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EARLY WARNING, RISK AREAS AND VECTOR MANAGEMENT OF THE PINE WOOD NEMATODE IN SPAIN III. Risk levels and susceptible areas for pine wilt disease in Castile and Leon. Aimed to an improved monitoring and sampling, it was decided to establish distinct risk levels for development of pine wilt disease and to determine those forest areas where these levels occurred. The program was applied to the Autonomous Community of Castile and Leon, using a geographical information system (GIS), where risk levels were based on two factors: 1- Species-specific susceptibilities of Pinus, based on the susceptibility for pine species after Evans et al. (1996): Highly susceptible: P. pinaster, P.sylvestris, P. nigra. Low to moderately susceptible: P. pinea , P. halepensis , P. radiata Stands were then classed in two types based on species composition and susceptibility: A- Stands having highly susceptible species as main or 2 nd order species. B- Stands having low to moderately susceptible species as main or 2 nd order species, including those with susceptible species in 3 rd or 4 nd order. 2- Mean July temperatures. A map was created showing the isotherms for July every 2ºC intervals, from <16ºC to >24ºC. Based on thermal estimations on disease development (Rutherford and Webster, 1987; Rutherford et al., 1990), four th ermal zones of risk were established in decreasing order, having mean July temperatures of 1:>22ºC; 2: 20 - 22ºC; 3: 18 - 20 ºC; 4: <18 ºC. Finally, six levels of risk for disease development were defined combining both parameters (Table 2). Besides, areas of higher probability of introduction of the pine wood nematode were delimited on these maps, considering as such those pine forest within 5 km of distance (maximum distance of dispersal for M. alternatus 3,3 km, Kobayashi et al., 1984) from every mill yard importing w ood and along the Portuguese border. Results showed than 31,4% of the more than one million ha of pine forests in Castile and Leon were classed as having high or very high risk for pine wilt disease development, if the nematode and the vector were present (Fig 4). Considering each of the pine species individually (Fig 5), P. pinaster resulted the most endangered (64,3% high or very high risk), followed by P. nigra (22,2%) and P. sylvestris (7%). However it should be noted that Scots pine presented moderate risk on 56,4% of 395.000 ha, due to these st ands occurring in the thermal zone between 18 and 20 ºC of mean July temperature. It must be stressed that a small increment of summer temperatures would greatly increase the risk for this species. Gerardo SANCHEZ PEÑA 1 , Juan A. PAJARES ALONSO 2 , Fernando IBEAS MILL ÁN 2 , Gema PÉREZ ESCOLAR 2 , Julio J. DÍEZ CASERO 2 , Mercedes FERNÁNDEZ FERNÁNDEZ 2 , Pablo MARTÍN PINTO 2 , Diego GALLEGO CAMBRONERO 2 1 Servicio de Protección Contra Agentes Nocivos, Dirección General para la Biodiversidad. Ministerio de Medio Ambiente. Gran Vía de San Francisco, 4. 28005 Madrid. Spain. [email protected] 2 Dpto. Producci ón Vegetal y Recursos Forestales. Escuela Técnica Superior de Ingenierías Agrarias, Universidad de Valladolid. Avd. Madrid, 44. 34004 Palencia. Spain. [email protected] Once M. galloprovincialis was confirmed as the vector of B. xylophilus in Portugal (Sousa et al., 2001) it was considered of great interest to develop an effective method to monitor and control this beetle. Many wood borers infecting dying conifers are known to be attracted by host odors. In Japan, M. alternatus resulted attracted to a blend of host monoterpenes, with ethanol increasing attraction (Ikeda et al., 1980; 1981), and turpentine or α-pinene, plus ethanol were attractive to North American M. carolinensis (Fatzinger, 1985) and M. scutellatus (Chénier and Philogène, 1989) respectively. Recent work (Allison et al., 2001; 2003) have confirmed earlier findings that several North American Monochamus species present a kairomonal response to some components of bark beetle (Ips spp.) aggregation pheromones. These semiochemicals resulted highly synergistic to α-pinene and ethanol in the attraction of M. clamator and M. scutellatus. Also, several trap designs have been tested for capturing large wood borers. It has been shown that traps with black silhouettes are more effective in capturing M. scutellatus than traps with clear vanes (De Groot and Nott, 2001). Multiple-funnel traps resulted less efficient that black cross- vane traps for capturing several North American Monochamus species (McIntosh et al., 2001) but when escape from the collection cup was avoided by killing the insects, they resulted similarly effective (Morewood et al, 2002). I.Introduction The pinewood nematode (PWN), Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle (Fig 1), is the causal agent of pine wilt disease, a serious threat that has caused extensive mortality of native pines in Japan and East Asia since 1900s, and it is vectored to new host trees by cerambycid beetles in the genus Monochamus (pine sawyer beetles) (Fig 2). Healthy trees are inoculated during adult maturation feeding in the shoots by the sawyers. Nematodes emerge from the insect vector and enter the trees through the wounds. Oviposition by Monochamus females later occurs on dead trees and larvae burrow under the bark and enter into the wood to built a pupal chamber. Nematodes aggregate around the chamber and move onto the new adult of the beetle, that will emerge carrying them to new host trees. Recent discovery of the PWN causing death of P. pinaster trees in Portugal (Mota et al., 1999) (Fig 3) has created great concern in Europe (quarantine organism). Pest risk assessments had concluded that the nematode would survive in Europe and tree mortality would likely be restricted to the warmer southern countries (Evans et al. 1996). Due to such a threat, in Spain, several efforts have been undertaken, including an early warning system and intensive sampling for nematode detection, determination of susceptible areas for disease occurrence and development of efficient tools for monitoring and control of the insect vector. Fig 2: Monochamusgalloprovincialis Fig 1: Bursaphelenchus xylophilus (Mamiya and Kiyohara , 1972) Fig 7: Catches of M. galloprovincialis in Sierra de la Culebra (Spain) to cross -vane (CRV) or to multiple-funnel (MF) traps baited with host blend (HB1 or HB2) alone or s upplemented with bark beetle blends (BB1 or BB2). Bars followed by the same letter are not significantly different (data log10(x+1) transformed, Tuckey’s test, p>0,05) IV. Trapping methods for Monochamus galloprovincialis Fig 8 : Catches of M. galloprovincialis in Sierra Espuña (Spain) to multiple- funnel traps baited with host blend alone (HB2) and/or with a single (BS) or a blend (BB3) of bark beetle pheromone components. Bars followed by the same letter are not significantly different (data log10(x+1) transformed, Tuckey’s test, p>0,05) Acknowledgements We thank J.M. Sierra, A. Martín (Centro de Sanidad Forestal de Calabazanos, Junta de Castilla y León) and J.D. Cabezas (Parque Regional de Sierra Espuña, Murcia) for access to field sites and to José Mª Cobos (Ministerio de Agricultura). Financial support was provided by a grant from Dirección General para la Biodiversidad (DGB), Ministerio de Medio Ambiente, Spain. References ALLISON, J.D.; BORDEN, J.H.; McINTOSH , R.L., GROTT, P.; GRIES, R.,2001. Kairomonalresponses by four Monochamus species ( Coleoptera: Cerambycidae) to bark beetle pheromones. Journal of Chemical Ecology, 27: 633-646 ALLISON, J., D.; MOREWOOD, W.D.; BORDEN, J. H.; HEIN, K.E. WILSON, I.M. 2003. Differential bio- activity of Ips and Dendroctonus (Coleoptera: Scolytidae) pheromone components for Monochamus clamator and M. scutellatus (Coleoptera: Cerambycidae). Environmental Entomology, 32 (1): 23-30. CHÉNIER, J. V., PHILOGÈNE, B.J. 1989. Field responses of certain forest Coleopterato conifer monoterpenes and ethanol. Journal of Chemical Ecology, 15: 1729-1745 EVANS, K. F., McNAMARA , D.G., BRAASCH, H., CHADOEUF, J., MAGNUSSON, C. 1996. Pest risk anayses (PRA) for the territories of the European Union (as PRA area) on Bursaphelenchus xylophilus and its vectors in the genus Monochamus. EPPO Bulletin 26: 199-249. FATZINGER, C. W. 1985. Attraction of the black turpentine beetle ( Coleoptera: Scolytidae) and other forest coleoptera to turpentine- baited traps. Environmental Entomology, 14: 768-775. GROOT, P. de; NOTT, R., 2001. Evaluation of traps of six different designs to capture pine sawyer beetles ( Coleoptera: Cerambycidae). Agricultural and Forest Entomology, 3 : 107-111. IKEDA, T., ENDA, N., YAMANE, A., ODA, K., TOYODA, T. 1980. Attractants for the Japanese pine sawyer, Monochamus alternatus Hope ( Coleoptera: Cerambycidae). Applied Entomology and Zoology, 15: 358-361. IKEDA, T. MIYAZAKI, M., ODA, K., YAMANE, A., ENDA, N. 1980. The chemical ecology of Monochamus alternatus Hope on the relationship whit pine wood nematodes and host tree. Proc. XVII IUFRO World Congress, Division II, pp. 297-303 MAMIYA, Y. KIYOHARA, T. 1972. Description of Bursaphelenchus lignicolus n.sp . (Nematoda: Aphelenchoididae) from pine wood and histopathology of nematode-infested trees. Nematologia,18: 120-124. McINTOSH , R. L., KATINIC, P. J., ALLISON, J. D., BORDEN, J.H., DOWNEY, D . L., 2001. Comparative efficacy of five types of trap for woodborers in the Cerambycidae, buprestidae and Siricidae. Agricultural and Forest Entomology, 3: 113-120. MOREWOOD, W.D. ; HEIN, K.E.; KATINIC, P.J.; BORDEN, J.H., 2002. An improved trap for large wood- boring insects, with special reference to Monochamus scutellatus (Coleoptera: Cerambycidae). Canadian Journal of Forest Research, 32: 519-525 MOTA, M.M., BRAASCH, H., BRAVO, M.A., PENAS, A.C., BURGERMEITER, W., METGE, K., SOUSA, E. 1999. Forest report of Bursaphelenchus xylophilus in Portugal and in Europe. Nematology, 1: 727-734. RUTHERFORD, T.A., WEBSTER, J.M. 1987. Distribution of pine wilt disease with respect to temperature in North America, Japan and Europe.Canadian Journal of Forest Research 17, 1050-1059 RUTHERFORD, T.A., MAMIYA, Y., WEBSTER, J.M. 1990.Nematode-induced pine wilt disease: factors influencing its occurrence and distribution. Forest Science, 36: 145-155 SOUSA, E., BRAVO, M.A., PIRES, J. NAVES, P., PENAS, A.C., BONIFACIO, L., MOTA. M.M. 2001. Bursaphelenchus xylophilus (Nematoda; Aphelenchoididae) associated with Monochamus galloprovincialis (Coleoptera: Cerambycidae) in Portugal. Nematology, 3: 89-91. 802 232 665 111 TOTAL 118 32 43 43 VALENCIA 46 19 18 9 PAIS VASCO 19 6 13 NAVARRA MURCIA 47 23 23 1 MADRID 19 7 12 LA RIOJA 363 20 310 33 GALICIA 120 60 60 EXTREMADURA 54 81 31 5 CATALU ÑA 76 19 57 CASTILLA LE ÓN 10 5 5 CASTILLA LA MANCHA 9 9 CANTABRIA 17 17 BALEARES 13 5 6 2 ASTURIAS 38 13 25 ARAGÓN 85 5 62 18 ANDALUCÍA Total P. pinaster stants Mill yards and other wood industries Entry areas (port, airport, etc.) Autonomous Communities NUMBER OF SAMPLES II. Surveys for presence of the nematode Table 2: Risk levels for disease development Fig 4: Risk of pine wilt disease development in Castile and León pine forests. Fig 5: Risk of disease development for each pine species Fig 6: MF and CRV traps An early warning system was soon adopted to detect the presence of B. xylophilus in Spain. Visual surveys were conducted annually from 1999 to 2003, following itineraries along the plots included in systematic grid of the ICP Forest program, in areas close to the Portuguese border (Fig 4). Also, the plots in the grid with occurrence of P. pinaster trees all over Spain were included. Surveys consisted of tree observation for acute symptoms of decline (wilting, dieback, etc) on P. pinaster trees. When a tree was found to show symptoms and these could not be assigned to any known causing agent (i.e. bark beetles), wood samples were then taken and sent to the laboratory for nematode analysis. Besides, intensive sampling was carried out within areas of higher risk of nematode introduction (entry ports and airports, mill yards, wood industries) and randomly in other P. pinaster forest. About 800 wood samples were collected and analyzed for nematodes annually (Table 1). All these surveys were coordinated by joint effort between the Forest Health Services in the Agriculture and Environment Ministries (DGB) and were carried out by the autonomous forest administration in the different regions. As result, several non-pathogenic Bursaphelenchus species have been detected, such as B. mucronatus, B. sexdentati and B. pinasteri, but no B. xylophilus has been found in any of the surveys and samplings so far conducted from 1999 to now. Fig 4 : Annual visual survey (1999- 2003) Table 1: Number of samples collected in each Autonomous Community (2003) 0 1 2 3 4 5 6 HB1/CRV HB1/MF HB1+BB1/CRV HB1+BB1/EM Treatment Beetles captured (Means+SE) a a b b 0 2 4 6 8 10 12 HB2/CRV HB2/MF HB2+BB2/CRV HB2+BB2/MF Treatment Female Male a a b b M. galloprovincialis captured Fig 3: P. pinaster trees showingsymptoms ofpinewilt disease in Portugal < 18 ºC A, B VI. Very low 20-22 ºC B V. Low > 22 ºC B IV. Moderate to low 18-20 ºC A III. Moderate 20-22 ºC A II. High > 22 ºC A I. Very high Thermal zone Stand type Risk level P. pinaster stands P. pinaster present P. pinaster absent Survey itinerary ANNUAL VISUAL SURVEY (1999-2003) Several field experiments were carried out to determine if M. galloprovincialis would also present a kairomonal response to bark beetle pheromones that could be used for monitoring and control, and to compare the effectiveness of black-panel cross-vane traps versus multiple-funnel traps in capturing this species.Two host volatile blends (HB) based on turpentine, α- pinene and ethanol, and three different blends of bark beetle semiochemicals (BB) were tested in complete randomized block designs (N=7), deployed whether in 80x30 black-panel cross -vane traps (CRV) or in 12-unit multiple-funnel traps (MF) (Fig 6). Collection cups were provided with a piece of DDVP insecticide to avoid escape of the trapped beetles. Results (Fig 7) showed that catches were significantly greater in traps baited with host blends (HB1 or HB2) plus bark beetles blends (BB1 or BB2) than in traps baited with host blends alone. The later stimuli resulted poorly attractive. No differences in catches were found between cross-vane and multiple-funnel traps, either when they were lured with host volatiles alone or when supplemented by bark beetle semiochemicals . This shows that both types of traps are equally effective in capturing M. galloprovincialis , providing the insects can not escape from the collecting receptacle. In another experiment (Fig 8) a net synergistic effect between h ost volatiles (HB2) an a bark beetle single pheromone component (BS) was clearly demonstrated. Traps releasing the BS alone trapped six times more beetles than those releasing the host blend alone, and when both stimuli were emitted together, catches were two times higher than the sum of individual catches. Notably, addition of a third blend of bark beetle semiochemicals (BB3) to host volatiles significantly raised the catches 3,6 ti mes compared to the BS plus HB bait. This very high number of catches (54 insect/trap in 46 days) is more remarkable considering than M. galloprovincialis population in the area was estimated of moderate to low levels.This result is very promising and might imply practical consequences in the management of this beetle. Operational monitoring and trapping of M. galloprovincialis could be already feasible using this attractive blend, though further improvement and refining of the lure is currently under study. M. galloprovincialis captured 0 10 20 30 40 50 60 70 HB2 BS HB2+BS HB2+BB3 Treatment Beetles captured (Mean+SE) Female Male a b c d
