7), across all seven sampled soil textures, with variable organic matter content (0.73%-5.92%). The ecological trends and the presence of Pristionchus may affect the occurrence of EPNs and their prospective use as biological control agents against L. cinnamomeus in black truffle plantations.

Infectivity of Steinernema carpocapsae and S. feltiae to Larvae and Adults of the Hazelnut Weevil, Curculio nucum: Differential Virulence and Entry Routes.

We investigated the existing susceptibility differences of the hazelnut weevil, Curculio nucum L. (Coleoptera:, Curculionidae) to entomopathogenic nematodes by assessing the main route of entry of the nematodes, Steinernema carpocapsae strain B14 and S. feltiae strain D114, into larvae and adult insects, as well as host immune response. Our results suggested that S. carpocapsae B14 and S. feltiae D114 primarily entered adult insects and larvae through the anus. Larvae were more susceptible to S. feltiae D114 than S. carpocapsae B14 and adults were highly susceptible to S. carpocapsae B14 but displayed low susceptibility to S. feltiae D114. Penetration rate correlated with nematode virulence. We observed little evidence that hazelnut weevils mounted any cellular immune response toward S. carpocapsae B14 or S. feltiae D114. We conclude the differential susceptibility of hazelnut weevil larvae and adults to S. carpocapsae B14 and S. feltiae D114 primarily reflected differences in the ability of these two nematodes to penetrate the host.

Sex-related differences in the susceptibility of Periplaneta americana and Capnodis tenebrionis to the entomopathogenic nematode Steinernema carpocapsae.

Entomopathogenic nematodes have a broad range of insect hosts, but efficacy varies depending on many factors including insect species and life stage of the host. In this study we examined the susceptibility of males and females of two insect species, the American cockroach Periplaneta americana and the flatheaded rootborer Capnodis tenebrionis to Steinernema carpocapsae by exposing adults to different concentrations. A separate study examined the routes of entry used by nematodes into males and females. Males of both species were more susceptible than females, with greater than 97% infection when a concentration of 50 IJs/cm(2) was applied, whereas mortalities lower than 58% were achieved against females 14 days post-infection. Infective juveniles were detected in similar number on the surface of both sexes, but when compared among internal regions the number of nematodes was significantly higher in gonads and ducts of males than of females for P. americana (5.49 in males; 0.02 in females) and C. tenebrionis (6.88 in males; 0.00 in females).

Ecological characterization of entomopathogenic nematodes isolated in stone fruit orchard soils of Mediterranean areas.

Entomopathogenic nematodes in the families Steinernematidae and Heterorhabditidae were isolated from stone-fruit orchards in two Mediterranean regions of Spain. A total of 630 soil samples (210 sites) from Catalonia and 90 soil samples (30 sites) from Murcia were evaluated resulting in 5.2% and 20% of the soils testing positive for nematodes, respectively. Ten steinernematid isolates and three heterorhabditid isolates were recovered using the Galleria mellonella baiting method. Based on morphometric data, molecular data, and cross-breeding experiments the nematode species were identified as Steinernemafeltiae and Heterorhabditis bacteriophora. Environmental tolerance to heat, desiccation and hypoxia, the effect of temperature on infectivity and reproduction and nematode migration in sand columns were compared among isolates and one Steinernema carpocapsae strain. Results showed differences among species and a great variability within species. Beneficial traits for each strain were added up to identify a superior candidate to control Mediterranean flat-headed rootborer, Capnodis tenebrionis. When all analyzed factors were considered, three S. feltiae isolates (Bpa, Sor and M116) obtained the best scores, and when hypoxia was removed, two of the strains (Bpa and Sor) continued ranking superior to other strains.