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.

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.

IDENTIFICAÇÃO POLIFÁSICA DE GONGRONELLA SP. E DE RHIZOPUS SP. DEGRADADORES DO FUNGICIDA SISTÉMICO METALAXIL

A capacidade destes fungos degradarem compostos xenobióticos e recalcitrantes é uma característica biotecnologicamente importante tornando-os potencialmente úteis para processos de biorremediação. Gongronella sp. e Rhizopus sp. foram isolados do solo de vinhas da região do Alentejo, Portugal. Estes isolados mostraram elevada capacidade de degradarem o fungicida acilalanina metalaxil. No presente estudo, para a identificação polifásica de Gongronella sp. e Rhizopus sp., presuntivamente identificado como R. stolonifer, várias linhagens de referência da ordem Mucorales (Absidia, Circinella, Gongronella e Rhizopus) foram incluídas. A abordagem polifásica combinou a análise das macro- e micromorfologias, a análise da sequência inteira da região ITS ribossomal (i.e., ITS1/5.8S rDNA/ITS2) e ainda o uso da espectrometria de massas por Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF).

First detection of Bursaphelenchus luxuriosae associated with Pinus pinaster in Portugal

During a field survey carried out on symptomatic maritime pine trees (Pinus pinaster) in Góis, central Portugal, Bursaphelenchus luxuriosae was isolated for the first time in Portugal, and in Europe. Identification of the nematodes was based on morphological characters and molecular analyses for this species. The general morphology of both females and males is in agreement with the original description for B. luxuriosae, namely the typical morphology of the male spicules and the conspicuous morphology of female tail. Species identification was confirmed through sequencing of the ITS rDNA region and the fragment spanning the D2/D3 domain of the 28S rDNA gene. This species belongs to the xylophilus-group and is the third species in this group known in Portugal. The nematodes were retrieved in small numbers (<100 nematodes/100 g dry wood), and no insect vector association could be established.

Genetic diversity of Bursaphelenchus cocophilus in South America

Molecular characterisation of Bursaphelenchus cocophilus, the causal agent of ‘red ring disease’, is imperative for efficient identification procedures in Brazil and Colombia, because quarantine species such as B. xylophilus and B. mucronatus are already listed in both countries. ITS-1/2 region and D2-D3 segment of LSU rDNA were used to characterise isolates of B. cocophilus obtained from coconut plantations in Brazil and Colombia. Results from ITS-1/2 and LSU rDNA regions showed that all isolates of B. cocophilus from Brazil and Colombia formed a monophyletic group. The LSU rDNA region indicated that all isolates formed a single monophyletic group with high Bayesian posterior probability (100%). This is the first study on ITS-1/2 for the characterisation of B. cocophilus populations. A species-specific primer was designed for identification of B. cocophilus.

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.

Cytogenetic characterization of the Passiflora edulis Sims x Passiflora cincinnata Mast. interspecific hybrid and its parents.

The genus Passiflora L. consists of approximately 530 widely distributed species, including Passiflora edulis, which has drawn interest because of its commercial and agronomic value. Passiflora cincinnata is another important species owing to its long flowering period and resistance or tolerance to diseases and pests. In the present study, the meiotic segregation and pollen viability of an interspecific hybrid (P. edulis x P. cincinnata) and its parents were analyzed. The genomic contents were characterized using chromomycin A3 (CMA3)/40-60-diamidino-2-phenylindole (DAPI) staining, fluorescent in situ hybridization with 5S/45S ribosomal DNA (rDNA), genomic in situ hybridization (GISH), and inter-simple sequence repeat (ISSR) markers. The results indicated the diploid chromosome number for the parents and interspecific hybrid was 2n = 18. We also observed regular meiosis, one pair of S rDNA sites, and two pairs of 45S rDNA sites that colocalized with two pairs of CMA3 /DAPI- bands. The GISH data revealed three distinct chromosomal groups in the hybrid. The genetic origins of the interspecific hybrid, and its relationship with its parents were also confirmed using ISSR markers.

Mycosphaerella areola - the Teleomorph of Ramularia areola of cotton in Brazil, and its epidemiological significance.

2016