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The rice root-knot nematode (RRKN), Meloidogyne graminicola Golden and Birchfield 1965, is a dangerous crop pest that affects rice production on a global scale. The largest rice-producing countries struggle with the impacts of RRKN infestation, namely, underdeveloped plants and a reduction in rice grain that can reach up to 70% of crop yield. In addition, the shift to strategies of sustainable pest management is leading to a withdrawal of some of the most effective pesticides, given the dangers they pose to the environment and human health. Volatile metabolites produced by plants can offer safer alternatives. The present study characterized the nematicidal activity of volatile phytochemicals against the RRKN and compared the most active with commercial nematicides concerning their safety to the environment and human health. Rice plants were used to grow large numbers of RRKNs for direct-contact bioassays. Mortality induced by the volatiles was followed for four days on RRKN second-stage juveniles. Of the 18 volatiles tested, carvacrol, eugenol, geraniol, and methyl salicylate showed the highest mortalities (100%) and were compared to traditional nematicides using (eco)toxicological parameters reported on freely available databases. While methyl salicylate had a faster activity, carvacrol had more lasting effects. When compared to synthetic nematicides, these volatile phytochemicals were reported to have higher thresholds of toxicity and beneficial ecotoxicological parameters. Ultimately, finding safer alternatives to traditional pesticides can lower the use of damaging chemicals in farming and leverage the transition to agricultural practices with a lower impact on biodiversity.

The combination of a sensitive and specific magnetoresistive sensing device with an easy DNA extraction method and a rapid isothermal amplification is presented here targeting the on-site detection of Globodera pallida , a potato endoparasitic nematode. FTA-cards were used for DNA extraction, LAMP was the method developed for DNA amplification and a nanoparticle functionalized magnetic-biosensor was used for the detection. The combinatorial effect of these three emerging technologies has the capacity to detect G. pallida with a detection limit of one juvenile, even when mixed with other related species. This combined system is far more interesting than what a single technology can provide. Magnetic biosensors can be combined with any DNA extraction protocol and LAMP forming a new solution to target G. pallida . The probe designed in this study consistently distinguished G. pallida (∆V ac binding /V ac sensor above 1%) from other cyst nematodes (∆V ac binding /V ac sensor below 1%). It was confirmed that DNA either extracted with FTA-cards or Lab extraction Kit was of enough quantity and quality to detect G. pallida whenever present (alone or in mixed samples), ensuring probe specificity and sensitivity. This work provides insights for a new strategy to construct advanced devices for pathogens in-field diagnostics. LAMP runs separately but can be easily integrated into a single device., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Camacho, Albuquerque, Inácio, Martins, Mota, Freitas and de Andrade.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Rusinque, Camacho, Serra, Nóbrega and Inácio.)

Control of the pinewood nematode (PWN), the causal agent of pine wilt disease, can be achieved through the trunk injection of nematicides; however, many pesticides have been linked to environmental and human health concerns. Essential oils (EOs) are suitable alternatives due to their biodegradability and low toxicity to mammals. These complex mixtures of plant volatiles often display multiple biological activities and synergistic interactions between their compounds. The present work profiled the toxicity of eight EOs against the PWN in comparison to their 1:1 mixtures, to screen for successful synergistic interactions. Additionally, the main compounds of the most synergistic mixtures were characterized for their predicted environmental fate and toxicity to mammals in comparison to emamectin benzoate, a commercial nematicide used against PWN. The mixtures of Cymbopogon citratus with Mentha piperita and of Foeniculum vulgare with Satureja montana EOs showed the highest activities, with half-maximal effective concentrations (EC 50 ) of 0.09 and 0.05 µL/mL, respectively. For these, complete PWN mortality was reached after only ca. 15 min or 2 h of direct contact, respectively. Their major compounds had a higher predicted affinity to air and water environmental compartments and are reported to have very low toxicity to mammals, with low acute oral and dermal toxicities. In comparison, emamectin benzoate showed lower nematicidal activity, a higher affinity to the soil and sediments environmental compartments and higher reported oral and dermal toxicity to mammals. Overall, uncovering synergistic activities in combinations of EOs from plants of different families may prove to be a source of biopesticides with optimized toxicity against PWNs.

The potato cyst nematode (PCN), Globodera pallida, has acquired significant importance throughout Europe due to its widespread prevalence and negative effects on potato production. Thus, rapid and reliable diagnosis of PCN is critical during surveillance programs and for the implementation of control measures. The development of innovative technologies to overcome the limitations of current methodologies in achieving early detection is needed. Lab-on-a-chip devices can swiftly and accurately detect the presence of certain nucleotide sequences with high sensitivity and convert the presence of biological components into an understandable electrical signal by combining biosensors with microfluidics-based biochemical analysis. In this study, a specific DNA-probe sequence and PCR primers were designed to be used in a magnetoresistive biosensing platform to amplify the internal transcribed spacer region of the ribosomal DNA of G. pallida. Magnetic nanoparticles were used as the labelling agents of asymmetric PCR product through biotin−streptavidin interaction. Upon target hybridization to sensor immobilized oligo probes, the fringe field created by the magnetic nanoparticles produces a variation in the sensor’s electrical resistance. The detection signal corresponds to the concentration of target molecules present in the sample. The results demonstrate the suitability of the magnetic biosensor to detect PCR target product and the specificity of the probe, which consistently distinguishes G. pallida (DV/V > 1%) from other cyst nematodes (DV/V < 1%), even when DNA mixtures were tested at different concentrations. This shows the magnetic biosensor’s potential as a bioanalytical device for field applications and border phytosanitary inspections.

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.

Plant-parasitic nematodes (PPNs) are among the most notorious and underrated threats to food security and plant health worldwide, compromising crop yields and causing billions of dollars of losses annually. Chemical control strategies rely heavily on synthetic chemical nematicides to reduce PPN population densities, but their use is being progressively restricted due to environmental and human health concerns, so alternative control methods are urgently needed. Here, we review the potential of bacterial and fungal agents to suppress the most important PPNs, namely Aphelenchoides besseyi , Bursaphelenchus xylophilus , Ditylenchus dipsaci , Globodera spp., Heterodera spp., Meloidogyne spp., Nacobbus aberrans , Pratylenchus spp., Radopholus similis , Rotylenchulus reniformis , and Xiphinema index .

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Vicente, Soares, Faria, Espada, Mota, Nóbrega, Ramos and Inácio.)

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.

The pinewood nematode (PWN), Bursaphelenchus xylophilus , is the causal agent of pine wilt disease (PWD) and a quarantine organism in many countries. Managing PWD involves strict regulations and heavy contingency plans, and present climate change scenarios predict a spread of the disease. The urgent need for sustainable management strategies has led to an increasing interest in promising biocontrol agents capable of suppressing the PWN, like endoparasitic nematophagous fungi of the Esteya genus. Here, we review different aspects of the biology and ecology of these nematophagous fungi and provide future prospects.

Rice ( Oryza sativa L.) is one of the main cultivated crops worldwide and represents a staple food for more than half of the world population. Root-knot nematodes (RKNs), Meloidogyne spp., and particularly M. graminicola , are serious pests of rice, being, probably, the most economically important plant-parasitic nematode in this crop. M. graminicola is an obligate sedentary endoparasite adapted to flooded conditions. Until recently, M. graminicola was present mainly in irrigated rice fields in Asia, parts of the Americas, and South Africa. However, in July 2016, it was found in northern Italy in the Piedmont region and in May 2018 in the Lombardy region in the province of Pavia. Following the first detection in the EPPO region, this pest was included in the EPPO Alert List as its wide host range and ability to survive during long periods in environments with low oxygen content, represent a threat for rice production in the European Union. Considering the impact of this nematode on agriculture, a literature review focusing on M. graminicola distribution, biology, identification, and management was conducted.

Pine wilt disease (PWD) is a complex disease that severely affects the biodiversity and economy of Eurasian coniferous forests. Three factors are described as the main elements of the disease: the pinewood nematode (PWN) Bursaphelenchus xylophilus , the insect-vector Monochamus spp., and the host tree, mainly Pinus spp. Nonetheless, other microbial interactors have also been considered. The study of mycoflora in PWD dates back the late seventies. Culturomic studies have revealed diverse fungal communities associated with all PWD key players, composed frequently of saprophytic fungi (i.e., Aspergillus , Fusarium , Trichoderma ) but also of necrotrophic pathogens associated with bark beetles, such as ophiostomatoid or blue-stain fungi. In particular, the ophiostomatoid fungi often recovered from wilted pine trees or insect pupal chambers/tunnels, are considered crucial for nematode multiplication and distribution in the host tree. Naturally occurring mycoflora, reported as possible biocontrol agents of the nematode, are also discussed in this review. This review discloses the contrasting effects of fungal communities in PWD and highlights promising fungal species as sources of PWD biocontrol in the framework of sustainable pest management actions.

The potato cyst nematode (PCN) Globodera pallida has acquired significant importance throughout Europe due to its nefarious effects on potato production. Rapid and reliable diagnosis of PCN is critical during the surveillance programs and for the implementation of control measures. Molecular DNA-based methods are available, but they require expensive laboratory facilities, equipment and trained technicians. Moreover, there is an additional need of time for sample shipment and testing. In this work, we have developed a new and simple assay which reliably discriminates G. pallida from other cyst nematodes in less than 40 min. This assay may be applied either on cysts or juveniles with the ability to detect a single juvenile of G. pallida in a sample of at least 40 juveniles of the non-target species G. rostochiensis . This test should be a tool to improve the performance of the laboratory and has the potential to be performed on-site.

Morphological and molecular studies were conducted to characterize the specific identity of 36 isolates of potato cyst nematodes (PCNs) recovered from soil samples collected in several potato producing areas of Algeria. Morphometric data revealed that 44% of isolates contained Globodera pallida alone, 28% contained Globodera rostochiensis alone and 28% mixtures of the two species. Morphometric values of cysts and second-stage juveniles were generally distributed with slight differences in the expected ranges for both Globodera species. Inter- and intraspecific morphometric variability in nematode isolates was noted. Molecular analysis using conventional multiplex PCR with species-specific primers and TaqMan real-time PCR confirmed the morphological identification. In addition, the distribution of both potato cyst nematode species throughout various parts of the country was investigated. In the central areas, the isolates of G. pallida alone dominate, whereas isolates of G. rostochiensis alone are more frequent in the southern areas. In the eastern regions, mixed isolates are more representative. Most isolates examined in the western areas are mixtures of the two species or G. rostochiensis alone. Comparatively, G. pallida remains the most widely distributed species in its geographic range. This study confirms the presence of two PCN species, G. pallida and G. rostochiensis , in Algeria and provides additional information on their biogeographic distribution.

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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Camacho, de Andrade, Mota, Nobrega, Vicente, Rusinque and Inácio.)

In Winter 2016, root and soil samples were collected from the rhizosphere soil at 10 to 15 cm depth of turfgrass, in the yard of a complex of houses in Caxias, a region near Lisbon, Portugal. The grass (dominated by tall fescue) showed yellow patches, stunting, and poor growth. Several specimens of a ring nematode (50-60 nematodes/100 ml soil) were recovered from soil and identified as Mesocriconema xenoplax (peach ring nematode) based on morphological and morphometrical analysis of females. The observed morphological features were with previous descriptions. Species identification was confirmed through sequencing of the fragment spanning D2/D3 domain of the 28S rDNA gene. Phylogenetic analysis revealed that the Portuguese isolate grouped with M. xenoplax isolates (98% similarity), supporting its identification as M. xenoplax . This is the first report of M. xenoplax from turfgrass in Portugal and in Europe contributing with additional information on the distribution of this phytoparasite., In Winter 2016, root and soil samples were collected from the rhizosphere soil at 10 to 15 cm depth of turfgrass, in the yard of a complex of houses in Caxias, a region near Lisbon, Portugal. The grass (dominated by tall fescue) showed yellow patches, stunting, and poor growth. Several specimens of a ring nematode (50-60 nematodes/100 ml soil) were recovered from soil and identified as Mesocriconema xenoplax (peach ring nematode) based on morphological and morphometrical analysis of females. The observed morphological features were with previous descriptions. Species identification was confirmed through sequencing of the fragment spanning D2/D3 domain of the 28S rDNA gene. Phylogenetic analysis revealed that the Portuguese isolate grouped with M. xenoplax isolates (98% similarity), supporting its identification as M. xenoplax . This is the first report of M. xenoplax from turfgrass in Portugal and in Europe contributing with additional information on the distribution of this phytoparasite.

Platypus cylindrus is an important wood borer of cork oak trees (Quercus suber) in the Mediterranean region, namely Portugal, Morocco and Algeria where its presence has drastically increased in the past few decades. On the contrary, the insect is not a relevant pest in Tunisia. The aim of this work is to analyze morphological and genetic differences among Tunisian and Portuguese populations in order to understand their role in the diverse population dynamics (e.g., aggressiveness) of the insect. The information could be used as a novel tool to implement protective measures. Insects were collected from cork oak stands in Tunisia (Ain Beya, Babouch and Mzara) and Portugal (Chamusca and Crato). Morphological traits of female and male mycangial pits were determined, using scanning electron microscopy but no significant differences were found. Genetic differences were analyzed using nuclear (internal simple sequence repeat polymerase chain reaction) and mitochondrial (cytochrome oxidase I (COI)) molecular markers. The results showed a very low level of intraspecific polymorphism and genetic diversity. The alignment of COI sequences showed high percentage of identical sites (99%) indicating a very low variation in nucleotide composition. Other variables related with the ecology of the insect and its associated fungi must be studied for a better understanding of the differences in the insect population's dynamic in Mediterranean countries.

Background: The pinewood nematode (PWN) Bursaphelenchus xylophilus is an important conifer disease worldwide. It is the direct cause of the death of millions of pines in south-east Asia (mainly Japan, China and Korea) and has been established in Portugal since 1999. The phasing out of methyl bromide has created an urgent need for alternative treatment of wood packaging materials. The effect of sulfuryl fluoride (SF), a broad-spectrum fumigant used to control insects, was tested in Pinus pinaster boards naturally infested by PWN., Results: Boards were fumigated for 24 h at three different temperatures (15, 20 and 30 °C) with dosage ranges of 3169-4407, 1901-4051 and 1385-2141 gh m(-3) respectively. Treated wood was sampled for nematode identification and counting, before treatment and after 24 h, 72 h and 21 days. No survival was found in the 15 °C and 30 °C treatments, while at 20 °C the mortality ranged from 94.06 to 100%. Some reasons for the survival at 20 °C are presented., Conclusion: Results confirm SF to be an effective quarantine treatment for PWN at 15 and 30 °C. Further studies are needed to obtain the most effective dosage at 20 °C, and to determine the toxicity of SF fumigation on B. xylophilus at other temperatures, especially at 25 °C., (© 2013 Society of Chemical Industry.)

Ambrosia fungi live associated with beetles (Scolytidae and Platypodidae) in host trees and act as a food source for the insects. The symbiotic relation is important to the colonizing strategies of host trees by beetles. Ambrosia fungi are dimorphic: they grow as ambrosial form and as mycelium. The fungi are highly specialized, adapted to a specific beetle and to the biotope where they both live. In addition other fungi have been found such as tree pathogenic fungi that may play a role in insects host colonization success. Saprophytic fungi are also present in insects galleries. These may decompose cellulose and/or be antagonistic to other less beneficial fungi. This paper summarizes the importance of ambrosia fungi and the interaction with insects and hosts. The possibility of the transport of pathogenic fungi by Platypus cylindrus to cork oak thus contributing for its decline is discussed.