Bioassays against pinewood nematode: assessment of a suitable dilution agent and screening for bioactive essential oils

Acetone was investigated and found to be an appropriate alternative to Triton X-100 as a solvent of essential oils in bioassays aimed to investigate their effects on pinewood nematode (Bursaphelenchus xylophilus) mortality. Therefore it was used as dilution agent to screen the effectiveness of fifty two essential oils against this pest. Thirteen essential oils were highly effective, resulting in more than 90% pinewood nematode mortality at 2 mg/mL, with six of them resulting in 100% mortality. LC100 values ranged between 0.50 mg/mL and 0.83 mg/mL for the essential oils of Origanum vulgare and Satureja montana, respectively. Essential oils were submitted to gas chromatography and gas chromatography-mass spectrometry analysis and their chemical composition established. Data from essential oils with 100% mortality at 2 mg/mL and other essential oils previously found to have LC100 ≤ 2 mg/mL was combined, their chemical profiles investigated by correspondences analysis plus automatic classification.

Resposta do Olival Intensivo e Super-Intensivo a Dois Regimes de Rega: Parâmetros Fisiológicos, Produção e Qualidade

O olival de regadio tem tido grande expansão nos últimos anos no Alentejo, sendo a administração da rega mais adequada às necessidades hídricas um dos fatores determinantes da sua boa gestão. No presente trabalho, avaliou-se a resposta de duas variedades de Olea europaea, Cobrançosa e Arbequina, em regime intensivo e super- intensivo, respetivamente, a duas dotações de rega, a normalmente utilizada pelo agricultor (RA) e outra experimental, com dotações acima (RA+) ou abaixo (RA−) das praticados em RA. Mediram-se os principais parâmetros hídricos das plantas e o teor em clorofilas, e registou-se a assinatura espectral em folhas adultas e jovens, ao meio- dia solar, em três épocas do ano, primavera, final do verão e inverno de 2011. Em Outubro foi feita a colheita, tendo-se quantificado a produção em termos de produção total e teor de óleo na matéria seca, e a qualidade do azeite em termos de acidez e oxidação. Face aos resultados, conclui-se que no olival intensivo de Cobrançosa, na rega experimental (RA+), acima da praticada pelo agricultor, não se verificou diferenças significativas na produção total nem no teor de óleo na matéria seca. Não se verificaram também diferenças significativas entre as regas nos parâmetros hídricos avaliados. Quanto ao olival super-intensivo de Arbequina, a rega experimental (RA−), deficitária relativamente à do agricultor (RA), acarretou menor produção, associada a menor teor relativo de água nas folhas, potenciais hídricos mais negativos e menor condutância estomática no final do verão e inverno, mantendo-se no entanto o teor de óleo nos frutos. O teor em clorofilas e alguns índices de vegetação foram influenciados pelo regime de rega apenas em algumas das datas. Nos dois olivais, as regas experimentais não influenciaram a qualidade do azeite, tendo-se obtido azeites extra virgem com propriedades semelhantes aos das modalidades RA. O estudo prossegue em 2012.

Pegada hídrica de um olival super-intensivo sob clima mediterrânico, utilizando medições de campo e deteção remota

A pegada hídrica de uma cultura representa o volume de água necessário para produzir, relacionando as necessidades hídricas da cultura com a produção. As suas componentes, pegadas hídricas azul, verde e cinzenta, referem-se respectivamente aos volumes de água superficial e subterrânea, precipitação e de água necessária para assimilar a poluição utilizados pela cultura. A determinação das pegadas hídricas azul e verde é normalmente conseguida através da estimativa da evapotranspiração cultural, aplicando coeficientes culturais a uma evapotranspiração de referência, calculada a partir de dados meteorológicos. No presente estudo foram utilizadas medições da evapotranspiração para estimar a pegada hídrica de um olival super-intensivo na região de Évora. As necessidades hídricas foram medidas utilizando um método de fluxo de seiva para determinar a transpiração e o método micrometeorológico das flutuações instantâneas para medir directamente a evapotranspiração. Esta técnica foi utilizada durante um período de tempo limitado, enquanto as medições do fluxo de seiva, que foram efectuadas para períodos alargados, permitiram a extensão dos registos. A evapotranspiração medida directamente apresentou valores de cerca de 3 mm d-1 e o quociente entre evapotranspiração real e evapotranspiração de referência é próximo de 0,6 para o mesmo período. Comparou-se a estimativa da pegada hídrica obtida com o procedimento habitual com a resultante de medições in-situ e utilizando técnicas de deteção remota. A pegada hídrica do olival sob estudo foi inferior às simulações encontradas na literatura, o que pode ser explicado por diferenças na densidade de plantação, produção e gestão da rega. O olival em estudo obteve uma produção elevada, com um azeite que preencheu as características essenciais à classificação de azeite extra virgem, o mais valorizado, o que contraria o efeito do elevado consumo de água, resultando numa pegada hídrica inferior à de olivais não regados ou com menor densidade de plantação.

POPULATION DYNAMICS OF PLANT PARASITIC NEMATODES AND THEIR RELATIONSHIPS WITH SOIL PHYSICO-CHEMICAL PARAMETERS DURING THE FALLOW PERIOD AFTER SUGARBEET AND LEADING INTO TOMATO CULTIVATION

The main objective of this research was the study of the soil nematode community, and in particular plant parasitic nematodes (PPN), from a field located in Portugal’s southern region, used for sugarbeet production. The study was performed from February to July 2003, covering part of the fallow period previous to tomato cultivation, the alternative crop in the rotation. The end of the fallow period in March and the soil preparation period in May were marked by a significant reduction in the numbers of PPN, whereas their numbers increased on the following tomato crop. The genus Helicotylenchus stood out as the most representative group, forming 90% of all PPN counted each month. The genus Heterodera was relatively abundant in the months following the previous sugarbeet crop, and numbers of the genus Meloidogyne increased during the tomato crop. The correlations between these group and environmental parameters show that, apart from the direct influence of the host, pH, organic matter, temperature and soil moisture significantly influenced nematode abundance and community composition.

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.

Computerized key of the genus genus Laimaphelenchus Fuchs, 1937

The Order Aphelenchida contains several genera of economic importance, namely Aphelenchoides and Bursaphelenchus. Nematode species belonging to these 2 genera frequently co-habit with other genera such as Laimaphelenchus. It is therefore important to clearly distinguish them, as well as understand the group´s biodiversity. A computerized, or e-key, for the genus Laimaphelenchus Fuchs has been developed in the BiKey Identification system (Dianov & Lobanov, 1996-2004). The e-key includes 14 species and 34 characters (from 2 to 6 character states each). It also includes the built-in algorithm ranging characters according their diagnostic values to minimize the number of the diagnosis steps (average number of steps is 2.7; values are re-calculated at each step). The most important characters (as calculated by BiKey) are: length of posterior branch of the female genital system; excretory pore position; vulval anterior flap shape; number pairs of mail tale papillae; male bursa shape (ventral view); number of tail tip setae in female; female tail tip stub shape; presence of mucro on tail tip in male. Key is pictorial (image-operating), multientry, as other BiKey products.

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.

Poly(vinyl alcohol) with sulfonic acid groups as catalyst for the alpha-pinene alkoxylation

The alkoxylation of a-pinene was carried out over poly(vinyl alcohol) containing sulfonic acid groups, as catalyst. The main product of a-pinene alkoxylation was a-terpinyl methyl ether being also formed bornyl methyl ether, fenchyl methyl ether, limonene and terpinolene as by-products. The absorption band at 1037 cm-1 in the FTIR spectrum of PVA_SSA40 (PVA membrane containing 40% of –OH groups esterified with SSA) which does not appear in the spectrum of PVA, indicates the presence of the sulfonic acid groups. The catalytic activity of PVA with sulfonic acid groups increases when the amount of sulfosuccinic acid used in the polymer crosslinking is increased from 5% to 40%. However, when the crosslinking degree increases from 20% to 40%, the conversion of a-pinene increases only slightly. Good values of selectivity to a-terpinyl methyl ether were obtained over poly(vinyl alcohol) with sulfonic acid groups. A simple kinetic model, which fits experimental concentration data quite well, was developed.

A synopsis of the genus Bursaphelenchus Fuchs, 1937 (Aphelenchida: Parasitaphelenchidae) with keys to species

The 75 valid species of the genus Bursaphelenchus are listed together with their synonyms. Diagnostic characters and their states are discussed and illustrated. Tabular and traditional text keys are provided for the genus. Two new subspecies are proposed to distinguish populations of B. piniperdae and B. poligraphi, as described by Rühm (1956), from the original descriptions of these species published by Fuchs (1937). Known records of Bursaphelenchus species with their associated natural vectors, plants and plant families are given. Dendrograms of species relationships (UPGMA, standard distance: mean character difference) based on combined taxonomic characters and also on spicule characters only, are provided. Discussion as to whether the species groups are natural or artificial (and therefore purely diagnostic) is based on their relationships in the dendrogram and the vector and associated plant ranges of the species. Of the six species groups distinguished, two appear to represent natural assemblages, these being the xylophilus-group (with ten species) and the hunti-group (seven species), of which two, B. cocophilus and B. dongguanensis, form the cocophilus-cluster which is separated on the dendrogram from the main clusters. The remaining four species groups appear to be artificial and purely diagnostic in function, namely the aberrans-group (four species); the eidmanni-group (six species); the borealis-group (five species), and the piniperdae-group (43 species). Two new subspecies, both in the piniperdae-group, viz. B. piniperdae ruehmpiniperdae n. subsp. and B. poligraphi ruehmpoligraphi n. subsp., are proposed and diagnosed from B. piniperdae piniperdae and B. poligraphi poligraphi the respective type subspecies. Bursaphelenchus dongguanensis is regarded as being a valid member of the genus and its transfer to Parasitaphelenchus is rejected.

Chromosome structure and behaviour in Bursaphelenchus xylophilus (Nematoda: Parasitaphelenchidae) germ cells and early embryo

Chromosome structure and behaviour in both meiosis of the germ cells and mitosis of the embryo from fertilisation to the two-cell stage in Bursaphelenchus xylophilus were examined by DAPI staining and three-dimensional reconstruction of serial-section images from confocal laser-scanning microscopy. By this method, each chromosome’s shape and behaviour were clearly visible in early embryogenesis from fertilisation through the formation and fusion of the male and female pronuclei to the first mitotic division. The male pronucleus was bigger than that of the female, although the oocyte is larger and richer in nutrients than the sperm. From the shape of the separating chromosomes at anaphase, the mitotic chromosomes appeared to be polycentric or holocentric rather than monocentric. Each chromosome was clearly distinguishable in the male and female germ cells, pronuclei of the one-cell stage embryo, and the early embryonic nuclei. The haploid number of chromosomes (N) was six (2n = 12), and all chromosomes appeared similar. The chromosome pair containing the ribosomal RNA-coding site was visualised by fluorescence in situ hybridisation. Unlike the sex determination system in Caenorhabditis elegans (XX in hermaphrodite and XO in male), the system for B. xylophilus may consist of an XX female and an XY male.

Bursaphelenchus antoniae sp. n. (Nematoda: Parasitaphelenchidae) associated with Hylobius sp. from Pinus pinaster in Portugal

Bursaphelenchus antoniae sp. n. is described and illustrated. Dauer juveniles were isolated from the body of the large pine weevil, Hylobius sp., collected from maritime pine (Pinus pinaster) stumps, in Portugal. Bursaphelenchus antoniae sp. n. was reared and maintained in P. pinaster wood segments and on Petri dish cultures of the fungi Botrytis cinerea and Monilinia fructicola. The new species is characterised by a relatively small body length of ca 583 μm (females) and 578 μm (males), a lateral field with two incisures, presence of a small vulval flap and a conoid female tail with a rounded or pointed terminus. Males have stout spicules with a disc-like cucullus and seven caudal papillae arranged as a single midventral precloacal papilla, one precloacal pair and two postcloacal pairs. In the character of the lateral field, B. antoniae sp. n. comes close to B. abietinus, B. rainulfi and B. hylobianum, whilst spicule characters place it within the piniperdae-group sensu Ryss et al. Morphologically, B. antoniae sp. n. is closest to B. hylobianum; the spicules of these two species having flattened, wing-like, alae on the distal third of the lamina. Bursaphelenchus antoniae sp. n. is distinguished from B. hylobianum on the arrangement of the caudal papillae (two vs three pairs). ITS-RFLP profiles and the failure to hybridise support the separation of the two species. Phylogenetic analysis of the new species, based on the 18S rDNA sequence, supports the inclusion of this new species in the B. hylobianum-group sensu Braasch. Sequence analysis of the 28S rDNA D2/D3 domain did not place the new species in a definite group.

Satellite DNA as a target for TaqMan real-time PCR detection of the pinewood nematode, Bursaphelenchus xylophilus

The pinewood nematode (PWN), Bursaphelenchus xylophilus , is a major pathogen of conifers, which impacts on forest health, natural ecosystem stability and international trade. As a consequence, it has been listed as a quarantine organism in Europe. A real-time PCR approach based on TaqMan chemistry was developed to detect this organism. Specific probe and primers were designed based on the sequence of the Msp I satellite DNA family previously characterized in the genome of the nematode. The method proved to be specific in tests with target DNA from PWN isolates from worldwide origin. From a practical point of view, detection limit was 1 pg of target DNA or one individual nematode. In addition, PWN genomic DNA or single individuals were positively detected in mixed samples in which B. xylophilius was associated with the closely related non-pathogenic species B. mucronatus , up to the limit of 0.01% or 1% of the mixture, respectively. The real-time PCR assay was also used in conjunction with a simple DNA extraction method to detect PWN directly in artificially infested wood samples. These results demonstrate the potential of this assay to provide rapid, accurate and sensitive molecular identification of the PWN in relation to pest risk assessment in the field and quarantine regulation.

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.