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

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

Formulación bioinsecticida a partir del aceite esencial de ambrosia arborescens mill (altamisa) de aplicación canina

La presente investigación se planteó reemplazar el uso de insecticidas sintéticos, formulando un champú bioinsecticida de aplicación canina mediante la acción biocida del aceite esencial deAmbrosia arborescens Mill (Altamisa). La planta se recolectó en las laderas del rio Tomebamba, cercanas al Campus Balzay de la Universidad de Cuenca Parroquia San Joaquín. La recolección se realizó durante los meses de Enero a Marzo del 2016. El desarrollo y formulación del producto se realizó en el Laboratorio de Biotecnología, Facultad de Ciencias Químicas de la Universidad de Cuenca. La obtención del aceite esencial de A. arborescens se realizó mediante hidrodestilación por el método Clevenger, con un rendimiento del 0,14%. La actividad biocida se estableció en un ensayo “in vitro” ante el nematodo Panagrellus redivirus, determinándose la dosis letal (DL50) de 250 uL/mL. Debido a la moderada DL50y bajo rendimiento, se planteó como estrategia, determinar el DL50 del extracto orgánico de A. arborescens, el cual se obtuvo mediante una extracción con metanol, consiguiendo un rendimiento del 2 % y DL50de 31,25 uL/mL. De acuerdo estos resultados se procedió a realizar pruebas en pulgas de perros(Ctenocephalides canis) con el extracto de A. arborescens, estableciendo una efectividad del 100 % a la concentración de 46,875 mg/mL en el periodo de tiempo más corto, siendo esta la dosis aplicada para la formulación del champú. El extracto metanólico de A. arborescens presentó elevada actividad biocida, comparado con el aceite esencial. Esta sustancia activa es promisoria en la formulación de bioinsecticidas para mascotas.

Evaluation of the reaction of watermelon parent and F1 plants to Meloidogyne enterolobii.

The aim of this study was to evaluate the performance of progenies from Citrullus lanatus var. lanatus (cultivated watermelons) when crossed with progenies from C. lanatus var. citroides (fodder watermelon with a historic of resistance to the nematode Meloidogyne enterolobii). The parents and their F1s were evaluated for resistance to this nematode. In the initial stages of eleven treatments, watermelon seedlings plantlets were transplanted to plastic bags of six kilograms once the first leaves developed. Ten inoculated plants with 5,200 eggs in the soil near the stem of the plant and four non-inoculated ones were used in each treatment, in a complete block design. Sixty-two days after sowing, the following characteristics were evaluated: the length of the aerial part of the plant (LAP, in m), fresh mass of the aerial part (FMAP, in g), root fresh mass (RFM, in g), egg number (EN) and reproduction factor (RF). A comparison between the averages of inoculated and non-inoculated plants was performed using Scott-Knott test at 5% and the diallelic analysis was performed using the GENES program. The morphological characteristics did not allow for the identification of the parent plants or the F1s with respect to nematode resistance, but the variables EN and RF were useful for such identification. The analyses of the general and specific combining abilities indicate highly significant effects with respect to this resistance, showing additive gene effects as well as dominance and epistatic gene effects, allowing for identification of parents and F1s that can be used in watermelon breeding programs to improve resistance to the M. enterolobii.

Genome selection in fruit breeding: application to table grapes.

Genomic selection (GS) has recently been proposed as a new selection strategy which represents an innovative paradigm in crop improvement, now widely adopted in animal breeding. Genomic selection relies on phenotyping and high-density genotyping of a sufficiently large and representative sample of the target breeding population, so that the majority of loci that regulate a quantitative trait are in linkage disequilibrium with one or more molecular markers and can thus be captured by selection. In this study we address genomic selection in a practical fruit breeding context applying it to a breeding population of table grape obtained from a cross between the hybrid genotype D8909-15 (Vitis rupestris × Vitis arizonica/girdiana), which is resistant to dagger nematode and Pierce?s disease (PD), and ?B90-116?, a susceptible Vitis vinifera cultivar with desirable fruit characteristics. Our aim was to enhance the knowledge on the genomic variation of agronomical traits in table grape populations for future use in marker-assisted selection (MAS) and GS, by discovering a set of molecular markers associated with genomic regions involved in this variation. A number of Quantitative Trait Loci (QTL) were discovered but this method is inaccurate and the genetic architecture of the studied population was better captured by the BLasso method of genomic selection, which allowed for efficient inference about the genetic contribution of the various marker loci. The technology of genomic selection afforded greater efficiency than QTL analysis and can be very useful in speeding up the selection procedures for agronomic traits in table grapes.

Espécies de Meloidogyne em hortaliças e outras culturas provenientes de áreas periurbanas da África Subsaariana

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Fitopatologia, Programa de Pós-Graduação em Fitopatologia, 2016.

Multiplicação do nematoide Meloidogyne paranaensis e velocidade de enraizamento de estacas caulinares em sete espécies de plantas medicinais

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Agronomia e Medicina Veterinária, Programa de Pós-Graduação em Agronomia, 2016.

Nematóide (Heterodera glycines Ichinohe) formador de cisto em soja.

O nematóide Heterodera glycines Ichinohe é considerado o parasita mais destrutivo da soja (Glycine max (L.) Merrill). A doença causada pela infestação desse nematóide, primeiramente obserada no Japão, recebeu a denominção de "nanismo amarelo", devido aos sintomas por ela produzidos nas plantas de soja infecatadas.

Screening of the 'pathogen box' identifies an approved pesticide with major anthelmintic activity against the barber's pole worm

There is a substantial need to develop new medicines against parasitic diseases via public-private partnerships. Based on high throughput phenotypic screens of largely protozoal pathogens and bacteria, the Medicines for Malaria Venture (MMV) has recently assembled an open-access 'Pathogen Box' containing 400 well-curated chemical compounds. In the present study, we tested these compounds for activity against parasitic stages of the nematode Haemonchus contortus (barber's pole worm). In an optimised, whole-organism screening assay, using exsheathed third-stage (xL3) and fourth-stage (L4) larvae, we measured the inhibition of larval motility, growth and development of H. contortus. We also studied the effect of the 'hit' compound on mitochondrial function by measuring oxygen consumption. Among the 400 Pathogen Box compounds, we identified one chemical, called tolfenpyrad (compound identification code: MMV688934) that reproducibly inhibits xL3 motility as well as L4 motility, growth and development, with IC50 values ranging between 0.02 and 3 μM. An assessment of mitochondrial function showed that xL3s treated with tolfenpyrad consumed significantly less oxygen than untreated xL3s, which was consistent with specific inhibition of complex I of the respiratory electron transport chain in arthropods. Given that tolfenpyrad was developed as a pesticide and has already been tested for absorption, distribution, excretion, biotransformation, toxicity and metabolism, it shows considerable promise for hit-to-lead optimisation and/or repurposing for use against H. contortus and other parasitic nematodes. Future work should assess its activity against hookworms and other pathogens that cause neglected tropical diseases.

Development of a novel molecular tool for the rapid assessment of the biodiversity changes of benthic nematodes assemblages. Mares Conference Marine Ecosystems Helath and conservation

The molecular profiling system was developed using directed terminal-restriction fragment length polymorphism (dT-RFLP) to characterize soil nematode assemblages by relative abundance of feeding guilds and validation by comparison to traditional morphological method. The good performance of these molecular tools applied to soil nematodes assemblages create an opportunity to develop a novel approach for rapid assessment of the biodiversity changes of benthic nematodes assemblages of marine and estuarine sediments. The main aim of this research is to combine morphological and molecular analysis of estuarine nematodes assemblages, to establish a tool for fast assessment of the biodiversity changes within habitat recovery of Zostera noltii seagrass beds; and validate the dT-RFLP as a high-throughput tool to assess the system recovery. It was also proposed to develop a database of sequences related to individuals identified at species level to develop a new taxonomic reference system. A molecular phylogenetic analysis of the estuarine nematodes has being performed. After morphological identification, barcoding of 18S rDNA are being determined for each nematode species and the results have shown a good degree of concordance between traditional morphology-based identification and DNA sequences. The digest strategy developed for soil nematodes is not suitable for marine nematodes. Then five samples were cloned and sequenced and the sequence data was used to design a new dT-RFLP strategy to adapt this tool to marine assemblages. Several solutions were presented by DRAT and tested empirically to select the solution that cuts most efficiently, separating the different clusters. The results of quantitative PCR showed differences in nematode density between two sampling stations according the abundance of the nematode density obtained by the traditional methods. These results suggest that qPCR could be a robust tool for enumeration of nematode abundance, saving time.

Assessment of the Availability of Forest Biomass for Bioenergy Production in Southwest Portugal.

In 2014, Portugal was the seventh largest pellets producer in the World. Since the shortage of raw material is one of the major obstacles that the Portuguese sellets market faces, the need for a good assessment of biomass availability for energy purposes at both country and regional levels is reinforced. This work uses a Geographical Information System environment and remote sensing data to assess the availability and sustainability of forest biomass residues in a management unit with around 940 ha of maritime pine forest. The period considered goes from 2004 to 2015. The study area is located in Southwestern Portugal, close to a pellets factory; therefore the potential Contribution of the residual biomass generated in the management unit to the production of pellets is evaluated. An allometric function is used for the estimation of maritime pine above ground biomass. With this estimate, and considering several forest operations, the residual biomass available was assessed, according to stand composition and structure. This study shows that, when maritime pine forests are managed to produce wood, the amount of residues available for energy production is small (an average of 0.37 t ha -1  year -1 were generated in the study area between 2004 and 2015). As a contribution to the sustainability of the Portuguese pellets industries, new management models for maritime pine forests may be developed. The effect of the pinewood nematode on the availability of residual biomass can be clearly seen in this study. In the management unit considered, cuts were made to prevent dissemination of the disease. This contributes to a higher availability of forest residues in a specific period of time, but, in the medium term, they lead to a decrease in the amount of residues that can be used for energy purposes.

Benthic nematodes biodiversity patterns along the gradient of the Tagus estuary (Western Coast of Portugal): Link with abundances distribution pattern of the nonindigenous bivalve Ruditapes philippinarum

The non-indigenous bivalve Ruditapes philippinarum is native to the western Pacific Ocean and it has been one of the most widely introduced species for aquaculture purposes in Europe. In Portugal its presence is known in several estuaries for more than two decades and its populations have increased greatly along the last years1. Currently it’s one of the dominant benthic species in some areas of the Tagus estuary (264,1±470,8 ind./30s tow). Studies on the impacts of invasive bivalves on meiofaunal communities are scarce and restricted to the harvesting effect. Meiofauna of the Tagus estuary is poorly known and possible impacts of the introduction of R. philippinarum were never assessed.

Fixing, storage time and DNA extraction applied to dT-RFLP for ecological and molecular studies of marine nematodes assemblages

The application of molecular methods offers an alternative faster than traditional methods based on morphology It is nearly impossible to process all the samples in short period using traditional methods, and the deterioration of marine sediments rapidly occurs The dT-RFLP (directed Terminal-Restriction Fragment Length Polymorphism) allows a rapid assessment of biodiversity changes of nematodes assemblages The use of a not suitable fixing, storage time and DNA extraction could be a limitation in molecular analysis like dT-RFLP and real time PCR.Objetives: the best fixative •the level of DNA degradation over the time •the best DNA extraction method for marine nematodes and suitable for dT-RFLP analysis

Development of molecular approaches to assess the diversity and abundance of marine nematodes assemblages for assessment of Good Environmental Status of the estuarine and marine ecosystems

In Europe, the concerns with the status of marine ecosystems have increased, and the Marine Directive has as main goal the achievement of Good Environmental Status (GES) of EU marine waters by 2020. Molecular tools are seen as promising and emerging approaches to improve ecosystem monitoring, and have led ecology into a new era, representing perhaps the most source of innovation in marine monitoring techniques. Benthic nematodes are considered ideal organisms to be used as biological indicator of natural and anthropogenic disturbances in aquatic ecosystems underpinning monitoring programmes on the ecological quality of marine ecosystems, very useful to assess the GES of the marine environment. dT-RFLP (directed Terminal-Restriction Fragment Length Polymorphism) allows to assess the diversity of nematode communities, but also allows studying the functioning of the ecosystem, and combined with relative real-time PCR (qPCR), provides a high-throughput semi-quantitative characterization of nematode communities. These characteristics make the two molecular tools good descriptors for the good environmental status assessment. The main aim of this study is to develop and optimize the dT-RFLP and qPCR in Mira estuary (SW coast, Portugal). A molecular phylogenetic analysis of marine and estuarine nematodes is being performed combining morphological and molecular analysis to evaluate the diversity of free-living marine nematodes in Mira estuary. After morphological identification, barcoding of 18S rDNA and COI genes are being determined for each nematode species morphologically identified. So far we generated 40 new sequences belonging to 32 different genus and 17 families, and the study has shown a good degree of concordance between traditional morphology-based identification and DNA sequences. These results will improve the assessment of marine nematode diversity and contribute to a more robust nematode taxonomy. The DNA sequences are being used to develop the dT-RFLP with the ability to easily process large sample numbers (hundreds and thousands), rather than typical of classical taxonomic or low throughput molecular analyses. A preliminary study showed that the digest enzymes used in dT-RFLP for terrestrial assemblages separated poorly the marine nematodes at taxonomic level for functional group analysis. A new digest combination was designed using the software tool DRAT (Directed Terminal Restriction Analysis Tool) to distinguished marine nematode taxa. Several solutions were provided by DRAT and tested empirically to select the solution that cuts most efficiently. A combination of three enzymes and a single digest showed to be the best solution to separate the different clusters. Parallel to this, another tool is being developed to estimate the population size (qPCR). An improvement in qPCR estimation of gene copy number using an artificial reference is being performed for marine nematodes communities to quantify the abundance. Once developed, it is proposed to validate both methodologies by determining the spatial and temporal variability of benthic nematodes assemblages across different environments. The application of these high-throughput molecular approaches for benthic nematodes will improve sample throughput and their implementation more efficient and faster as indicator of ecological status of marine ecosystems.

Bioactivity of Ruta graveolens and Satureja montana Essential Oils on Solanum tuberosum Hairy Roots and Solanum tuberosum Hairy Roots with Meloidogyne chitwoodi Co-cultures

As a nematotoxics screening biotechnological system, Solanum tuberosum hairy roots (StHR) and S. tuberosum hairy roots with Meloidogyne chitwoodi co-cultures (StHR/CRKN) were evaluated, with and without the addition of the essential oils (EOs) of Satureja montana and Ruta graveolens. EOs nematotoxic and phytotoxic effects were followed weekly by evaluating nematode population density in the co-cultures as well as growth and volatile profiles of both in vitro cultures types. Growth, measured by the dissimilation method and by fresh and dry weight determination, was inhibited after EO addition. Nematode population increased in control cultures, while in EO-added cultures numbers were kept stable. In addition to each of the EOs main components, and in vitro cultures constitutive volatiles, new volatiles were detected by gas chromatography and gas chromatography coupled to mass spectrometry in both culture types. StHR with CRKN co-cultures showed to be suitable for preliminary assessment of nematotoxic EOs.

Evidences for an opportunistic and endophytic lifestyle of the Bursaphelenchus xylophilus -associated bacteria Serratia marcescens PWN146 isolated from wilting Pinus pinaster

Pine wilt disease (PWD) results from the interaction of three elements: the pathogenic nematode, Bursaphelenchus xylophilus; the insect-vector, Monochamus sp.; and the host tree, mostly Pinus species. Bacteria isolated from B. xylophilus may be a fourth element in this complex disease. However, the precise role of bacteria in this interaction is unclear as both plant-beneficial and as plant-pathogenic bacteria may be associated with PWD. Using whole genome sequencing and phenotypic characterization, we were able to investigate in more detail the genetic repertoire of Serratia marcescens PWN146, a bacterium associated with B. xylophilus. We show clear evidence that S. marcescens PWN146 is able to withstand and colonize the plant environment, without having any deleterious effects towards a susceptible host (Pinus thunbergii), B. xylophilus nor to the nematode model C. elegans. This bacterium is able to tolerate growth in presence of xenobiotic/organic compounds, and use phenylacetic acid as carbon source. Furthermore, we present a detailed list of S. marcescens PWN146 potentials to interfere with plant metabolism via hormonal pathways and/or nutritional acquisition, and to be competitive against other bacteria and/or fungi in terms of resource acquisition or production of antimicrobial compounds. Further investigation is required to understand the role of bacteria in PWD. We have now reinforced the theory that B. xylophilus-associated bacteria may have a plant origin.