Bettering Humanity through Biology

Biology is arguably referred to as the “science of the XXIth century”. This prestigious title intrinsically contains a huge responsibility. For many centuries, Biology has contributed directly or indirectly to bettering humankind, although its obvious and objective effects have only been made evident since the XIXth century. There are three main domains to which Biology has made significant contributions: Agriculture, Environment and Medicine. Several scientific disciplines connected to Biology have been involved such as Genetics (mendelian and molecular), Cell Biology, Ecology, Microbiology, and what was known for a long time as “Natural History” (today we would include these roughly within Botany and Zoology). Agronomy, a relatively recent science, has made a tremendous impact by providing knowledge on growing plants and animals, and developing new and better crops. One specific moment in time, following WW II, known as the “Green Revolution” benefitted humanity immensely, by combating hunger in countries such as India and Mexico. The “father” of the Green Revolution, Norman Borlaug, was awarded the 1970 Peace Nobel Prize for such achievements. In the XXIth century, biologists and agronomists are working hard to develop new and better crops to feed almost 8 billion people. In the medical field, the contributions are inumerable, from the discovery and development of vaccines (Jenner and Pasteur), to antibiotics (Fleming) and combatting diseases. This has increased the average life expectancy of humans from around 30-40 in the beginning of the XXth century, to a present value of around 75 (depending on the country). These achievements have been recognized by society, through dozens of Nobel Prizes in Medicine. All these successes have been made possible through Biology. In the past 30-40 years, numerous voices have been raised alerting for the environmental degradation of our planet, its land and oceans, its biomes and ecosystems. We have been depleting our planet at an incredible rate. But today, biologists and environmental scientists have the knowledge and tools to better the planet. We know how the ecosystems function and what causes harm them. There is still time, together with a strong public opinion, to halt the damage. Once again, Biology is a principal actor

Culturing Bursaphelenchus cocophilus in vitro and in vivo.

Red ring disease (RRD) is of particular importance in many African oil palms- and coconut-producing regions in Central and South America and the Caribbean. Its causal agent, the nematode Bursaphelenchus cocophilus (Cobb) Baujard, causes extensive damage to tissues in the plant trunk that typically leads to plant death within months. Nearly 100 years after its first report RRD remains understudied largely because the nematode cannot be cultured in vivo or in vitro, what hinders sustained research efforts on basic and applied aspects of the pathosystem. To overcome this problem we attempted in vivo culturing in coconut seedlings, paying attention to aspects that had been overlooked in previous trials. We also attempted in vitro culturing on several fungi endophytic to healthy and RRD-affected coconut trees. In the two in vivo assays performed we were able to recover hundreds of nematodes from the seedlings up to 60 days after inoculation, but the nematodes seemed unable to sustain parasitism in most seedlings. No nematode was recovered from the endophytic fungal cultures in neither of the two assays performed. Hence B. cocophilus continues to stand as the only obligatory plant-parasitic species of the speciose genus Bursaphelenchus and a somewhat intractable plant-parasitic nematode to study on

The History of Expansion of the Genus Bursaphelenchus (Nematoda: Aphelenchida: Parasitaphelenchidae)

Because of globalization and removal of geographical barriers, frequent biological invasions of introduced species become an urgent environmental problem. According to the Convention on Biological Diversity (CBD), precise identification of dangerous aggressive species at the early stages of their invasion to new regions is the most important component of the environmental control and monitoring. To resist the potential environmental hazard, the precise data are required on the current distribution and history of expansion of pests that are of global economic importance

A taxonomic database for the pinewood nematode Bursaphelenchus xylophilus, and other Bursaphelenchus species

The main objective of this project is to develop an information database with the inclusion of text (research articles, keys, bibliography, etc.), images (jpeg files), video clips (mpeg files), in order to collect in one physical format (CD-ROM) all relevant information related to the taxonomy of pine wood nematode (PWN), Bursaphelenchus xylophilus, as well as other Bursaphelenchus species. Initially, all major research papers will be scanned in ‘pdf’ format. All available images of quality and importance to the subject will also be scanned from either journals, bulletins, reports, or obtained privately from colleagues’ image collections. In certain cases, image quality (brightness, contrast, colour) may be enhanced with Adobe Photoshop. Short video clips will be compressed to ‘mpeg’ format for inclusion in the CD-ROM. An updated and broad bibliography will be included, as well as an intuitive taxonomic key, based on classical dicotomous keys but with a better and more intuitive presentation. Pre-existing images will be used in most cases; however, nematode specimens may need to be observed and photographed (jpeg image) in the lab. All the incorporated elements will be interlinked for ease of search from text to image to video. The total collection of images may be viewed individually using ‘Extensis Portfolio’. The final product will be PC and Mac compatible. Future editions/updates will be developed. Ultimately, researchers, technicians and political decision-makers will have at their disposal a practical and readily available source of information on this extremely important pest and pathogen. Colleagues working with PWN are encouraged to provide materials for this task

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

Molecular characterization of Portuguese populations of the pinewood nematode Bursaphelenchus xylophilus using cytochrome b and cellulase genes

Bursaphelenchus xylophilus is the causal agent of pine wilt disease and a worldwide pest with high economic impact. Since its first diagnosis in Portugal in 1999, it has been subjected to quarantine measures with impact on forest health and ecosystem stability, significantly affecting international trade of wood products. The disease was detected in the north and centre of continental Portugal and, since 2008, the whole country has been considered an affected area. Recently, it was detected in Madeira Island. In order to avoid new outbreaks, it has become of major importance to understand the patterns of spread, introduction points and to characterize the new populations from continental Portugal and Madeira Island. Mitochondrial cytochrome b (cytb) and parasitic cellulase gene sequences were used to evaluate the genetic relationships among isolates that could indicate possible origins of the new outbreaks. Portuguese isolates were compared with isolates from USA, China, Japan and South Korea, in order to investigate possible infection pathways and disease spread patterns in Portugal. Phylogenetic trees based on both genes show that Portuguese isolates group with Asian isolates. Isolates from USA are in a separate position in both gene trees. However, the phylogenetic tree based on the cellulase gene sequences shows higher differentiation among Portuguese isolates than that of cytb. These results agree with those previously obtained using inter-simple sequence repeats (ISSR). This was the first study to use cytb and cellulase genes to characterize pinewood nematode (PWN) populations. This study suggests that cellulase is a better marker than cytb to study genetic diversity in B. xylophilus

Perspectives on the use of plant growth promoting bacteria as biocontrol agents for the pine wilt disease

Pine Wilt Disease (PWD) has been considered one of the major threats affecting conifer forests and forestry economics throughout the whole world. This complex disease is caused by the pinewood nematode (PWN) Bursaphelenchus xylophilus, through an intricate interaction with other partners like nematode associated bacteria and an insect vector that is responsible for nematode tree-to-tree transportation and dissemination. The first report of PWD dates to 1905 in Japan. Later, the PWD spread out to China (1982) and Korea (1988). In Europe, PWD was firstly reported in Portugal in 1999. Since then the disease has spread throughout the country and recently new PWD focus were found in Madeira Island (2008) and Spain (2010), thus indicating an elevated risk to other European countries (including the Scandinavia region) and forestlands. Although efforts for controlling and understanding PWD have been made, till now there are no significant solutions rather than the destruction of infected trees and forestlands, leading to huge losses, i.e 26 million m3 of timber in Japan, which is both economic and ecologically unacceptable. Therefore, new solutions are needed in order to control PWD. Some of the disease control methods developed rely on the use of chemicals with nematicidal effects. Plant breeding programs are also being conducted in order to obtain resistant pine trees. Yet, these approaches can endure as also they can be non-environmental friendly. In other plant diseases caused by plant parasitic nematodes the use of plant growth promoting bacteria (PGPB) as biocontrol agents is considered to be an interesting and effective green-approach. In this sense, the results obtained in our lab indicate that the inoculation of Pinus pinaster (maritime pine) seedlings with PGPB leads to a reduction of PWD development, as also to an increased plant growth. These results suggest that the inoculation of pine seedlings with PGPB in a “nursery” system can be a useful alternative in obtaining PWN resilient pine trees

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

Nematoxic effect of essential oils and their fractions against the pinewood nematode, Bursaphelenchus xylophilus

The pinewood nematode (PWN) Bursaphelenchus xylophilus is a highly pathogenic plant parasite that greatly affects pine forests. In Portugal, the most affected species is Pinus pinaster Aiton. Despite great efforts, since its fist detection in 1999, the PWN has spread through the country, including Madeira Island, having been recently detected in Spain [1,2]. Containing this pest is of the utmost importance for European pine forest safeguard. Since most synthetic chemicals used to control phytoparasites are toxic to humans and animals, and can accumulate in the soil and in food plants [3], in the present work, the nematoxic potential of over 80 essential oils (EOs), isolated from the Portuguese flora, were assessed against the PWN. EOs were isolated by hydrodistillation and analysed by GC and GC-MS [3]. EOs hydrocarbon and oxygen-containing fractions were obtained as in [4]. Direct-contact assays, adapted from [3], were performed by adding EOs/methanol stock-solutions to 50-100 mixed-stage PWN suspensions. After 24h in darkness, dead and live nematodes were counted under an inverted microscope. Assays were repeated at least 10 times in two series. Mortalities ≥96% were obtained with 2μL/mL of the EOs isolated from Cymbopogon citratus, Eucalyptus citriodora, Mentha arvensis, Origanum virens, Origanum vulgare, Ruta graveolens, Satureja montana, Syzygium aromaticum, Thymbra capitata, Thymus caespititius (carvacrol and/or thymol-rich), Thymus vulgaris and Thymus zygis. These EOs were further tested at 1, 0.5 and 0.25μL/mL. Minimum lethal concentrations (LC100) <0.4μL/mL, were obtained for the 2-undecanone-rich R. graveolens EO and the carvacrol and γ-terpinene-rich S. montana and T. capitata EOs. Assays with EO fractions revealed that the monoterpene-rich nematoxic EOs control PWN through their combined hydrocarbon and oxygen-containing fractions through additive and/or synergic relations. As complex mixtures of active components, EOs may prove to be effective nematoxic age nts

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