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.