Metodologia para o Estudo de Fármacos Vegetais

O mecanismo de actuação, a toxicidade e os efeitos clínicos permanecem desconhecidos para a maioria dos fármacos vegetais, assentando o seu uso no conhecimento tradicional. É porém frequente encontrar plantas tóxicas que, quando ingeridas inapropriadamente e sem acompanhamento técnico, podem ser letais. A identificação micromorfológica é útil na inventariação da diversidade e no reconhecimento de caracteres diferenciadores de fármacos vegetais. O recurso a metodologias no âmbito da biologia molecular é uma mais-valia em relação ao complexo, moroso e dispendioso estudo fitoquímico. Usando quantidades de material muito pequenas, elas constituem também um avanço na certificação da qualidade, segurança e reprodutibilidade da composição de fármacos. Como exemplo da sua aplicação, apresentamos um estudo realizado com plantas endémicas de Cabo Verde, usadas na medicina tradicional, Tornabenea insularis e T. annua. Estas espécies apresentam problemas taxonómicos, o que também levanta questões na sua correcta utilização. Através da combinação dos microcaracteres foliares e dos frutos bem como da extracção de DNA e amplificação por PCR da região ITS e do gene 26S rDNA, os nossos resultados não permitem a separação dos dois taxa, não confirmando assim a actual classificação

species identification, distribution, and new host records in Portugal

Funding Information: This research was supported by the “Fundação para a Ciência e a Tecnologia” (FCT, Portugal) and the European Social Funds, through the “Programa Operacional Regional Centro”, under the Ph.D. fellowship 2020.05541.BD and through the R&D Unit, UIDB/04551/2020 (GREEN-IT—Bioresources for Sustainability). Acknowledgments Funding Information: This research was supported by the “Fundação para a Ciência e a Tecnologia” (FCT, Portugal) and the European Social Funds, through the “Programa Operacional Regional Centro”, under the Ph.D. fellowship 2020.05541.BD and through the R&D Unit, UIDB/04551/2020 (GREEN-IT—Bioresources for Sustainability). Publisher Copyright: Copyright © 2023 Rusinque, Camacho, Serra, Nóbrega and Inácio.Considered one of the most devastating plant parasitic nematodes worldwide, Meloidogyne spp. (commonly known as the root-knot nematodes (RKNs)) are obligate sedentary endoparasites that establish in the roots, causing hyperplasia and hypertrophy of surrounding cells, triggering the formation of galls. These galls will affect root development and physiology, leading to substantial yield losses. During 2017–2022, an extensive survey of Meloidogyne species was undertaken in Portugal (mainland and islands). A total of 1,071 samples were collected by the National Plant Protection Organization (DGAV) and private farmers from different regions of the country and were analysed at the Laboratory of Nematology (NemaINIAV). Samples in which the presence of Meloidogyne sp. was detected were used to perform bioassays to obtain females and juveniles for further studies. Since the accurate identification of RKNs is an important aspect of crop management, morphological and biochemical characterisation was performed. The most common morphological features were observed, showing consistency with previous descriptions of the genus. The biochemical identification using the esterase (EST) phenotype revealed the phenotypes of Meloidogyne arenaria, M enterolobi, M. hispanica, M. hapla, M. incognita, M javanica, and M. luci. Meloidogyne incognita and M. javanica were found to be the most prevalent species in the different regions followed by M. arenaria and M. hapla. This is the first distribution report performed in Portugal on RKNs, contributing to the development of management strategies and to updated information on the status of these pests in Europe.publishersversionpublishe

Root-Knot Nematode Species Associated with Horticultural Crops in the Island of Azores, Portugal

Plant-parasitic nematodes (PPN) are an economically important group of plant pests present throughout the world. In particular, root-knot nematodes (RKN), sedentary endoparasites of a wide variety of economically important hosts with the ability to survive in temperate regions. During 2020–2021 an extensive survey of Meloidogyne spp. was undertaken on the island of São Miguel Azores, Portugal. A total of 80 samples comprising 23 species of plants were collected from 13 localities in 4 districts of the island. Samples included field and greenhouse vegetable crops. Bioassays were carried out to obtain females and juveniles for morphological and biochemical characterization of the isolates. The observed morphological features showed high similarity and consistency with previous descriptions of the genus. Concerning the biochemical characterization, the esterase (EST) phenotype displayed the patterns described for M. incognita, M. arenaria, and M. javanica. Meloidogyne incognita was found to be the most prevalent species with respect to both natural host range and geographical distribution, followed by M. arenaria and M. javanica. This is the first field survey that assesses the species of root-knot nematodes associated with horticultural crops in the Azores Island, contributing additional information on the distribution of this genusinfo:eu-repo/semantics/publishedVersio

Genetic diversity assessment among Corema album (L.) D. Don (Ericaceae) genotypes based on ISSR markers and agro-morphological traits

Research ArticleCorema album (L.) D. Don is the only species of the genus Corema growing naturally on sand dunes throughout the Atlantic coast of the Iberian Peninsula, noted for the white coloured berries and nutritional value. The lack of information on genetic studies of the species is one of the most limitations for the establishment of this species as a new culture. Thus, this study focuses on the assessment of the genetic diversity based on inter-simple sequence repeats (ISSR) molecular markers and morphological traits. Seventy-one female plants, from four different sampled sites, were evaluated using six ISSR loci and eight morphological traits. Fifty polymorphic loci were detected. The dendrogram based on the UPGMA method and the principal coordinate analysis classified the 71 C. album genotypes into distinct clusters. The analyses revealed that accessions from the same geographical area were generally, but not entirely, clustered into the same group. Analysis of molecular variance was higher among populations, than within populations. The analysis of morphological traits revealed that there is no distinct separation among the C. album genotypes grown in different geographic areas. To our knowledge, this is the first study on the assessment of the genetic diversity in this speciesinfo:eu-repo/semantics/publishedVersio

Nematode-Mycobiota interactions in Pine Wilt Disease

Pine wilt disease is one of the most important diseases for conifer forests worldwide. This complex disease involves the interaction between three primary biological elements - the plant parasitic nematode Bursaphelenchus xylophilus; the insect-vector Monochamus sp., and the host tree Pinus spp. – and other secondary elements such as endophytic bacteria and fungi. The development of B. xylophilus is strongly associated with fungi that colonize the declining trees, with special impact in their reproduction and number of individuals carried by the vector. In light of previous knowledge, we are focused in obtaining a detailed characterization of the structure and dynamics of the nematode-fungi interactions through culturable and non-culturable approaches with particular emphasis in metagenomics analysis. Our aim is to understand if nematode-associated mycobiota plays a key-role in the development of the disease, in interaction with nematode and insect-vector, and into which extend it can be used to disrupt the disease cycle

PineEnemy - Exploring the NEmatode-MYcobiota interactions in Pine Wilt Disease

Pine wilt disease (PWD) is one of the most important threats to conifer forests in Europe [1] and worldwide [2], causing severe economic and environmental damages. This complex disease results from the interaction between three biological elements: the pathogenic agent Bursaphelenchus xylophilus, also known as pinewood nematode (PWN); the insect-vector (cerambycid beetles of the genus Monochamus) [3], and the host tree, mostly from Pinus spp. [4]. In 1999, B. xylophilus was firstly identified in maritime pines (Pinus pinaster) in Portugal and in Europe [1, 5]. Despite a nationwide monitoring system, enforcement of strict phytosanitary measures and constraints implemented after PWN detection, this devastating problem has spread quickly in Portuguese mainland, Madeira Island [5] and Northern Spain [6]. As a migratory endoparasite, once inside susceptible tree, PWN has the ability to feed on the living parenchyma of the epithelial cells in the resin canals, causing a reduction in water flux and ultimately cessation of resin flow. In the later stages of the disease, PWN assumes a mycetophagous phase. The development of the PWN population appears to be strongly associated with fungi that colonize the declining trees [3]. Fungi harboured in weakened pines are seen as essential for the ongoing development and completion of the PWN life-cycle, affecting not only PWN reproduction [8] but also the number of individuals carried by the insect-vector [9-10]. Through a spatio-temporal analysis, PineEnemy will focus on the characterization of the structure and dynamics of the nematode-fungi interactions through culturable and non-culturable approaches with special emphasis in metagenomics analysis. Our aim is to understand if PWN-associated mycobiota plays a key-role in the development of PWD, in interaction with PWN and insect-vector, and into which extend can be targeted to disrupt the disease cycle

Fungal Communities of the Pine Wilt Disease Complex: Studying the Interaction of Ophiostomatales With Bursaphelenchus xylophilus

Original ResearchConsidered one of the most devastating plant–parasitic nematodes worldwide, Bursaphelenchus xylophilus (commonly known as pinewood nematode, PWN) is the causal agent of the pine wilt disease in the Eurasian coniferous forests. This migratory parasitic nematode is carried by an insect vector (Monochamus spp.) into the host tree (Pinus species), where it can feed on parenchymal cells and reproduce massively, resulting in the tree wilting. In declining trees, PWN populations are strongly dependent on fungal communities colonizing the host (predominantly ophiostomatoid fungi known to cause sapwood blue-staining, the blue-stain fungi), which not only influence their development and life cycle but also the number of individuals carried by the insect vector into a new host. Our main aim is to understand if PWN-associated mycobiota plays a key role in the development of PWD, in interaction with the PWN and the insect vector, and to what extent it can be targeted to disrupt the disease cycle. For this purpose, we characterized the fungal communities of Pinus pinaster trees infected and non-infected with PWN in three collection sites in Continental Portugal with different PWD temporal incidences. Our results showed that non-infected P. pinaster mycoflora is more diverse (in terms of abundance and fungal richness) than PWN-infected pine trees in the most recent PWD foci, as opposed to the fungal communities of long-term PWD history sites. Then, due to their ecological importance for PWN survival, representatives of the main ophiostomatoid fungi isolated (Ophiostoma, Leptographium, and Graphilbum) were characterized for their adaptative response to temperature, competition in-between taxa, and as food source for PWN. Under the conditions studied, Leptographium isolates showed promising results for PWN control. They could outcompete the other species, especially O. ips, and significantly reduce the development of PWN populations when compared to Botrytis cinerea (routinely used for PWN lab culturing), suggesting this to be a natural antagonist not only for the other blue-stain species but also for the PWNinfo:eu-repo/semantics/publishedVersio

Fungal communities of the Pine Wilt Disease Complex: Studying the Interaction of Ophiostomatales with Bursaphelenchus xylophilus

Considered one of the most devastating plant–parasitic nematodes worldwide, Bursaphelenchus xylophilus (commonly known as pinewood nematode, PWN) is the causal agent of the pine wilt disease in the Eurasian coniferous forests. This migratory parasitic nematode is carried by an insect vector (Monochamus spp.) into the host tree (Pinus species), where it can feed on parenchymal cells and reproduce massively, resulting in the tree wilting. In declining trees, PWN populations are strongly dependent on fungal communities colonizing the host (predominantly ophiostomatoid fungi known to cause sapwood blue-staining, the blue-stain fungi), which not only influence their development and life cycle but also the number of individuals carried by the insect vector into a new host. Our main aim is to understand if PWN-associated mycobiota plays a key role in the development of PWD, in interaction with the PWN and the insect vector, and to what extent it can be targeted to disrupt the disease cycle. For this purpose, we characterized the fungal communities of Pinus pinaster trees infected and non-infected with PWN in three collection sites in Continental Portugal with different PWD temporal incidences. Our results showed that non-infected P. pinaster mycoflora is more diverse (in terms of abundance and fungal richness) than PWN-infected pine trees in the most recent PWD foci, as opposed to the fungal communities of long-term PWD history sites. Then, due to their ecological importance for PWN survival, representatives of the main ophiostomatoid fungi isolated (Ophiostoma, Leptographium, and Graphilbum) were characterized for their adaptative response to temperature, competition in-between taxa, and as food source for PWN. Under the conditions studied, Leptographium isolates showed promising results for PWN control. They could outcompete the other species, especially O. ips, and significantly reduce the development of PWN populations when compared to Botrytis cinerea (routinely used for PWN lab culturing), suggesting this to be a natural antagonist not only for the other blue-stain species but also for the PWN

How parasitism genes are regulated: a motif to search for genes regulators in the plant parasitic nematode Bursaphelenchus xylophilus

Plant-parasitic nematodes threaten global agricultural and forestry systems. The search for new control strategies in line with the EU’s sustainability goals highlight significant knowledge gaps. Like all other plant pathogens, plant-parasitic nematodes deliver several parasitism proteins (effectors) into the host plant to cause disease. NemaWAARS project focuses on mechanism(s) of regulation and gene control expression of parasitism genes in pinewood nematode, Bursaphelenchus xylophilus. From the previous transcriptomic data derived from the pharyngeal gland cells (considered a specialized tissue potentially related to parasitism) we have identified a non-coding DNA motif - STATAWAARS - associated in the promotor region of highly abundant and secreted expressed genes. Given that this non-coding genetic signature unifies many sequences of unrelated parasitism genes, it implies the existence of a potential major regulator(s), that binds to this sequence to control the expression of downstream genes. We hypothesize that by disrupting this regulator(s), it would be possible to simultaneously disrupt the expression of many associated parasitism-related genes. To test the hypothesis the project aims to identify proteins (or complex of proteins) that bind in the promoter regions of parasitism-related genes (in vivo) or identify other regulatory candidates for master regulators of parasitism-related genes expression that are enriched in the pharyngeal gland cell tissues. For the best candidate regulatory proteins, an RNAi approach will target the selected gene candidates and evaluate the regulatory role in effector genes expression and in interaction with the host (in planta). Under an ongoing national and international collaborative network, the strategy in NemaWAARS will include innovative approaches to explore the regulators that govern effector gene expression applied in B. xylophilus research

First report of Pratylenchus penetrans (Nematoda: Pratylenchidae) associated with amaryllis (Hippeastrum x hybrid), in Portugal

The world flower business is a highly dynamic industry and in Portugal yields around EUR 500 million a year (INE 2013). Root lesion nematodes (RLNs), Pratylenchus spp., are considered the third most important group of plant parasitic nematodes (PPNs) worldwide due to their large distribution, wide host range, and impact on economically important crops (Castillo and Vovlas 2007). The most recent report of RLNs parasitizing amaryllis (Hippeastrum spp.) was the amaryllis lesion nematode, Pratylenchus hippeastri, in Florida (U.S.A.) (Gozel et al. 2007). In 2016, soil and root samples of amaryllis hybrid Hippeastrum × hybridum cv. Benfica were collected from potted plants produced in greenhouses in Montijo (Setúbal Peninsula, Central Portugal), 50 km from Lisbon. Nematodes were extracted from soil samples according to the protocol PM 7/119 (1) (EPPO 2013) and identified to genus level based on morphometrical traits of 10 females and 11 males. DNA extraction was conducted from 10 nematodes separately, followed by PCR amplification and sequencing of the D2/D3 expansion segment of the 28S rRNA and 18S rRNA genes using the primers D2A and D3B (Nadler et al. 1999) and 1813F and 2646R (Holterman et al. 2006). The nucleotide sequences were deposited as MN756841 and MN756509 at the NCBI. RLNs were detected in all five samples in a concentration of around 100 nematodes/100 cm3 of soil. Some primary roots of affected plants showed small, elliptical, reddish-brown lesions and poor growth. More severely affected plants presented stunted, chlorotic foliage and had the cortical lesions enlarged to the point that rotting was severe and many roots were easily detached from the bulb; in some cases, there was not emergence from bulbs. The female body was moderately slender, almost straight when killed by gentle heat, lateral field with four straight lines, body annules distinct, lip with three annules, stylet knobs round, median bulb round, spermatheca round and filled with sperm, tail tapering to rounded terminus, sometimes with two striae. Body length was 558.99 ± 48.50 μm, body width 22.45 ± 2.02 μm, stylet length 18.52 ± 1.02 μm, and tail length 32.91 ± 7.50 μm. Values of a, c, and c′ ratios were 25.95 ± 2.53, 17.62 ± 3.45, and 38.49 ± 4.42; V (%) value was 80.92 ± 1.69. Males were smaller than females with body length 511.37 ± 29.90 μm, stylet length 17.55 ± 1.04 μm, and spicule length 18.53 ± 1.17 μm. No other PPN was found in the suspensions extracted from the soil samples. BLAST hits showed that partial sequence of D2/D3 rRNA loci (540 bp) was 96.2% identical (e-value 0.0) to Pratylenchus penetrans isolate MU2 (KP161612) and voucher T143 (KY828357). The partial sequence of 18S rRNA (650 bp) was 100% identical to P. penetrans isolate ILVO-Pp (MH983023) and 99.81% to P. penetrans Praty-A (AB661627). In the corresponding phylogenetic trees, both P. penetrans sequences clustered together with other species from the penetrans group (P. fallax, P. convallariae, and P. oleae) with good support (Janssen et al. 2017). Nevertheless, although all species within this group share many morphological characteristics (Subbotin et al. 2008), our specimens presented higher similarity, both morphological and molecular, with P. penetrans. So far, this RLN has been found associated mainly with potato fields in Portugal (Abrantes et al. 1987; Esteves et al. 2015); thus, to our knowledge, this is the first report of P. penetrans parasitizing amaryllis, in Portugal