Root-knot nematode assessment: species identification, distribution, and new host records in Portugal.

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.

Can Beauveria bassiana (Bals.-Criv.) Vuill. Control the Key Fruit Pests of the European Chestnut Tree, under Field Conditions?

The chestnut moth (Cydia splendana Hübner) and the chestnut weevil (Curculio elephas Gyllenhal) cause serious damage to European producers and companies that transform and market the chestnut. The objective of the present work was to evaluate, under field conditions, the possibilities of Beauveria bassiana (Bals.-Criv.) Vuill. to infect and kill the larvae of the two main carpophagous pests of European chestnut, in treatments directed at the soil. For this purpose, the surfaces of vases were sprayed with two concentrations of conidia/mL 5 × 107 (T1) and 1 × 108 (T2). The control (T0) was sprayed with distilled water. Larval mortality and infection were evaluated on five dates (D8 to D220). Confirmation of the fungus present in the larva was performed by molecular analysis. The results obtained are promising for the use of B. bassiana as a biological control agent against these key pests of the chestnut crop. There were no significant differences in mortality between the T1 and T2 modalities, however, they were significantly higher than the control. In the case of total mortality (dead and infected larvae), no significant differences were observed for C. elephas either. In the case of C. splendana, the T2 modality obtained better results in terms of total mortality.

The Northern Root-Knot Nematode Meloidogyne hapla: New Host Records in Portugal.

Root-knot nematodes (RKNs), Meloidogyne spp., are a group of plant-parasitic nematodes (PPNs) of great economic significance worldwide. The northern root-knot nematode, Meloidogyne hapla, is one of the most important species of RKNs occurring in cold regions. In Portugal so far, M. hapla has been found parasitizing potato and fig trees. During surveys carried out in 2019-2022 in fields for horticultural and ornamental production, soil and root samples were collected. Roots were observed under a stereomicroscope to determine the presence of galls. Nematodes were extracted from the soil. Morphological features showed a high similarity and consistency with previous descriptions of the genus. For molecular analysis, total genomic DNA was isolated from single nematodes and used to amplify in multiplex reaction using the species-specific primers JMV1, JMV2 and JMVhapla, and for sequencing of the ITS region with the primers TW81/AB28. Multiplex PCR amplification produced a specific fragment of 440 bp and PCR amplification of the ITS region yielded a single fragment of 550 bp, as expected. The obtained sequences showed a similarity ranging from 99.8% to 100% with the sequences of M. hapla available in the database. The phylogenetic tree revealed that the isolates grouped with M. hapla isolates. From the 690 samples collected, M. hapla was detected in three different hosts (grapevine, eucalyptus and potato) in four districts of mainland Portugal and on Madeira Island. To our knowledge, this is the first report of M. hapla infecting the grapevine and eucalyptus in Portugal.

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.

Ceratocystiopsis quercina sp. nov. Associated with Platypus cylindrus on Declining Quercus suber in Portugal.

Platypus cylindrus is the most common ambrosia beetle in stands of Quercus suber in Portugal. This insect farms specialized fungi in sapwood galleries, using its mycangia to carry and store these organisms. Some ectosymbiotic fungi carried by P. cylindrus are phytopathogenic and cause extensive tree mortality and severe economic losses. To understand the role of P. cylindrus fungal symbionts in stands of Q. suber we examined beetle galleries present in declining and/or dying cork oak trees during field surveys. Logs with active galleries were obtained in situ and from captured emerging beetles. Insects were aseptically dissected, and their mycangia and intestine were retrieved. Morphological and molecular profiles of fungal isolates obtained from cultured insect parts were carried out to accurately characterize and identify isolated fungi. Molecular characterizations were performed with DNA sequence data from four loci, i.e., LSU, SSU, 5.8S-ITS2-28S, and TUB. Morphological results consistently showed a collection of Ophiostoma-like fungal axenic isolates, while phylogenies inferred that this collection constitutes an undescribed taxon reported herein for the first time in association with P. cylindrus in Portuguese cork oak stands. The novel species was erected as Ceratocystiopsis quercina sp. nov. and constitutes a new phytopathogenic fungal species associated with symptoms of vegetative cork oak decline.

First Detection of Meloidogyne luci (Nematoda: Meloidogynidae) Parasitizing Potato in the Azores, Portugal.

Potato is the third most important crop in the world after rice and wheat, with a great social and economic importance in Portugal as it is grown throughout the country, including the archipelagos of Madeira and the Azores. The tropical root-knot nematode (RKN) Meloidogyne luci is a polyphagous species with many of its host plants having economic importance and the ability to survive in temperate regions, which pose a risk to agricultural production. In 2019, M. luci was detected from soil samples collected from the council of Santo António in Pico Island (Azores). Bioassays were carried out to obtain females, egg masses, and second-stage juveniles to characterize this isolate morphologically, biochemically, and molecularly. The observed morphological features and morphometrics showed high similarity and consistency with previous descriptions. Concerning the biochemical characterization, the esterase (EST) phenotype displayed a pattern with three bands similar to the one previously described for M. luci and distinct from M. ethiopica. Regarding the molecular analysis, an 1800 bp region of the mitochondrial DNA between cytochrome oxidase subunit II (COII) and 16S rRNA genes was analyzed and the phylogenetic tree revealed that the isolate grouped with M. luci isolates (99.17%). This is the first report of M. luci parasitizing potato in the Azores islands, contributing additional information on the distribution of this plant-parasitic nematode.

Potato Cyst Nematodes: Geographical Distribution, Phylogenetic Relationships and Integrated Pest Management Outcomes in Portugal.

The identification and phylogenetic relationships of potato cyst nematodes (PCN) were studied to assess the potential value of geographical distribution information for integrated pest management of potato production in Portugal. This research focused on PCN species, Globodera pallida and Globodera rostochiensis. From 2013 until 2019, 748 soil samples from the rhizosphere of different potato cultivars were surveyed in the Portuguese mainland to detect and identify both species and track their location. PCN are widespread invasive species throughout Portugal. In fact, during the survey period an incidence of 22.5% was estimated for the tested samples. The patterns of infestation vary among regions, increasing from south to north, where PCN were first detected. Currently, both species are present in all potato producing regions of the country, with a greater incidence of G. pallida. Phytosanitary control measures are influencing to the observed results. The use of potato cultivars resistant to G. rostochiensis led to a decrease of this species but had no influence on G. pallida detections, which continues its reproduction freely since there are no effective resistant cultivars for this species. The relationship between the presence, infestation rate, spread and geographical distribution of PCN is discussed in terms of behavioral responses of the potato cultivars and the implications for developing new integrated crop protection measures.

Characterization of the cork formation and production transcriptome in Quercus cerris × suber hybrids.

Cork oak is the main cork-producing species worldwide, and plays a significant economic, ecological and social role in the Mediterranean countries, in particular in Portugal and Spain. The ability to produce cork is limited to a few species, hence it must involve specific regulation mechanisms that are unique to these species. However, to date, these mechanisms remain largely understudied, especially with approaches involving the use of high-throughput sequencing technology. In this study, the transcriptome of cork-producing and non-cork-producing Quercus cerris × suber hybrids was analyzed in order to elucidate the differences between the two groups of trees displaying contrasting phenotypes for cork production. The results revealed the presence of a significant number of genes exclusively associated with cork production, in the trees that developed cork. Moreover, several gene ontology subcategories, such as cell wall biogenesis, lipid metabolic processes, metal ion binding and apoplast/cell wall, were only detected in the trees with cork production. These results indicate the existence, at the transcriptome level, of mechanisms that seem to be unique and necessary for cork production, which is an advancement in our knowledge regarding the genetic regulation behind cork formation and production.

The draft genome sequence of cork oak.

Cork oak (Quercus suber) is native to southwest Europe and northwest Africa where it plays a crucial environmental and economical role. To tackle the cork oak production and industrial challenges, advanced research is imperative but dependent on the availability of a sequenced genome. To address this, we produced the first draft version of the cork oak genome. We followed a de novo assembly strategy based on high-throughput sequence data, which generated a draft genome comprising 23,347 scaffolds and 953.3 Mb in size. A total of 79,752 genes and 83,814 transcripts were predicted, including 33,658 high-confidence genes. An InterPro signature assignment was detected for 69,218 transcripts, which represented 82.6% of the total. Validation studies demonstrated the genome assembly and annotation completeness and highlighted the usefulness of the draft genome for read mapping of high-throughput sequence data generated using different protocols. All data generated is available through the public databases where it was deposited, being therefore ready to use by the academic and industry communities working on cork oak and/or related species.

A comprehensive assessment of the transcriptome of cork oak (Quercus suber) through EST sequencing.

BACKGROUND: Cork oak (Quercus suber) is one of the rare trees with the ability to produce cork, a material widely used to make wine bottle stoppers, flooring and insulation materials, among many other uses. The molecular mechanisms of cork formation are still poorly understood, in great part due to the difficulty in studying a species with a long life-cycle and for which there is scarce molecular/genomic information. Cork oak forests are of great ecological importance and represent a major economic and social resource in Southern Europe and Northern Africa. However, global warming is threatening the cork oak forests by imposing thermal, hydric and many types of novel biotic stresses. Despite the economic and social value of the Q. suber species, few genomic resources have been developed, useful for biotechnological applications and improved forest management. RESULTS: We generated in excess of 7 million sequence reads, by pyrosequencing 21 normalized cDNA libraries derived from multiple Q. suber tissues and organs, developmental stages and physiological conditions. We deployed a stringent sequence processing and assembly pipeline that resulted in the identification of ~159,000 unigenes. These were annotated according to their similarity to known plant genes, to known Interpro domains, GO classes and E.C. numbers. The phylogenetic extent of this ESTs set was investigated, and we found that cork oak revealed a significant new gene space that is not covered by other model species or EST sequencing projects. The raw data, as well as the full annotated assembly, are now available to the community in a dedicated web portal at http://www.corkoakdb.org. CONCLUSIONS: This genomic resource represents the first trancriptome study in a cork producing species. It can be explored to develop new tools and approaches to understand stress responses and developmental processes in forest trees, as well as the molecular cascades underlying cork differentiation and disease response.

Acorn selection by the wood mouse Apodemus sylvaticus: a semi-controlled experiment in a Mediterranean environment.

Fruits are highly important food resources for mammals in Mediterranean Europe, and due to the dominance of oaks (Quercus sp.), acorns are among those used by a vast array of species, including rodents. The metabolic yield of acorn intake may determine a selection pattern: preference for fat, carbohydrate, and consequently energy-rich fruits; or avoidance of fruits containing high concentrations of secondary chemical compounds (e.g., tannic acid). We studied the acorn feeding selection pattern of wood mice (Apodemus sylvaticus) inhabiting a mixed oak woodland, southwest Portugal, using an experiment conducted in an open-air enclosure. We tested which variables associated with the wood mouse (e.g., sex) and acorns (e.g., size and nutrient content) from three oak species (holm Q. rotundifolia, Portuguese Q. faginea and cork Q. suber oak) could be constraining acorn consumption. Our results indicate that wood mice are selecting acorns of the most common oak species (Q. suber), probably due to their previous familiarization with the fruit due to its dominance in the ecosystem but probably also because its chemical characteristics (sugar contents). Rodent gender and acorn morphology (width) are also influential, with females more prone to consume acorns with smaller width, probably due to handling limitation. This selective behaviour may have consequences for dispersion and natural regeneration of the different oak species.