Biosecurity and Emerging Plant Health Problems in Turf Production and Maintenance

Recent research has supported the view that the distributions of many pests and diseases have extended towards higher latitudes over the last 50 years. Probably driven by a combination of climate change and trade, this extension to the ranges of hundreds of plant pathogens may have serious implications not only for agriculture, horticulture and forestry, but also for turf production &maintenance. Here we review our data relating to the current status of three emerging pest and disease problems across North West Europe (rapid blight, Labyrinthula sp. , the root knot nematode, Meloidogyne minor and the pacific stem gall nematode, Anguina pacificae ) and discuss the factors which may be involved in their spread and increasing impact on the turf industry. With turf production and maintenance becoming an increasingly international business, we ask if biosecurity and the promotion of plant health in turf production fields and associated sport facilities should be a greater priority for the industry. We also examine if a lack of effective biosecurity measures in the materials supply chain has led to increased plant health problems.

Passive Intermodulation in Distributed Circuits with Cascaded Discrete Nonlinearities

The principle aspects of passive intermodulation (PIM) characterisation in distributed printed circuits with cascaded lumped nonlinearities are presented. Mechanisms of PIM generations have been investigated experimentally and modelled using the formalism of X-parameters. The devised equivalent circuit models are applied to the analysis of microstrip lines with distributed and cascaded lumped sources of nonlinearity. The dynamic measurements have revealed that PIM generation rates in straight and meandered microstrip lines differ and significantly deviate from those expected for the respective discrete sources of nonlinearity. The obtained results indicate that multiple physical sources of nonlinearity contribute to PIM generation in printed circuits. Finally, it is demonstrated that the electrical discontinuities can have significant effect on the overall PIM response of the distributed passive circuits and cause PIM product leakage and parasitic coupling between isolated circuit elements.

Reproductive success of parasitized males in a marine reef fish

Parasitism is hypothesized to reduce reproductive success in heavily parasitized males because females
may preferentially mate with less parasitized males (parasite-mediated sexual selection) or parasites may compromise
male competitiveness. In marine systems, this hypothesis is largely unexplored. This paper provides the first confirmed record of a copepod ectoparasite (Caligus buechlerae Hewitt 1964) on the common triplefin (Forsterygion lapillum) and evaluates the hypothesis that males parasitized with C. buechlerae experience lower reproductive success than unparasitized males (as determined
by the presence and area of eggs within male nests). We found that 38 % of males we surveyed were infected with
at least one C. buechlerae, with a median of two individuals per infected male. About 32 % of males were defending
eggs, with 62.5 % of those males infected with at least one parasite. Males of greater total length (TL) were both
more likely to be infected and more likely to be defending eggs. However, when statistically accounting for the effects
of TL, parasite infection had no effect on the probability of defending eggs, or the average surface area of eggs when
present. Positive covariation in fish length, the presence of eggs and parasite infection observed here potentially suggest
that the importance of parasitic infection on reproductive success may depend upon the strength of selection for larger male body size. Our study is one of the few studies to investigate the effects of ectoparasites on reproductive success in reef fish and also provides a quantitative measure of infection for a widespread species within New Zealand.

Influência de fatores ambientais na condição corporal e produção de leite da cabra serrana – ecótipo ribatejano

A cabra Serrana destaca-se, no panorama das raças autóctones, pela sua considerável distribuição geográgica, expressão numérica e desempenho produtivo. A preservação destes recursos genéticos em sistemas produção rentáveis, requer um aprofundamento do conhecimento sobre os fatores que influenciam o seu desempenho produtivo. Observaram-se 51 animais de raça Serrana, ecótipo Ribatejano, inscritos no Registo Zootécnico, em 4ª ou 5ª lactação, provenientes de seis explorações, recolhendo-se dados realativos à idade ao parto, condição corporal e grau de parasitismo (estrongilídeos gastrintestinais, oocistos e larvas de primeiro estadio de estrongilídeos pulmonares). Utilizou-se a informação do contraste leiteiro das cabras, disponibilizada pela Associação de Criadores de Caprinos e Ovinos do Ribatejo e Oeste. A condição corporal foi analisada considerando os efeitos do criador, mês e tipo de parto, e como covariável, o efeito linear da produção de leite, eliminação parasitária e os efeitos linear e quadrático da idade ao parto. Os fatores ambientais idade ao parto, produção de leite e a eliminação de estrongilídeos gastrintestinais, influenciaram significativamente (p<0,05) a condição corporal. A condição corporal registou valores superiores em cabras com idades compreendidas entre 50 e 60 meses de vida, e foi influenciada negativamente pela elevação da produção de leite e pelo nível de estrongilídeos gastrintestinais. The Serrana stands out among autochthonous goat breeds for its wide geographical distribution, large inventory and considerable productive performances. The preservation of animal genetic resources of this kind requires a better knowledge of the factors affecting productive performance. Data has been collected on 51, 4th and 5th lactation registered Serrana goats of the Ribatejano ecotype, from six different herds. Parameters examined were age at kidding, body condition score and parasitic infection level (gastrointestinal strongyles, oocysts and first stage larvae of pulmonary strongyles). Lactation data from the official milk recording service have been supplied by the Associação de Criadores de Caprinos e Ovinos do Ribatejo e Oeste. Body condition score was analysed for the discrete effects of herd, kidding month and number of kids born per kidding, the covariant linear effects of milk yield and parasitic infection and the linear and quadratic effects of age at kidding. Age at kidding, milk yield and gastrointestinal strongyle fecal egg counts have significantly affected body condition scores (p<0,05). Body condition scores were higher for 50 to 60-month old goats, and were negatively influenced by milk yield and gastrointestinal strongyle infection level.

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.

First report of an entomopathogenic nematode from continental Portugal

In continental Portugal no information is available concerning the occurrence of entomopathogenic nematodes. During a survey in several different habitats from the southern regions of the country, several isolates where identified as Steinernema feltiae. This represents the first report of an entomopathogenic nematode genus for continental Portugal.

Bursaphelenchus Fuchs, 1937 (Nematoda: Parasitaphelenchidae) species associated with Pinus species in northern Turkey

A survey for Bursaphelenchus nematodes, associated with different conifer trees, was conducted in several forest areas in the northern regions of Turkey. Only pine trees (Pinus nigra, P. pinaster and P. sylvestris) yielded Bursaphelenchus specimens. Nematodes were identified using several morphological diagnostic characters of the genus (male spicule structure, number of lateral incisures, number and distribution of the male papillae, presence of female vulval flap), and confirmed by using RFLP analysis of the internal transcriber spacer (ITS) regions of ribosomal DNA. Three different species were identified from several sampled areas, namely B. mucronatus, B. pinophilus and B. sexdentati, representing a first report of the last two species for Turkey. The association of B. pinophilus with black pine (P. nigra) is herein reported for the first time.

Bursaphelenchus xylophilus : opportunities in comparative genomics and molecular host–parasite interactions

Most Bursaphelenchus species are fungal feeding nematodes that colonize dead or dying trees. However, Bursaphelenchus xylophilus , the pine wood nematode, is also a pathogen of trees and is the causal agent of pine wilt disease. B. xylophilus is native to North America and here it causes little damage to trees. Where it is introduced to new regions it causes huge damage. The most severely affected areas are found in the Far East but more recently B. xylophilus has been introduced into Portugal and the potential for damage here is also high. As incidence and severity of pine wilt disease are linked to temperature we suggest that climate change is likely to exacerbate the problems caused by B. xylophilus and, in addition, will extend (northwards in Europe) the range in which pine wilt disease can occur. Here we review what is currently known about the interactions of B. xylophilus with its hosts, including recent developments in our understanding of the molecular biology of pathogenicity in the nematode. We also examine the potential developments that could be made by more widespread use of genomics tools to understand interactions between B. xylophilus , bacterial pathogens that have been implicated in disease and host trees.

A rapid staining-assisted wood sampling method for PCR-based detection of pine wood nematode Bursaphelenchus xylophilus in Pinus massoniana wood tissue

For reasons of unequal distribution of more than one nematode species in wood, and limited availability of wood samples required for the PCR-based method for detecting pinewood nematodes in wood tissue of Pinus massoniana, a rapid staining-assisted wood sampling method aiding PCR-based detection of the pine wood nematode Bursaphelenchus xylophilus (Bx) in small wood samples of P. massoniana was developed in this study. This comprised a series of new techniques: sampling, mass estimations of nematodes using staining techniques, and lowest limit Bx nematode mass determination for PCR detection. The procedure was undertaken on three adjoining 5-mg wood cross-sections, of 0.5 · 0.5 · 0.015 cm dimension, that were cut from a wood sample of 0.5 · 0.5 · 0.5 cm initially, then the larger wood sample was stained by acid fuchsin, from which two 5-mg wood cross-sections (that adjoined the three 5-mg wood cross-sections, mentioned above) were cut. Nematode-staining-spots (NSSs) in each of the two stained sections were counted under a microscope at 100· magnification. If there were eight or more NSSs present, the adjoining three sections were used for PCR assays. The B. xylophilus – specific amplicon of 403 bp (DQ855275) was generated by PCR assay from 100.00% of 5-mg wood cross-sections that contained more than eight Bx NSSs by the PCR assay. The entire sampling procedure took only 10 min indicating that it is suitable for the fast estimation of nematode numbers in the wood of P. massonina as the prelimary sample selections for other more expensive Bx-detection methods such as PCR assay.

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.

Pine wilt disease: a worldwide threat to forest ecosystems

The pinewood nematode (PWN), Bursaphelenchus xylophilus, the causal agent of pine wilt disease (PWD), is a serious pest and pathogen of forest tree species, in particular among the genus Pinus. It was first reported from Japan in the beginning of the XXth century, where it became the major ecological catastrophe of pine forests, with losses reaching over 2 million m3/ year in the 1980s. It has since then spread to other Asian countries such as China, Taiwan and Korea, causing serious losses and economic damage. In 1999, the PWN was first detected in the European Union (EU), in Portugal, and immmediately prompted several government (national and EU) actions to assess the extent of the nematode’s presence, and to contain B. xylophilus and its insect vector (Monochamus galloprovincialis) to an area with a 30km radius in the Setúbal Peninsula, 20 km south of Lisbon. International wood trade, with its political as well as economic ramifications, has been seriously jeopardized. The origin of the population of PWN found in Portugal remains elusive. Several hypotheses may be considered regarding pathway analysis, basically from two general origins: North America or the Far East (Japan or China). World trade of wood products such as timber, wooden crates, palettes, etc… play an important role in the potential dissemination of the pinewood nematode. In fact, human activities involving the movement of wood products may be considered the single most important factor in spreading of the PWN. Despite the dedicated and concerted actions of government agencies, this disease continues to spread. Very recently (2006), in Portugal, forestry and phytosanitary authorities (DGRF and DGPC) have announced a new strategy for the control and ultimately the erradication of the nematode, under the coordination of the national program for the control of the pinewood nematode (PROLUNP). Research regarding the bioecology of the nematode and insect as well as new detection methods, e.g., involving real-time PCR, has progressed since 1999. International agreements (GATT, WTO) and sharing of scientific information is of paramount importance to effectively control the nematode and its vector, and thus protect our forest ecosystems and forest economy.

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).

Bursaphelenchus pinophilus Brzeski & Baujard, 1997 (Nematoda: Parasitaphelenchinae) associated with nematangia on Pityogenes bidentatus (Herbst, 1783) (Coleoptera: Scolytinae), from the Czech Republic

The occurrence of Bursaphelenchus species in the Czech Republic is poorly known, the first report of the genus being made by Kubátová et al. (2000) who reported the association of B. eremus with the hyphomycetous microfungus, Esteya vermicola, and the bark beetle, Scolytus intricatus, collected from Quercus robur, in central Bohemia. To date, four other species have been reported from the country, namely B. fungivorus (Braasch et al., 2002), B. hofmanni (see Braasch, 2001), B. mucronatus (see Braasch, 2001) and B. vallesianus (Gaar et al., 2006). More recently, a survey for Bursaphelenchus species associated with bark- and wood-boring insects in the Czech Republic identified B. pinophilus Brzeski & Baujard, 1997 from the Moravia region. Although this represents a new country record, it was also associated with nematangia on the hind wings of a new insect vector. A total of 404 bark- and wood-boring insects were collected from declining or symptomatic trees and screened for the presence of Bursaphelenchus. Bark and longhorn beetles were captured manually after debarking parts of the trunk displaying symptoms of insect attacks. Longhorn beetle larvae were also collected together with logs cut from the trunk. Logs were kept at room temperature in the laboratory until insect emergence. Each adult insect was individually dissected in water and examined for nematodes. All nematodes resembling dauer juveniles of Bursaphelenchus were collected and identified by molecular characterisation using a region of ribosomal DNA (rDNA) containing the internal transcribed spacer regions ITS1 and ITS2. ITS-RFLP analyses using five restriction enzymes (AluI, HaeIII, HinfI, MspI, RsaI) were performed to generate the species-specific profile according to Burgermeister et al. (2009). Species identification was also confirmed by morphological data after culture of the dauers on Botrytis cinerea Pers. ex Ft., growing in 5% malt extract agar. During this survey, only species belonging to the Curculionidae, subfamily Scolytinae, revealed the presence of nematodes belonging to Bursaphelenchus. Dauers of this genus were found aggregated under the elytra in nematangia formed at the root of the hind wings (Fig. 1). The dauers were identified from 12 individuals of Pityogenes bidentatus (Herbst, 1783) (Coleoptera: Scolytinae) collected under the bark of Pinus sylvestris trunks. Each insect carried ca 10-100 dauers. The ITS-RFLP patterns of the dauers so obtained confirmed the identification of B. pinophilus associated with this insect species. Bursaphelenchus pinophilus has been found mainly in Europe and has been reported from various countries such as Poland (Brzeski & Baujard, 1997), Germany (Braasch, 2001), and Portugal (Penas et al., 2007). The recent detection of this species associated with dead P. koraiensis in Korea (Han et al., 2009) expands its geographical distribution and potential importance. It has been found associated only with Pinus species, but very little is known about the insect vector. The bark beetle, Hylurgus ligniperda, was initially suggested as the insect vector by Pe-nas et al. (2006), although the nematode associated with this insect was later reclassified as B. sexdentati by morphological and molecular analysis (Penas et al., 2007). According to the literature, P. bidentatus has been cited as a vector of Ektaphelenchus sp. (Kakuliya, 1966) in Georgia, and an unidentified nematode species in Spain (Roberston et al., 2008). Interestingly, B. pinophilus was found in the nematangia formed at the root of the hind wings of P. bidentatus. Although this phenomenon is not so common in other Bursaphelenchus species, B. rufipennis has been found recently in such a structure on the hind wings of the insect Dendroctonus rufipennis (Kanzaki et al., 2008). Although other nematode species (e.g., Ektaphelenchus spp.) are frequently found associated within the same nematangia (see Kanzaki et al., 2008), in this particular case, only dauers of B. pinophilus were identified. The association between B. pinophilus and P. bidentatus represents the first report of this biological association and the association with the Scolytinae strengthens the tight and specific links between this group of Bursaphelenchus species and members of the Scolytinae (Ryss et al., 2005).