Battisti, A.

Chinellato, F.

Faccoli, M.

Hardwick, S.

Simonato, M.

Suckling, D. M.

English

JKI Open Journal Systems (Julius Kühn-Institut)

  IUFRO / REPHRAME International Conference on Pine Wilt Disease 2013  Berichte aus dem Julius Kühn-Institut 169. 2013 23 Chinellato F et al., Smart-traps combined with molecular on-site detection to monitor Monochamus spp. and associated pine wood nematode. In: Schröder, T. (ed.), Pine Wilt Disease Conference 2013, pp. 23-25, Braunschweig, ISSN: 1866-590X Smart-traps combined with molecular on-site detection to monitor Monochamus spp. and associated pine wood nematode Chinellato F.11 Department of Agronomy, Food, Natural resources, Animals and Environment, University of Padova, V.le dell’Università, 16 – 35020 Legnaro (PD), Italy. , Simonato M.1, Battisti A.1, Faccoli M.1, Hardwick S.2, Suckling D.M.3 2 AgResearch Limited, Lincoln, New Zealand. 3 The New Zealand Institute for Plant and Food Research Limited, Lincoln, New Zealand. Email: fabio.chinellato@studenti.unipd.it INTRODUCTION   The pine sawyer beetles Monochamus spp. (Coleoptera Cerambycidae) are the main vectors of the Pine Wood Nematode (PWN), Bursaphelenchus xylophilus, the agent of pine wilt disease in various parts of the world (Mamiya 1983). In Europe, M. galloprovincialis (Olivier) gained importance as a vector after the finding of the PWN in Portugal in 1999 (Sousa et al. 2001). An effective monitoring method based on early detection of both vector insects and associated nematode is needed in order to adopt appropriate phytosanitary measures (Rassati et al. 2012 and 2013).  MATERIALS AND METHODS  The present study shows a new technology for the remote detection of beetle catch combined with on-site molecular detection of both vector and nematode identity. A multi-funnel trap, baited with either specific or generic blend, and equipped with a specifically modified security camera (BioCam, Mi5 Security, Auckland, New Zealand), composed by a wide-angle lens, 1 or 3 MegaPixel sensor, rechargeable battery pack and internal modem for General Packet Radio Service (GPRS) connection was used. The interval between images taken by the camera can be programmed and saved in a Secure Digital (SD) memory card. The images can be stored in the same SD card and simultaneously sent to a safe repository accessible through the web, from which they are downloadable. On the same repository it is possible to check the level of battery charge of each camera and the GPRS coverage as well. When a target beetle is detected, an on-site visit is planned, during which a fragment of the thorax is analyzed using a Loop Mediated Isothermal Amplification (LAMP) portable   IUFRO / REPHRAME International Conference on Pine Wilt Disease 2013  Berichte aus dem Julius Kühn-Institut 169. 2013 24 device (Genie II, Optigene, UK) to identify the trapped species of Monochamus spp. and to detect the PWN possibly vectored by the beetles. Currently, primers were deloped for the endemic M. galloprovincialis and M. sutor, and for the exotic M. alternatus and M. carolinensis. For the beetles identified as Monochamus, presence of PWN is also tested with the same device using slighty modified LAMP primers from Kikuchi et al. (2009), specific for the nematode ITS1 region. A positive control for the nematode is included in the test. The technique allows amplifying target DNA in a few minutes visualizing the results immediately. RESULTS Images obtained by cameras are definitely adequate to visually recognize large longhorn beetles such as Monochamus spp.. All the main morphological traits of the species are detectable (Fig. 1). The system works also under sub-optimal light conditions. LAMP primers designed to amplify the ITS2 region of M. galloprovincialis, M. sutor, M. alternatus and M. carolinensis show to be specific, giving a positive result only for these species after 10-15 minutes after the test start (Fig. 2). On the other hand, no positive insects for PWN have been detected until now. CONCLUSIONS Both technologies are designed for quick and cheap on-site analyses, and can be used by non-expert staff with a short training. In case of positive samples, they must be taken to the laboratory and analyzed more accurately with standard protocols for official confirmation. REFERENCES Kikuchi T; Aikawa T; Oeda Y, Karim N, Kanzaki N (2009) A rapid and precise diagnostic method for detecting the pinewood nematode Bursaphelenchus xylophilus by Loop-Mediated Isothermal Amplification. Phytopathology 99, 1365-1369. Mamiya Y (1983). Pathology of the pine wilt desease caused by Bursaphelenchus xylophilus. Annual Review of Phytopathology 21, 201-220. Rassati D; Petrucco Toffolo E; Battisti A, Faccoli M (2012). Monitoring of the pine sawyer beetle Monochamus galloprovincialis by pheromone traps in Italy. Phytoparasitica 40, 329-336. Rassati D; Petrucco Toffolo E; Roques A; Battisti A; Faccoli M (2013) Trapping wood boring beetles in Italian ports: a pilot study. Journal of Pest Science DOI 10.1007/s10340-013-0499 Sousa E; Bravo MA; Pires J; Naves P; Penas AC; Bonifacio L; Mota MM (2001). Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae) associated with Monochamus galloprovincialis (Coleoptera: Cerambycidae) in Portugal. Nematology 3, 89-91.   IUFRO / REPHRAME International Conference on Pine Wilt Disease 2013  Berichte aus dem Julius Kühn-Institut 169. 2013 25   Figure 1  Picture taken by 3MP trap camera. One individual of Monochamus spp. is clearly recognizable on the left, together with several individuals of the longhorn beetle Acanthocinus griseus, one of the western seed bug Leptoglossus occidentalis (above) and several small bark beetles (right).    Figure 2.  Amplification profile for the LAMP assay carried on Monochamus spp. and other cerambycid beetles. Positive curves are obtained in 10-15 minutes only for M. galloprovincialis, M. sutor, M. alternatus and M. carolinensis. 

Smart-traps combined with molecular on-site detection to monitor Monochamus spp. and associated pine wood nematode

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Smart-traps combined with molecular on-site detection to monitor Monochamus spp. and associated pine wood nematode

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