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a b s t r a c tSatureja montana (winter savory) and Ruta graveolens (rue) nematotoxic essential oils (EOs) (0.5 l EO/ml culture medium) were assessed for the first time in Pinus pinaster in vitro shoot cultures (Ppi) and P. pinaster shoots with Bursaphelenchus xylophilus co-cultures (PpiBx). The EOs nematotoxic effect was evaluated on B. xylophilus population density in PpiBx co-cultures and the phytotoxic activity to the host was assessed by evaluating relative water content and volatile profiles both on Ppi cultures and on PpiBx co-cultures. Carvacrol-rich S. montana EO showed phytotoxicity, by inducing shoot chlorosis and drooping, whereas no major morphological changes were detected on R. graveolens EO-added Ppi and PpiBx in vitro cultures. Both EOs maintained the nematotoxicity during all experimental phases. R. graveolens EO proved to be an effective PWN antagonist to be further evaluated for pine wilt disease control, given its less phytotoxicity while maintaining nematoxicity.

Meloidogyne spp., commonly known as rootknot nematodes (RKNs), are economically important plant sedentary endoparasites that cause galls on susceptible hosts. The Columbia root-knot nematode (CRKN), M. chitwoodi, is a quarantine A2 type pest by the European and Mediterranean Plant Protection Organization since 1998. This nematode has been found associated with economically important crops such as potato and tomato, causing severe damage and making the agricultural products unacceptable for the fresh market and food processing. In vitro co-culture of host and parasite offers an advantageous experimental system for studying plant-RKN interactions. The structure, growth and production of volatiles of Solanum tuberosum hairy roots (HR) and of S. tuberosum HR/ CRKN co-cultures were compared. HR were induced by inoculation of aseptic potato tuber segments with Rhizobium rhizogenes. Co-cultures were initiated by inoculating HR with sterilized CRKN eggs. Infection with CRKN induced the RKN symptomatology in the HR and several nematode life stages were observed by light and scanning electron microscopy. Potato HR and HR/CRKN co-cultures exhibited similar growth patterns, evaluated by measuring fresh and dry weight and by the dissimilation method. Volatiles, isolated by distillation-extraction and analyzed by gas chromatography (GC) and gas chromatography coupled to mass spectrometry, revealed that palmitic acid (37-52 %), n-pentadecanal (10-16 %) and linoleic acid (2-16 %) were the main constitutive components of S. tuberosum HR, and of the HR/CRKN co-cultures (24-44, 8-22 and 4-18 %, respectively

Main conclusion Co-cultures of Pinus pinaster with Bursaphelenchus xylophilus were established as a biotechnological tool to evaluate the effect of nematotoxics addition in a host/parasite culture system.The pinewood nematode (PWN), Bursaphelenchus xylophilus, the causal agent of pine wilt disease (PWD), was detected for the first time in Europe in 1999 spreading throughout the pine forests in Portugal and recently in Spain. Plant in vitro cultures may be a useful experimental system to investigate the plant/nematode relationships in loco, thus avoiding the difficulties of field assays. In this study, Pinus pinaster in vitro cultures were established and compared to in vivo 1 year-old plantlets by analyzing shoot structure and volatiles production. In vitro co-cultures were established with the PWN and the effect of the phytoparasite on in vitro shoot structure, water content and volatiles production was evaluated. In vitro shoots showed similar structure and volatiles production to in vivo maritime pine plantlets. The first macroscopic symptoms of PWD were observed about 4 weeks after in vitro co-culture establishment. Nematode population in the culture medium increased and PWNs were detected in gaps of the callus tissue and in cavities developed from the degradation of cambial cells. In terms of volatiles main components, plantlets, P. pinaster cultures, and P. pinaster with B. xylophilus co-cultures were all b-and a-pinene rich. Cocultures may be an easy-to-handle biotechnological approach to study this pathology, envisioning the understanding of and finding ways to restrain this highly devastating nematode.

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As a nematotoxics screening biotechnological system, Solanum tuberosum hairy roots (StHR) and S. tuberosum hairy roots with Meloidogyne chitwoodi co-cultures (StHR/CRKN) were evaluated, with and without the addition of the essential oils (EOs) of Satureja montana and Ruta graveolens. EOs nematotoxic and phytotoxic effects were followed weekly by evaluating nematode population density in the co-cultures as well as growth and volatile profiles of both in vitro cultures types. Growth, measured by the dissimilation method and by fresh and dry weight determination, was inhibited after EO addition. Nematode population increased in control cultures, while in EO-added cultures numbers were kept stable. In addition to each of the EOs main components, and in vitro cultures constitutive volatiles, new volatiles were detected by gas chromatography and gas chromatography coupled to mass spectrometry in both culture types. StHR with CRKN co-cultures showed to be suitable for preliminary assessment of nematotoxic EOs.

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