Computerized key to the genus Bursaphelenchus Fuchs, analysis of species clusters based on morphology, using information of insect vectors and associated plants, with a revision of the genus.

The genus Bursaphelenchus includes B. xylophilus (Steiner et Buhrer, 1934) Nickle, 1981, which is of world economic and quarantine importance. Distinction among several species of the pinewood nematodes species complex (PWNSC) is often difficult. Besides standard morphology, morphometrics and molecular biology, new tools are welcome to better understand this group. The computerized (or e-) key of this genus, presented in this communication, includes 74 species (complete list of valid species of the world fauna) and 35 characters, that were used by the taxonomic experts of this group, in the original descriptions. Morphology of sex organs (male spicules and female vulval region) was digitized and classified to distinguish alternative types. Several qualitative characters with overlapping character states (expressions) were transformed into the morphometric indices with the discontinuous ranges (characters of ratios of the spicule dimensions). Characters and their states (expressions) were illustrated in detail and supplied by brief user-friendly comments. E-key was created in the BIKEY identification system (Dianov & Lobanov, 1996-2004). The system has built-algorithm ranging characters depending on their diagnostic values at each step of identification. Matrix of species and the character states (structural part of the e-key database) may be easily transformed using statistical packages into the dendrograms of general phenetic similarities (UPGMA, standard distance: mean character difference). It may be useful in the detailed analysis of taxonomy and evolution of the genus and in its splitting to the species groups based on morphology. The verification of the dendrogram using the information on the species links with insect vectors and their associated plants, provided an opportunity to recognize the five clusters (xylophilus, hunti, eremus sensu stricto, tusciae and piniperdae sensu stricto), which seem to be the natural species groups. The hypothesis about the origin and the first stages of the genus evolution is proposed. A general review of the genus Bursaphelenchus is presented.

Review of the genus Bursaphelenchus Fuchs, 1937 (Nematoda, Aphelenchida) with some conclusions on the host-parasite and vector-parasite evolution.

INTRODUCTION AND GOALS: Genus Bursaphelenchus includes several pests of the world importance for the rural economy, the most dangerous are the Bursaphelenchus xylophilus (the pinewood nematode caused decline of the pine trees in south Asia and in one spot area in Europe, Portugal, Peninsula de Setubal) and the Bursaphelenchus cocophilus, causing the decline of coco-palm plantations in Carribean and Latin American regions. The peculiarity of the host-parasite association of the genus that the nematode life cycle includes three trophic components: plant (mostly a tree), insect vector and a fungus. Goals of the presentation is to list all species of the world fauna and all efficient diagnostic characters, then create the identification tool and analyze the similarity of species and possible ways and causes of the host-parasite evolution of the group. RESULTS: Complete list of species with synonymy and a catalogue of all efficient diagnostic characters with their states, selected from papers of the most experienced taxonomists of the genus, are given for the genus Bursaphelenchus. List of known records of Bursaphelenchus species with names of natural vectors and plants and their families is given (for world pests the most important groups of trees and insects are listed). The tabular, traditional and computer-aided keys are presented. Dendrograms of species relationships (UPGMA, standard distance: mean character difference) based on all efficient taxonomic characters and separately on the spicule characters only, are given. Discussion whether the species groups are natural or purely diagnostic ones is based on the relationships dendrograms and the vector and associated plant ranges of Bursaphelenchus species; the xylophilus species group (B. xylophilus, B. abruptus, B. baujardi, B. conicaudatus, B. eroshenkii, B. fraudulentus, B. kolymensis, B. luxuriosae; B. mucronatus), the hunti group (B. hunti, B. seani, B. kevini and B. fungivorus) are probably the natural ones. CONCLUSIONS: The parasitic nematode association includes three trophic components: plant, insect vector and fungus. The initial insect-plant complex Scolytidae-Pinaceae is changeable and only in rare occasions the change of the preferred vector to Cerambycidae (the xylophilus group), Hymenoptera (the hunti group) led to formation of the natural species-groups. From the analysis it is clear that although the vector range is changeable it is comparatively more important for the evolution of the genus Bursaphelenchus than associations with plants at the family level. Data on the fungi species (3rd component in natural Bursaphelenchus associations) are insufficient for the detailed comparative analysis.

Morphobiometric and molecular characterization of Bursaphelenchus Fuchs, 1937 (Nematoda: Aphelenchoididae) species associated with Pinus pinaster Ainton in Portugal

Abstract - The genus Bursaphelenchus comprises almost 100 species mainly from the northern hemisphere, with conifers as the most important hosts. Among the various nematode species, the pine wood nematode (PWN), Bursaphelenchus xylophilus, is the casual agent of pine wilt disease (PWD), and the most important forest pest for pines worldwide, classified as an A1 quarantine organism within the European Union. In 1999 this nematode was detected for the first time in Portugal and Europa associated with maritime pine, Pinus pinaster. Following detection, a national program denominated "Programa Nacional de Luta contra o Nemátodo da Madeira do Pinheiro" (PROLUNP) was created to, among other objectives, determine the distribution of the PWN and its associated vector(s) and host(s), and therefore intensive surveys covering the entire country were conducted with thousands of wood samples and suspected insects being analyzed. This thesis presents the listing, distribution, frequency and the insects associated with Bursaphelenchus species found associated with maritime pine in Portugal, identifying and characterizing the various species by morphological, biometrical and molecular biology (ITS-RFLP and rDNA sequencing analysis) techniques. To achieve the objectives, a total of 4813 maritime pine wood samples and 3294 insects from 22 species and six families were individually analyzed. A total of nine Bursaphelenchus species were found, namely: B. antoniae, B. hellenicus, B. leoni, B. mucronatus, B. pinasteri, B. sexdentati, B. teratospicularis, B. tusciae and B. xylophilus, all of them (with the exception of B. xylophilus) being new records for Portugal. Some of the species appear to have a widespread distribution, such as B. leoni, B. teratospicularis and B. tusciae while others were very rarely found and apparently have a localized distribution range within the country, namely B. antoniae and B. mucronatus. The majority of the species is characteristic of the Mediterranean region and can also be found in countries such as Spain, Italy and Greece, reflecting the affinity of our fauna with those locations. The association of B. hellenicus and B. tusciae with maritime pine is here reported for the first time. Six of the Bursaphelenchus species were also found associated with insects, mainly from the family Scolytidae (Coleoptera). Some of these interactions were described for the first time, namely: B. hellenicus with both Ips sexdentatus and Hylurgus ligniperda, B. sexdentati with both H. ligniperda and Orthotomicus erosus and B. tusciae with H. ligniperda. The exclusive association of B. xylophilus with the cerambycid Monochamus galloprovincialis was also confirmed. The nematode's dauer juveniles were usually found in low numbers in the insect vectors (ca 10-100 per insect), although for B. xylophilus a few thousand specimens per insect were sometimes found. The location of the dauer juveniles differed according to the species, although they were more common under the elytra and wings of the adult insects. A species new to science was detected and formally described as B. antoniae, associated with Hylobius sp. (Coleoptera; Curculionidae) beetles. Morphologically, this new species is very similar to B. hylobianum, although it's distinct ITS-RFLP molecular pattern (with only the enzyme Haelll producing comparable restriction bands) and the failure of hybridization supported the two species as distinct entities. Additional phylogenetic analysis of the 18S rDNA sequence further supported the taxonomical proximity of B. antoniae with B. hylobianum. Concerning the PWN, detailed studies on the development and morphology of B. xylophilus were conducted, and comparative measurements of field-collected and laboratory-maintained populations demonstrated that nematodes from the second group displayed larger size in all morphometric parameters, which could derive from more adequate conditions of nourishment and/or temperature. Taxonomical studies on the development stages of B. xylophilus confirmed the existence of four propagative juvenile stages (J1,J2,J3 and J4), an adult stage with both sexes and two dispersal stages (jIII e jIV), with the measurements of the gonad length allowing the separation of the propagative stages. It is hoped that the acquired knowledge will be useful on future surveys of nematodes of the Bursaphelenchus genus collected from either wood material or insect vectors, and facilitate the correct distinction and identification of the various species which are now known to occur. ### Resumo - 0 género Bursaphelenchus compreende quase 100 espécies, distribuídas sobretudo nos países do hemisfério norte do globo terrestre. Embora algumas espécies já tenham sido detectadas em plantas herbáceas, os hospedeiros vegetais mais comuns deste género são as coníferas, particularmente pinheiros. 0 nemátode da madeira do pinheiro (NMP), Bursaphelenchus xylophilus, é considerado a espécie mais importante deste género uma vez que é o agente causal da doença da murchidão dos pinheiros ("pine wilt disease"). Originário dos Estados Unidos, onde não causa grande impacte, o NMP foi introduzido em alguns países da Ásia (China, Japão, Coreia e Taiwan) e mais recentemente na Europa (Portugal). Nestas regiões é responsável pela destruição de milhares de hectares de coníferas, assumindo uma elevada importância económica. Em Portugal, depois da sua detecção em 1999, associado a Pinus pinaster, foi implementado um programa nacional "Programa Nacional de Luta contra o Nemátodo da Madeira do Pinheiro" (PROLUNP) que permitiu determinar a área afectada pela praga (a sul do rio Tejo, península de Setúbal) bem como definir e implementar estratégias de controlo e prevenção da disseminação do NMP a outras zonas de Portugal. Recentemente, em Junho de 2008, foi confirmada a presença de B. xylophilus em outras regiões de Portugal levando as autoridades oficiais a definir todo o território continental como zona afectada e de restrição. As prospecções intensivas realizadas nos últimos anos incluíram a recolha e análise de milhares de amostras de madeira de pinheiro bem como de insectos associados ao pinheiro bravo conduzindo à identificação de várias espécies de Bursaphelenchus. Assim, os estudos conduzidos neste trabalho tiveram como objectivos efectuar uma caracterização morfológica, biométrica e molecular das espécies associadas a P. pinaster em Portugal bem como a sua distribuição geográfica e abundância. Os estudos biométricos foram realizados com populações extraídas directamente do meio natural. Foi ainda realizada uma pesquisa que permitiu identificar os insectos a que estão associadas essas espécies, os seus possíveis vectores. Foram analisadas no total 4813 amostras de P. pinaster e 3294 insectos (22 espécies pertencentes a seis famílias diferentes). Foram identificadas um total de nove espécies: B. antoniae n. sp., B. hellenicus, B. leoni, B. mucronatus, B. pinasteri, B. sexdentati, B. teratospicularis, B. tusciae e B. xylophilus. Foram realizados estudos morfológicos e biométricos de todas as espécies com excepção de B. mucronatus; o reduzido número de exemplares encontrados em apenas uma amostra foram utilizados para efectuar o diagnóstico molecular desta espécie (ITS-RFLP). Apesar de ter havido, sempre que possível, a confirmação molecular, na maioria dos casos a caracterização morfológica e biométrica permitiu a correcta identificação das espécies. Contudo, foi imprescindível a análise molecular em algumas amostras, nomeadamente para a identificação de B. xylophilus e B. sexdentati; dada a grande semelhança entre B. xylophilus e B. mucronatus e tendo sido encontradas algumas populações de B. xylophilus que possuíam fêmeas com cauda mucronada, foi necessária a realização da confirmação molecular. Com excepção de B. xylophilus, todas as outras espécies foram reportadas pela primeira vez em Portugal. Juntamente com B. xylophilus, B. pinasteri foi a espécie encontrada nas amostras de madeira de pinheiro com maior frequência. Algumas destas espécies como B. leoni, B. teratospicularis e B. tusciae foram reportadas em diferentes localidades do norte, centro e sul de Portugal, apresentando uma vasta distribuição geográfica; este resultado está em consonância com a forte associação destas espécies a climas mediterrânicos tal como acontece em Espanha, França, Itália e Grécia. Em oposição, espécies como B. antoniae e B. mucronatus foram encontradas apenas numa ocasião na região centro (Leiria) e norte (Figueira da Foz) do país, respectivamente. Bursaphelenchus mucronatus é igualmente pouco frequente em Espanha onde ocorre sobretudo na região norte, na Galiza. Esta espécie preferirá climas mais frios, ocorrendo com uma maior frequência nas regiões de latitude norte; esta análise é corroborada pela presença constante em países como Alemanha, Finlândia, França, Noruega, Rússia e Suécia. A nível mundial são descritas neste trabalho pela primeira vez as associações das espécies B. hellenicus e B. tusciae ao hospedeiro vegetal P. pinaster.

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.

Occurrence of the pinewood nematode, Bursaphelenchus xylophilus in Portugal and perspectives of the disease spread in Europe.

In 1999, the pinewood nematode (PWN) , Bursaphelenchus xylophilus was found and identified for the first time in Portugal and in Europe. Following detection, Portuguese authorities initiated the implementation of eradication measures during 1999 and 2000, following an alert provided to European Community officials; as a result, the nematode was confirmed to be confined in the Setúbal region, near Lisbon. A task force from the follow-up group (GANP) created by the Secretary of State for the Rural Development established a national eradication programme (PROLUNP) to (1) Contain PWN within the initial geographic limits; (2) Implement eradication measures; and (3) Monitor PWN at a national level. Research is presently being conducted both at universities as well as research institutes, focusing on the characterization ofBursaphelenchus species associated with maritime pine, as well as on the insect vector, Monochamus galloprovincialis. Recent reports indicate that the nematode may be present in Siberia (Russia), which would present a threat to Eastern European forestry. Efforts are presently being developed by several European countries to establish a research consortium to detect and study the possible presence of PWN, for a new PRAs. A recent workshop held in Portugal, in 2001, has been an oportunity for sharing experiences and techniques on detection and control. There is clearly a greater awareness of this issue in Europe.

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 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).