Base de dados e chaves electrónicas (ekeys): ferramentas úteis no diagnóstico e identificação de nemátodes fitoparasitas

Com o desenvolvimento das tecnologias de computação, o acesso à informação digital nos diversos domínios científicos tem revolucionado e facilitado a obtenção e utilização rápida de dados biológicos. Nos últimos anos, várias bases de dados (catálogos electrónicos) têm sido desenvolvidas, servindo de portal à biodiversidade existente em diversas áreas do globo (ex. Fauna Europaea, Tree of Life). Este tipo de informação permite a manutenção e a catalogação da biodiversidade existente a nível global; particularmente a biodiversidade de florestas, áreas cultivadas, ecossistemas aquáticos, bem como de espécies exóticas/invasivas encontradas nos diversos ecossistemas. Em 1999, foi detectada pela primeira vez, em Portugal e na Europa, o nemátode da madeira do pinheiro (Bursaphelenchus xylophilus, espécie de quarentena e classificada como A1 pela EPPO), associado ao pinheiro bravo (Pinus pinaster). Na sequência deste trabalho foram desenvolvidas diversas bases de dados, nomeadamente para as espécies do género Bursaphelenchus e outros géneros com espécies de elevado interesse económico (Aphelenchoides, Dolichodorus); bem como para outros grupos de espécies com especial interesse biológico (Laimaphelenchus, Parasitaphelenchus). A reunião deste tipo de informação possibilita a construção de matrizes, e a determinação dos caracteres diagnósticos mais importantes para a identificação das espécies, posteriormente utilizados na elaboração de chaves electrónicas politómicas (com base no sistema PICKEY, Dianov & Lobanov). A utilização e versatilidade deste sistema, nomeadamente para o diagnóstico e a identificação de nemátodes fitoparasitas de elevado interesse económico, é demonstrada como exemplo

The Pine Wood Nematode: a personal view

The first report of the disease (“pine wilt disease”) associated with the pinewood nematode, goes back to 1905, when Yano reported an unusual decline of pines from Nagasaki. For a long time thereafter, the cause of he disease was sought, but without success. Because of the large number of insect species that were usually seen around and on infected trees, it had always been assumed that the causal agent would prove to be one of these. However, in 1971, Kiyohara and Tokushike found a nematode of the genus Bursaphelenchus in infected trees. The nematode found was multiplied on fungal culture, inoculated into healthy trees and then re-isolated from the resulting wilted trees. The subsequent published reports were impressive: this Bursaphelenchus species could kill fully-grown trees within a few months in the warmer areas of Japan, and could destroy complete forests of susceptible pine species within a few years. Pinus densiflora, P. thunbergii und P. luchuensis were particularly affected. In 1972, Mamiya and Kiyohara described the new species of nematode extracted from the wood of diseased pines; it was a named Bursaphelenchus lignicolus. Since 1975, the species has spread to the north of Japan, with the exception of the most northerly prefectures. In 1977, the loss of wood in the west of the country reached 80%. Probably as a result of unusually high summer temperatures and reduced rainfall in the years 1978 and 1979, the losses were more than 2 million m3 per year. From the beginning, B. lignicolus was always considered by Japanese scientists to be an exotic pest. But where did it come from? That this nematode could also cause damage in the USA became clear in 1979 when B. lignicolus was isolated in great numbers from wood of a 39 year-old pine tree (Pinus nigra) in Missouri which had suddenly died after the colour of its needles changed to a reddish-brown colour (Dropkin und Foudin, 2 1979). In 1981, B. lignicolus was synonymised by Nickle et al. with B. xylophilus which had been found for the first time in the USA as far back as 1929, and reported by Steiner and Buhrer in 1934. It had originally been named Aphelenchoides xylophilus, the wood-inhabiting Aphelenchoides but was recognised by Nickle, in 1970,to belong in the genus Bursaphelenchus. Its common name in the USA was the "pine wood nematode" (PWN. After its detection in Missouri, it became known that B. xylophilus was widespread throughout the USA and Canada. It occurred there on native species of conifers where, as a rule, it did not show the symptoms of pine wilt disease unless susceptible species were stressed eg., by high temperature. This fact was an illuminating piece of evidence that North America could be the homeland of PWN. Dwinell (1993) later reported the presence of B. xylophilus in Mexico. The main vector of the PWN in Japan was shown to be the long-horned beetle Monochamus alternatus, belonging to the family Cerambycidae. This beetle lays its eggs in dead or dying trees where the developing larvae then feed in the cambium layer. It was already known in Japan in the 19th century but in the 1930s, it was said to be present in most areas of Japan, but was generally uncommon. However, with the spread of the pine wilt disease, and the resulting increase of weakened trees that could act as breeding sites for beetles, the populations of Monochamus spp. increased significantly In North America, other Monochamus species transmit PWN, and the main vector is M. carolinensis. In Japan, there are also other, less efficient vectors in the genus Monochamus. Possibly, all Monochamus species that breed in conifers can transmit the PWN. The occasional transmission by less efficient species of Monochamus or by some of the many other beetle genera in the bark or wood is of little significance. In Europe, M. galloprovincialis and M. sutor transmits the closely related species B. mucronatus. Some speculate that these two insect species are “standing by” and waiting for the arrival of B. xylophilus. In 1982, the nematode was detected and China. It was first found in dead pines near the Zhongshan Monument of Nanjing (CHENG et. al. 1983); 265 trees were then killed by pine wilt disease. Despite great efforts at eradication in China, the nematode spread further and pine wilt disease has been 3 reported from parts of the provinces of Jiangsu, Anhui, Guangdong, Shandong, Zhejiang and Hubei (YANG, 2003). In 1986, the spread of the PWN to Taiwan was discovered and in 1989, the nematode was reported to be present in the Republic of Korea where it had first been detected in Pinus thunbergii and P. densiflora. It was though to have been introduced with packing material from Japan. PWN was advancing. In 1984, B. xylophilus was found in wood chips imported into Finland from the USA and Canada, and this was the impetus to establish phytosanitary measures to prevent any possible spread into Europe. Finland prohibited the import of coniferous wood chips from these sources, and the other Nordic countries soon followed suit. EPPO (the European and Mediterranean Plant Protection Organization) made a recommendation to its member countries in 1986 to refuse wood imports from infested countries. With its Directive of 1989 (77/93 EEC), the European Community (later called the European Union or EU) recognised the potential danger of B. xylophilus for European forests and imposed restrictions on imports into the Europe. PWN was placed on the quarantine list of the EU and also of other European countries. Later, in 1991, a dispensation was allowed by the Commission of the EU(92/13 EEC) for coniferous wood from North America provided that certain specified requirements were fulfilled that would prevent introduction.

Pine wilt disease: global issues, trade and economic impact

Pine wilt disease (PWD) is perhaps the most serious threat to pine forests worldwide. Since it´s discovery in the early XXth century by Japanese forest researchers, and the relationship with its causative agent, the pinewood nematode (PWN) Bursaphelenchus xylophilus, in the 1970s, PWD has wreaked havoc wherever it appears. Firstly in the Far East (Japan, China and Korea) and now, more recently in 1999, in the EU (Portugal). The forest sector in Portugal plays a major role in the Portuguese economy with a 12% contribution to the industrial gross domestic product, 3.2% of the gross domestic product, 10% of foreign trade and 5% of national employment. Maritime pine (Pinus pinaster) is one of the most important pine productions, and industrial activity, such as the production of wood and resin, as well as coastal protection associated with sand dunes. Also, stone pine (Pinus pinea) plays an important role in the economy with a share derived from the exports of high-quality pineon seed. Thus, the tremendous economical and ecological impact of the introduction of a pest and pathogen such as the PWN, although as far as is known, the only species susceptible to the nematode is maritime pine. Immediately following detection, the research team involved (Univ. Évora, INIAP) informed the national plant quarantine and forest authorities, which relayed the information to Brussels and the appropriate EU authorities. A task force (GANP), followed by a national program (PROLUNP) was established. Since then, national surveys have been taking place, involving MADRP (Ministry of Agriculture), the University of Évora and several private corporations (e.g. UNAC). Forest growers in the area are particularly interested and involved since the area owned by the growers organizations totals 700 000 ha, largely affected by PWD. Detection of the disease has led to serious consequences and restrictions regarding exploration and commercialization of wood. A precautionary phytosanitary strip, 3 km-wide, has been recently (2007) established surrounding the affected area. The Portuguese government, through its national program PROLUNP, has been deeply involved since 1999, and in conjunction with the EU (Permanent Phytosanitary Committee, and FVO) and committed to controlling this nematode and the potential spread to the rest of the country and to the rest of the EU. The global impact of the presence of Bursaphelenchus xylophilus or the threat of its introduction and the resulting pine wilt disease in forested areas in different parts of the world is of increasing concern economically. The concern is exacerbated by the prevailing debate on climate change and the putative impact this could have on the vulnerability of the world’s pine forests to this disease. The scientific and regulatory approach taken in different jurisdictions to the threat of pine wilt disease varies from country to country depending on the perceived vulnerability of their pine forests to the disease and/or to the economic cost due to lost trade in wood products. Much of the research surrounding pine wilt disease has been located in the northern hemisphere, especially in southern Europe and in the warmer, coastal, Asian countries. However, there is an increased focus on this problem also in those countries in the southern hemisphere where plantations of susceptible pine have been established over the years. The forestry sector in Australia and New Zealand are on “high alert” for this disease and are practicing strict quarantine procedures at all ports of entry for wood products. As well, there is heightened awareness, as there is worldwide, for the need to monitor wood packaging materials for all imported goods. In carrying out the necessary monitoring and assessment of products for B. xylophilus and its vectors substantial costs are incurred especially when decisions have to be made rapidly and regardless of whether the outcome is positive or negative. Australia’s response recently to the appearance of some dying pines in a plantation illustrated the high sensitivity of some countries to this disease. Some $200,000 was spent on the assessment in order to save a potential loss of millions of dollars to the disease. This rapid, co-ordinated response to the report was for naught, because once identified it was found not to be B. xylophilus. This illustrates the particular importance of taking the responsibility at all levels of management to secure the site and the need of a rapid, reliable diagnostic method for small nematode samples for use in the field. Australia is particularly concerned about the vulnerability of its 1million hectares of planted forests, 80% of which are Pinus species, to attack from incursions of one or more species of the insect vector. Monochamus alternatus incursions in wood pallets have been reported from Brisbane, Queensland. The climate of this part of Australia is such that the Pinus plantations are particularly vulnerable to the potential outcome of such incursions, and the state of Queensland is developing a risk management strategy and a proactive breeding programme in response to this putative threat. New Zealand has 1.6 million hectares of planted forests and 89% of the commercial forest is Pinus radiata. Although the climate where these forests are located tends to be somewhat cooler than that in Australia the potential for establishment and development of the disease in that country is believed to be high. The passage alone of 200,000 m³/year of wood packaging through New Zealand ports is itself sufficient to require response. The potential incursion of insect vectors of pinewood nematode through the port system is regarded as high and is monitored carefully. The enormous expansion of global trade and the continued use of unprocessed/inadequately-processed wood for packaging purposes is a challenge for all trading nations as such wood packaging material often harbours disease or pest species. The extent of this problem is readily illustrated by the expanding economies and exports of countries in south-east Asia. China. Japan and Korea have significant areas of forestland infested with B. xylophilus. These countries too are among the largest exporting countries of manufactured goods. Despite the attempts of authorities to ensure that only properly treated wood is used in the crating and packaging of goods B. xylophilus and/or its insect vector infested materials is being recorded at ports worldwide. This reminds us, therefore, of the ease with which this nematode pest can gain access to forest lands in new geographic locations through inappropriate use, treatment or monitoring of wood products. It especially highlights the necessity to find an alternative to using low-grade lumber for packaging purposes. Lest we should believe that all wood products are always carriers of B. xylophilus and its vectors, it should be remembered that international trade of all kinds has occurred for thousands of years and that lumber-born pests and diseases do not have worldwide distribution. Other physico-biological factors have a significant role in the occurrence, establishment and sustainability of a disease. The question is often raised as to why the whole of southern Europe doesn’t already have B. xylophilus and pine wilt disease. European countries have traded with countries that are infested with B. xylophilus for hundreds of years. Turkey is an example of a country that appears to be highly vulnerable to pine wilt disease due to its extensive forests in the warm, southern region where the vector, Monochamus galloprovincialis, occurs. However, there is no record of the presence of B. xylophilus occurring there despite the importation of substantial quantities of wood from several countries In many respects, Portugal illustrates both the challenge and the dilemma. In recent times B. xylophilus was discovered there in the warm coastal region. The research, administrative and quarantine authorities responded rapidly and B. xylophilus appears to have been confined to the region in which it was found. The rapid response would seem to have “saved the day” for Portugal. Nevertheless, it raises again the long-standing questions, how long had B. xylophilus been in Portugal before it was found? If Lisbon was the port of entry, which seems very likely, why had B. xylophilus not entered Lisbon many years earlier and established populations and the pine wilt disease? Will the infestation in Portugal be sustainable and will it spread or will it die out within a few years? We still do not have sufficient understanding of the biology of this pest to know the answers to these questions.

Pine Wilt Disease: A Threat to Pine Forests in Turkey?

The pinewood nematode, is the causal agent of pine wilt disease, a serious threat to native pine forest in eastern Asia (Japan, Korea, China and Taiwan) and some parts of North America (USA, Canada and Mexico). In 1999, this nematode was found and identified for the first time in Portugal and in Europe. The detection of this quarantine pest in Portugal has indicated the need to know more about the distribution of Bursaphelenchus spp. in coniferous trees in Europe in order to describe the geographic range of the species and to act quickly in case of the nematode’s unwanted introduction into other European regions. Pine forest has a wide distribution in Turkey that increases the number of susceptible host trees for pinewood nematode. Because of these resaons, some regions of Turkey were surveyed for the presence of the nematode. Three different species of Bursaphelenchus were found. However, B. xylophilus was not detected. The detection of B. mucronatus, very similar to B. xylophilus biologically and morphologically, is very important. The presence of this species indicates that B. xylophilus could spread easly in conifer forests of Turkey. A study was conducted to determine the pathogenicity of B. mucronatus and 80% of seedlings of P. sylvestris were wilted. Biological characteristics of M. galloprovincialis were compared with M. carolinensis, Nort American vector, and some of them were found to be similar.

Developmental biology and cytogenetics of B. xylophilus

The pine wood nematode Bursaphelenchus xylophilus reproduces bisexually: a haploid sperm fertilizes a haploid oocyte, and the two pronuclei rearrange, move together, fuse, and begin diploid development. Early embryonic events taking place in the B. xylophilus embryo are similar to those of Caenorhabditis elegans, although the anterior-posterior axis appeares to be determined oppositely to that observed for C. elegans. Thai is, in the B. xylophilus embryo, the male pronucleus emerges at the future anterior end, whereas the female pronucleus appeares laterally. To understand the evolution of nematode developmental systems, we cloned the full length of Bx-tbb-1 (beta tubulin) from B. xylophilus cDNA and attempted to apply reverse genetics analysis to B. xylophilus. Several lengths of double stranded RNA (dsRNA) for the Bx-tbb-1 gene were synthesized by in vitro transcription, and both B. xylophilus and C. elegans were soaked in dsRNA for RNAi. Both nematodes could suck up the dsRNA, and we could detect the abnormal phenotypes caused by Bx-tbb-1 dsRNA in C. elegans, but not in B. xylophilus. We suspect that systemic RNAi might be suppressed in B. xylophilus and are attempting to establish other methods for functionally analyzing B. xylophilus genes.

Pine wilt disease and the pinewood nematode

Pine wilt disease (PWD) is one of the most damaging events affecting conifer forests (in particular Pinus spp.), in the Far East (Japan, China and Korea), North America (USA and Canada) and, more recently, in the European Union (Portugal). In Japan it became catastrophic, damaging native pine species (Pinus thunbergii and P. densiflora), and becoming the main forest problem, forcing some areas to be totally replaced by other tree species. The pine wilt nematode (PWN) Bursaphelenchus xylophilus, endemic, with minor damage, to North America, was introduced in Japan in the early XX century and then spread to Asia (China and Korea) in the 1980s. In 1999 it was detected for the first time in Portugal, where, due to timely detection and immediate government action, it was initially (1999-2008) contained to a small area 30 km SE of Lisbon. In 2008, the PWN spread again to central Portugal, the entire country now being classified as “affected area”. Being an A1 quarantine pest, the EU acted to avoid further PWN spreading and to eradicate it, by actions including financial support for surveyes and eradication, annual inspections and research programs. Experience from control actions in Japan included aerial spraying of insecticides to control the insect vector (the Cerambycid beetle Monochamus alternatus), injection of nematicides to the trunk of infected trees, slashing and burning of large areas out of control, beetle traps, biological control and tree breeding programs. These actions allowed some positive results, but also unsuccessful cases due to the PWN spread and virulence. Other Asian countries also followed similar strategies, but the nematode is still spreading in many regions. In Portugal, despite lower damage than Asia, PWD is still significant with high losses to the forestry industry. New ways of containing PWD include preventing movement of contaminated wood, cutting symptomatic trees and monitoring. Despite a national and EU legislative body, no successful strategy to control and eventually eradicate the nematode and the disease will prevail without sound scientific studies regarding the nematode and vector(s) bioecology and genetics, the ecology and ecophysiology of the pine tree species, P. pinaster and P. pinea , as well as the genomics and proteomics of pathogenicity (resistance/ susceptibility).

Molecular and biotechnological approaches to essential oils production in thymus caespititius

Tese de doutoramento, Biologia (Biotecnologia), Universidade de Lisboa, Faculdade de Ciências, 2014

Essential oils as anti-nematode agents and their influence on in vitro nematode

Tese de doutoramento, Biologia (Biotecnologia), Universidade de Lisboa, Faculdade de Ciências, 2015

Fenología y capacidad de dispersión de Monochamus galloprovincialis (Olivier 1795) en la Península Ibérica = Phenology and dispersal ability of Monochamus galloprovincialis (Olivier 1795) at Iberian Peninsula

La aparición y avance de la enfermedad del marchitamiento del pino (Pine Wilt Desease, PWD), causada por Bursaphelenchus xylophilus (Nematoda; Aphelenchoididae), el nematodo de la madera del pino (NMP), en el suroeste de Europa, ha puesto de manifiesto la necesidad de estudiar la fenología y la dispersión de su único vector conocido en Europa, Monochamus galloprovincialis (Col., Cerambycidae). El análisis de 12 series de emergencias entre 2010 y 2014, registradas en Palencia, València y Teruel, con material procedente de diversos puntos de la península ibérica, demostró una alta variabilidad en la fenología de M. galloprovincialis y la divergencia térmica respecto de las poblaciones portuguesas. Para éstas, el establecimiento de los umbrales térmicos de desarrollo de las larvas post-dormantes del vector (12,2 y 33,5ºC) permitió la predicción de la emergencia mediana para la fecha en la que se acumulaban de 822 grados-día. Ninguna de las series analizadas en este trabajo necesitó de dichos grados-día estimados para la emergencia mediana. Asimismo, la emergencia se adelantó en las regiones más calurosas, mientras que se retrasó en las zonas más templadas. Más allá de la posible variabilidad entre poblaciones locales peninsulares, se detectaron indicios de que la diferencia en la acumulación de calor durante el otoño puede afectar el grado de maduración de las larvas invernantes, y su posterior patrón temporal de emergencia. Por último, también fueron observados comportamientos de protandria en las emergencias. Respecto a la fenología de su vuelo, entre los años 2010 y 2015, fueron ejecutados un total de 8 experimentos de captura de M. galloprovincialis mediante trampas cebadas con atrayentes en diferentes regiones (Castellón, Teruel, Segovia y Alicante) permitiendo el seguimiento del periodo de vuelo. Su análisis permitió constatar la disminución de las capturas y el acortamiento del periodo de vuelo con la altitud, el inicio del vuelo en el mes de mayo/junio a partir de los 14ºC de temperatura media diaria, la influencia de las altas temperaturas en la disminución de las capturas estivales (potencial causante de perfiles bimodales en las curvas de vuelo en las zonas menos frías), la evolución de la proporción de sexos a lo largo del periodo de vuelo (que muestra una mayor captura de hembras al inicio y de machos al final) y el comportamiento diurno y ligado a las altas temperaturas del vuelo circadiano del insecto. Dos redes de muestreo sistemático de insectos saproxílicos instaladas en la Comunitat Valencia (Red MUFFET, 15 parcelas, año 2013) y en Murcia (Red ESFP, 20 parcelas, años 2008-2010) permitieron el estudio de la comunidad de insectos relacionada con M. galloprovincialis. Cada una de las parcelas contaba con una trampa cebada con atrayentes y una estación meteorológica. El registro de más de 250 especies de coleópteros saproxílicos demostró el potencial que tiene el empleo de redes de trampas vigía para la detección temprana de organismos exóticos, además de permitir la caracterización y evaluación de las comunidades de entomofauna útil, representando una de las mejores herramientas de la gestión integrada de plagas. En este caso, la comunidad de saproxílicos estudiada mostró ser muy homogénea respecto a la variación ambiental de las zonas de muestreo, y que pese a las pequeñas variaciones entre las comunidades de los diferentes ecosistemas, el rol que M. galloprovincialis desempeña en ellas a lo largo de todo el gradiente estudiado es el mismo. Con todo, el análisis mediante redes de interacción mostró su relevancia ecológica al actuar de conector entre los diferentes niveles tróficos. Por último, un total de 12 experimentos de marcaje-liberación-recaptura desarrollados entre 2009 y 2012 en Castellón, Teruel, Valencia y Murcia permitieron evaluar el comportamiento dispersivo de M. galloprovincialis. Las detecciones mediante trampas cebadas de los insectos liberados se dieron por lo menos 8 días después de la emergencia. La abundancia de población pareció relacionada con la continuidad, la naturalización de la masa, y con la afección previa de incendios. La dispersión no estuvo influida por la dirección ni la intensidad de los vientos dominantes. La abundancia de material hospedante (en lo referente a las variables de masa y a los índices de competencia) influyó en la captura del insecto en paisajes fragmentados, aunque la ubicación de las trampas optimizó el número de capturas cuando se ubicaron en el límite de la masa y en zonas visibles. Por último también se constató que M. galloprovincialis posee suficiente capacidad de dispersión como para recorrer hasta 1500 m/día, llegando a alcanzar distancias máximas de 13600m o de 22100 m. ABSTRACT The detection and expansion of the Pine Wilt Desease (PWD), caused by Bursaphelenchus xylophilus (Nematoda; Aphelenchoididae), Pine Wood Nematode (PWN), in southwestern Europe since 1999, has triggered off the study of the phenology and the dispersion of its unique vector in the continent, Monochamus galloprovincialis (Coleoptera, Cerambycidae). The analysis of 12 emergence series between 2010 and 2014 registered in Palencia, Teruel and Valencia (Spain), registered from field colonized material collected at several locations of the Iberian Peninsula, showed a high variability in the emergence phenology of M. galloprovincialis. In addition, these patterns showed a very acute thermal divergence regarding a development model fitted earlier in Portugal. Such model forecasted the emergence of 50% of M. galloprovincialis individuals in the Setúbal Peninsula (Portugal) when an average of 822 degree-days (DD) were reached, based on the accumulation of heat from the 1st of March until emergence and lower and upper thresholds of 12.2 ºC and 33,5 °C respectively. In our results, all analyzed series needed less than 822 DD to complete the 50% of the emergence. Also, emergency occurred earlier in the hottest regions, while it was delayed in more temperate areas. Beyond the possible variability between local populations, the difference in the heat accumulation during the fall season may have affected the degree of maturation of overwintering larvae, and subsequently, the temporal pattern of M. galloprovincialis emergences. Therefore these results suggest the need to differentiate local management strategies for the PWN vector, depending on the location, and the climatic variables of each region. Finally, protandrous emergence patterns were observed for M. galloprovincialis in most of the studied data-sets. Regarding the flight phenology of M. galloprovincialis, a total of 8 trapping experiments were carried out in different regions of the Iberian Peninsula (Castellón, Teruel, Segovia and Alicante) between 2010 and 2015. The use of commercial lures and traps allowed monitoring of the flight period of M. galloprovincialis. The analyses of such curves, helped confirming different aspects. First, a decline in the number of catches and a shortening of the flight period was observed as the altitude increased. Flight period was recorded to start in May / June when the daily average temperature went over 14 ° C. A significant influence of high temperatures on the decrease of catches in the summer was found in many occasions, which frequently lead to a bimodal profile of the flight curves in warm areas. The evolution of sex ratio along the flight period shows a greater capture of females at the beginning of the period, and of males at the end. In addition, the circadian response of M. galloprovincialis to lured traps was described for the first time, concluding that the insect is diurnal and that such response is linked to high temperatures. Two networks of systematic sampling of saproxylic insects were installed in the Region of Valencia (Red MUFFET, 15 plots, 2013) and Murcia (Red ICPF, 20 plots, 2008-2010). These networks, intended to serve the double purpose of early-detection and long term monitoring of the saproxylic beetle assemblies, allowed the study of insect communities related to M. galloprovincialis. Each of the plots had a trap baited with attractants and a weather station. The registration of almost 300 species of saproxylic beetles demonstrated the potential use of such trapping networks for the early detection of exotic organisms, while at the same time allows the characterization and evaluation of useful entomological fauna communities, representing one of the best tools for the integrated pest management. In this particular case, the studied community of saproxylic beetles was very homogeneous with respect to environmental variation of the sampling areas, and despite small variations between communities of different ecosystems, the role that M. galloprovincialis apparently plays in them across the studied gradient seems to be the same. However, the analysis through food-webs showed the ecological significance of M. galloprovincialis as a connector between different trophic levels. Finally, 12 mark-release-recapture experiments were carried out between 2009 and 2012 in Castellón, Teruel, Valencia and Murcia (Spain) with the aim to describe the dispersive behavior of M. galloprovincialis as well as the stand and landscape characteristics that could influence its abundance and dispersal. No insects younger than 8 days were caught in lured traps. Population abundance estimates from mark-release-recapture data, seemed related to forest continuity, naturalization, and to prior presence of forest fires. On the other hand, M. galloprovincialis dispersal was not found to be significantly influenced by the direction and intensity of prevailing winds. The abundance of host material, very related to stand characteristics and spacing indexes, influenced the insect abundance in fragmented landscapes. In addition, the location of the traps optimized the number of catches when they were placed in the edge of the forest stands and in visible positions. Finally it was also found that M. galloprovincialis is able to fly up to 1500 m / day, reaching maximum distances of up to 13600 m or 22100 m.

Melhoramento genético florestal e o desafio imposto pela doença da murchidão do pinheiro

Mestrado em Engenharia Florestal e dos Recursos Naturais - Instituto Superior de Agronomia - UL

Bacterial community associated to the pine wilt disease insect vectors Monochamus galloprovincialis and Monochamus alternatus

Monochamus beetles are the dispersing vectors of the nematode Bursaphelenchus xylophilus, the causative agent of pine wilt disease (PWD). PWD inflicts significant damages in Eurasian pine forests. Symbiotic microorganisms have a large influence in insect survival. The aim of this study was to characterize the bacterial community associated to PWD vectors in Europe and East Asia using a culture-independent approach. Twenty-three Monochamus galloprovincialiswere collected in Portugal (two different locations); twelve Monochamus alternatus were collected in Japan. DNA was extracted from the insects’ tracheas for 16S rDNA analysis through denaturing gradient gel electrophoresis and barcoded pyrosequencing. Enterobacteriales, Pseudomonadales, Vibrionales and Oceanospirilales were present in all samples. Enterobacteriaceae was represented by 52.2% of the total number of reads. Twenty-three OTUs were present in all locations. Significant differences existed between the microbiomes of the two insect species while for M. galloprovincialis there were no significant differences between samples from different Portuguese locations. This study presents a detailed description of the bacterial community colonizing the Monochamus insects’ tracheas. Several of the identified bacterial groups were described previously in association with pine trees and B. xylophilus, and their previously described functions suggest that they may play a relevant role in PWD.

Protein markers of B. xylophilus Steiner & Buhrer, 1934 (Nickle, 1970) populations using quantitative proteomics and character compatibility.

The Pine Wood Nematode (PWN) Bursaphelenchus xylophilus is a severe forest pathogen in countries where it has been introduced and is considered a worldwide quarantine organism. In this study, protein markers for differentiating populations of this nematode were identified by studying differences among four selected Iberian and one American population. These populations were compared by quantitative proteomics (iTRAQ). From a total of 2860 proteins identified using the public database from the B. xylophilus genome project, 216 were unambiguous and significantly differentially regulated in the studied populations. Comparisons of their pairwise ratio were statistically treated and supported in order to convert them into discrete character states, suggesting that 141 proteins were not informative as population specific markers. Application of the Character Compatibility methodology on the remaining 75 proteins (belonging to families with different biological functions) excludes 27 which are incompatible among them. Considering only the compatible proteins, the method selects a subset of 30 specific unique protein markers which allowed the compared classification of the Iberian isolates. This approach makes it easier search for diagnostic tools and phylogenetic inference within species and populations of a pathogen exhibiting a high level of genetic diversity.