In Vitro Co-Cultures of Pinus Pinaster with Bursaphelenchus Xylophilus: a Biotechnological Approach to Study Pine Wilt Disease

Planta (2015) 241:1325–1336 DOI 10.1007/s00425-015-2257-9 ORIGINAL ARTICLE In vitro co-cultures of Pinus pinaster with Bursaphelenchus xylophilus: a biotechnological approach to study pine wilt disease Jorge M. S. Faria • Ineˆs Sena • Ineˆs Vieira da Silva • Bruno Ribeiro • Pedro Barbosa • Lia Ascensaõ • Richard N. Bennett • Manuel Mota • A. Cristina Figueiredo Received: 17 November 2014 / Accepted: 31 January 2015 / Published online: 13 February 2015 Ó Springer-Verlag Berlin Heidelberg 2015 Abstract PWD were observed about 4 weeks after in vitro co-culture Main conclusion Co-cultures of Pinus pinaster with establishment. Nematode population in the culture medium Bursaphelenchus xylophilus were established as a increased and PWNs were detected in gaps of the callus biotechnological tool to evaluate the effect of nemato- tissue and in cavities developed from the degradation of toxics addition in a host/parasite culture system. cambial cells. In terms of volatiles main components, plantlets, P. pinaster cultures, and P. pinaster with B. xy- The pinewood nematode (PWN), Bursaphelenchus xylophilus co-cultures were all b- and a-pinene rich. Co- lophilus, the causal agent of pine wilt disease (PWD), was cultures may be an easy-to-handle biotechnological ap- detected for the first time in Europe in 1999 spreading proach to study this pathology, envisioning the under- throughout the pine forests in Portugal and recently in standing of and finding ways to restrain this highly Spain. Plant in vitro cultures may be a useful experimental devastating nematode. system to investigate the plant/nematode relationships in loco, thus avoiding the difficulties of field assays. In this Keywords Maritime pine Monoxenic culture Á Á study, Pinus pinaster in vitro cultures were established and Pinewood nematode Relative water content Shoots compared to in vivo 1 year-old plantlets by analyzing shoot structure Volatiles Á Á structure and volatiles production. In vitro co-cultures were Á established with the PWN and the effect of the phy- Abbreviations toparasite on in vitro shoot structure, water content and BAP 6-Benzylaminopurine volatiles production was evaluated. In vitro shoots showed DAI Days after inoculation similar structure and volatiles production to in vivo mar- EPPO European and Mediterranean Plant Protection itime pine plantlets. The first macroscopic symptoms of Organization J. M. S. Faria I. Sena I. Vieira da Silva L. Ascensaõ B. Ribeiro P. Barbosa M. Mota A. C. FigueiredoÁ (&) Á Á Á NemaLab/ICAAM,Á InstitutoÁ de Cieˆncias Agra´rias e Ambientais Universidade de Lisboa, Faculdade de Cieˆncias de Lisboa, DBV, Mediterraˆnicas, Universidade de E´ vora, Nućleo da Mitra, CESAM, Centro de Biotecnologia Vegetal, C2, Campo Grande, Ap. 94, 7002-554 E´ vora, Portugal 1749-016 Lisbon, Portugal e-mail: [email protected] e-mail: [email protected] P. Barbosa J. M. S. Faria e-mail: [email protected] e-mail: [email protected] M. Mota I. Sena e-mail: [email protected] e-mail: [email protected] R. N. Bennett I. Vieira da Silva Universidade de Tra´s-os-Montes e Alto Douro, Quinta dos e-mail: [email protected] Prados, Apartado 1013, 5000-911 Vila Real, Portugal L. Ascensaõ e-mail: [email protected] e-mail: [email protected] 123 1326 Planta (2015) 241:1325–1336 GC Gas chromatography Once inside the host plant, the nematodes reproduce and GC–MS Gas chromatography coupled to mass spectrometry multiply at a very high rate in the resin canals, consuming IBA Indole-3-butyric acid the epithelial cells (phytophagous phase), thus damaging LM Light microscopy internal pine structure. As infection progresses, embolized PAS Periodic acid–Schiff’s reagent tracheids rapidly enlarge and water potential decreases PWD Pine wilt disease ultimately leading to abrupt cavitation in the whole xylem PWN Pinewood nematode (Bursaphelenchus area (Umebayashi et al. 2011). At this stage, cavitation xylophilus) effects appear to be promoted by increase in production of RI Retention index terpenes by ethylene cues (Wang et al. 2010). RWC Relative water content As the tree very quickly begins displaying the charac- SEM Scanning electron microscopy teristic wilting symptoms, ‘‘drying out’’ and yellowing of SH Schenk and Hildebrandt medium the pine needles, the oleoresin exudation decreases and as a SHe Schenk and Hildebrandt elongation medium consequence nematodes are able to move freely through SHm Schenk and Hildebrandt multiplication medium the dying tree (Ikeda and Oda 1980; Kuroda 2008). t Trace Although stem anatomy is thought to be linked to varia- tions in pine susceptibility, for e.g. the arrangement of the resin canals (Kuroda 2004) or lignification of infected pine Introduction cell walls (Kusumoto et al. 2014), it is not yet established which anatomy characteristics influence PWN progression. The pine wilt disease (PWD) is caused by the pinewood The trees showing intensified wilting and yellowing of the nematode (PWN), Bursaphelenchus xylophilus (Steiner & needles may collapse within 1–4 months (EPPO 2012). Buhrer) Nickle, which is a highly pathogenic, migratory, The decaying trees are hosts to the oviposition of female facultative endoparasite which generally infects some Pi- beetles and the remaining life cycle progresses as described nus species. In Portugal, maritime pine, Pinus pinaster above (Mota and Vieira 2008). Aiton, is highly susceptible to infection. In 1999, the ne- The effect of nematotoxic compounds on this phy- matode was detected in Portugal (Mota et al. 1999) en- toparasite has been well-documented, mainly using direct dangering European pine forests and has progressed contact bioassays (Choi et al. 2007; Barbosa et al. 2010, throughout large areas of the country (Mota and Vieira 2012; Andre´s et al. 2012; Faria et al. 2013). However, 2008). In 2010, it was also found in Madeira island (Fon- research is commonly performed on the nematode species seca et al. 2012), and in 2011 for the first time in Spain alone and very seldom on the host–parasite system, not (Abelleira et al. 2011). It was classified as an A2 type taking into account the cytotoxicity to the plant host or the quarantine pest by the European and Mediterranean Plant plant’s capability to metabolize or biotransform the ne- Protection Organization (EPPO 2012). matotoxic active substances. The PWN dispersal and life cycle are dependent on By co-culturing host and parasite at the same time, vectors, cerambycid Monochamus spp., that include M. simulating the host-pathogen conditions, in vitro culture alternatus in East Asia, M. saltuarius in Japan, M. caro- can be a useful system to study plant/nematode interac- linensis in North America and M. galloprovincialis, tions, since it allows (a) eliminating variables due to en- abundant in the Portuguese pine forest (Mota and Vieira vironmental conditions, (b) having a contaminant-free 2008, Petersen-Silva et al. 2014). After feeding on the system, which, by being in a monoxenic culture, excludes fungus growing on dead or decaying wood (mycophagous the diverse-associated microbiota (Amerson and Mott phase), the nematodes molt into dispersal ‘‘third-stage 1982; Vicente et al. 2012), (c) manipulating single vari- dauer juvenile’’, JIII, able to outstand adverse conditions. ables, making possible the direct observation of plant/ne- Gathering around the developing insect, ‘‘fourth-stage matode responses in a controlled environment, which is dauer juvenile’’ (JIV) enters the tracheal system of the very difficult to achieve in greenhouse or in field condi- emerging young callow adult through its spiracles. In- tions, and also (d) attaining more biomass using fewer fection of susceptible Pinus spp. occurs in the dispersal resources. phase when adult beetles transmit the JIV to other trees The present study aimed at developing a reliable hos- while feeding on young tree branches (Futai 2013). At this t/pathogen system for PWD phytopathological research. To stage, PWNs are attracted to pine volatile cues that seem accomplish this, in vitro P. pinaster and in vitro P. pina- to determine changes in their development, particularly ster/B. xylophilus co-cultures were established. PWN major terpenes ratio (Zhao et al. 2007) and/or b-myrcene density in the co-culture medium was followed as well as content, as well as internal PWN neutral lipid energy in vitro pine relative water content. Healthy 1-year-old reserves (Stamps and Linit 2001). plantlets, pine in vitro cultures and pine/PWN co-culture 123 Planta (2015) 241:1325–1336 1327 structure and volatile production were also determined. The were maintained as described above and subculture was present work proposes maritime pine/PWN co-cultures as performed monthly. Elongation rate was followed monthly an adequate biotechnological tool to study the PWD, ca- by measuring individual shoot length, for 32 months. A pable of simulating many conditions of the ex vitro ne- minimum of 30 in vitro shoots were measured per month. matode infection. The data were statistically analyzed using Microsoft Excel 2013. Materials and methods Pinus pinaster with Bursaphelenchus xylophilus co- cultures (co-cultures) In vitro cultures establishment Bursaphelenchus xylophilus (isolate BxPt51T, retained at Pinus pinaster cultures (shoots) NemaLab and available on request) was obtained as described by Faria et al. (2013). Surface sterilization was Seeds from maritime pine trees grown at Mata Nacional do performed in aliquots of 500 ll, with 3250 ± 250 mixed- Escaroupim, Portugal, were washed with running tap water stage PWNs in ultrapure water. In asepsis, nematodes were for 5 min, then immersed in a commercial detergent (sur- suspended in a 50 % ethanol/ultrapure sterile water solu- factants: anionic C15 and \30 %, non-ionic C5 and tion (v/v) (20 ml), for 5 min in a 20 lm mesh sieve, and \15 %, disinfectant: triclosan 0.1 %) solution (10 drops then washed 5 times in ultrapure sterile water, 20 ml each, per 100 ml of distilled water) for 10 min and dipped in an resuspended in 1 ml sterile water.

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