Root-knot nematodes (RKN, Meloidogyne spp.) cause substantial agricultural losses worldwide, and sustainable alternatives to synthetic nematicides are needed. This study evaluated the potential bionematicidal effects of two formulations based on micronized and nanoformulated lignin extracted from grape stalks via alkaline extraction and deep eutectic solvent (DES) methods against the RKN M. luci. In vitro assays revealed that micronized lignin achieved moderate second-stage juveniles (J2) mortality (maximum 66.5% at 30 mg/mL). Lignin nanoparticles (LigNPs) markedly improved efficiency, causing 68.6% J2 mortality at 0.250 mg/mL (representing a 99% reduction in concentration). Alkaline LigNPs demonstrated high J2 hatching inhibition (87.8% at 0.468 mg/mL), while DES LigNPs induced 44.6% inhibition at 0.250 mg/mL. Soil assays, performed to confirm in vitro results, revealed differential performance: J2 mortality decreased substantially (31.8% for DES at 0.5 mg/mL, negligible for alkaline at 0.94 mg/mL), likely due to adsorption in soil and reduced bioavailability. However, DES LigNPs maintained significant J2 hatching inhibition in soil (66.9% at 0.5 mg/mL), suggesting their potential for preventive applications. Hypothetical nematicidal mechanisms are discussed. Although requiring higher concentrations than metallic nanoparticles, lignin offers advantages including biodegradability, agricultural waste valorisation, and environmental safety. This first demonstration of LigNPs as bionematicides establishes a foundation for developing new sustainable RKN management strategies based on agricultural byproducts.

Plant-parasitic nematodes (PPNs) are major constraints to agricultural productivity worldwide, causing estimated annual crop losses exceeding USD 150 billion. Among PPNs, root-knot nematodes (RKNs; Meloidogyne spp.) are particularly damaging due to their broad host range, high reproductive capacity, and limited availability of effective and sustainable strategies of management. This study evaluated the nematicidal activity of essential oils (EOs) from six plant species on second-stage juveniles (J2s) mortality and hatching of Meloidogyne chitwoodi and M. incognita. The EOs from Aloysia citriodora, Corymbia citriodora, Cymbopogon citratus, Cymbopogon winterianus, Elionurus muticus, and Lippia alba were tested at concentrations ranging from 0.75 to 18.0 μL mL−1, prepared in 1% Tween 80. Tween 80 (1%) and sterilized distilled water served as controls. A concentration of 3.0 μL mL−1 was selected to assess the efficacy against J2 mortality and hatching inhibition of both RKN species, in soil. Experiments were conducted twice with eight replicates per treatment, and data were subjected to Probit analysis to estimate the concentrations causing 50% and 90% of J2 mortality and hatching inhibition. All EOs exhibited nematicidal activity under in vitro conditions. Among them, Corymbia citriodora and Cymbopogon citratus EOs were the most effective (3 μL/mL), significantly suppressing J2 survival and hatching of M. chitwoodi and M. incognita in soil. These results indicate that plant-derived EOs represent promising biorational tools for RKN management and support further research on formulation development and field validation for use in sustainable crop protection systems.

Plant-parasitic nematodes (PPNs) cause yield losses in various crops worldwide. Damage due to PPNs can be severe, causing billions of dollars of crop losses across the globe annually. Information about PPNs occurrence in Mozambique is limited. Based on the literature, twenty-five genera of PPNs have been reported to affect several economically important crops, including root-knot nematodes (RKNs, Meloidogyne spp.), Scutellonema spp., root-lesion nematodes (RLNs, Pratylenchus spp.), spiral nematodes (Helicotylenchus spp.), and the dagger nematode (Xiphinema spp.), which are commonly associated with crops such as banana (Musa spp.), cassava (Manihot esculenta), cowpea (Vigna unguiculata), maize (Zea mays), sugarcane (Saccharum officinarum), and sunflower (Helianthus annuus). Dissemination of these nematodes is not yet fully understood, but the importation of plants, roots, rhizomes, and/or seeds likely contributes to the introduction and spread of PPNs. Although the implementation of PPN-mitigation strategies is crucial to crop production, their application is still limited in Mozambique, with quite a few reported uses of nematicides in the Manica and Maputo provinces. Therefore, adopting integrated management strategies that combine two or more practices, such as biological control, crop rotation, organic amendments, soil solarization, and, as a last resort, chemical nematicides, may be an option to effectively reduce the population of PPNs. This review gathers information on the occurrence and management of PPNs, as reported to date in Mozambique.

The increasing frequency of extreme weather events affects ecosystems and threatens food production. The reduction of chemical pesticides, together with other ecological approaches, is crucial to more sustainable agriculture. Plant-parasitic nematodes (PPN), especially root-knot nematodes (RKN), Meloidogyne spp., are responsible for extensive damage to a wide range of economically important crops, leading to yield losses and reduced quality of the products. This study aims to show the potential of native potato-growing soil bacterial strains as biological control agents in a more sustainable agriculture perspective. After screening thirty bacterial strains, a bacterial consortium, composed of B. amyloliquefaciens UC_2.4, P. capeferrum UC_21.3 A.1, and P. capeferrum UC_21.30 A.1, was defined and investigated in more detail due to their potential for plant growth-promoting bacteria (PGPB), fungicidal, and nematicidal activities. The genomes of the strains were sequenced and analyzed for PGPB traits, and phenotypic assays were also performed. The nematicidal activity of these strains towards PPN and the model organism Caenorhabditis elegans was assessed. Their potential as PGPB and for controlling PPN on soil was evaluated in pot assays with tomato plants cv. Coração de Boi, by using bacterial strains alone and as a consortium. Here, the bacterial consortium showed some PGPB traits verified by genome mining and phenotypic assays in vitro and pot assays with plants. It was able to act as nematicidal agents with 100% efficacy towards PPN but not against C. elegans, indicating a highly targeted action mechanism, which might be attributed to the surfactin, fengycin, and lipopeptides, not affecting other non-target organisms that play essential roles in soil health. The bacterial consortium reduced the infectivity of PPN in plants by threefold. This bacterial consortium was established for the first time and has the potential to serve as a new tool for managing RKN in a more sustainable agricultural environment.

A “monumental” centennial Pinus pinea L. tree of public interest with severe wilting symptoms was felled in Coimbra, Portugal. A survey was carried out to detect Aphelenchoididae nematodes (including Bursaphelenchus spp.) in the tree tissues. Nematode isolates were characterised/identified based on species-specific morphological characteristics, and on molecular data. Bursaphelenchus xylophilus was not detected. Three other Bursaphelenchus species (B. arthuri, B. fungivorus and B. sexdentati Type II) were found co-occurring in the tree. Other Aphelenchoididae nematodes, Potensaphelenchus stammeri and Cryptaphelenchus sp., were also identified. An annotated checklist of Bursaphelenchus spp. on P. pinea is also presented, demonstrating the wide variability of Bursaphelenchus species in this host. These results have shown that centennial wilted pine trees can be reservoirs of nematode diversity impacting forest health.

Nematodes with morphological characters of Bursaphelenchus arthuri, a species of the Fungivorus group, first described in 2005 in China from imported coniferous packaging wood, were extracted from a centennial Pinus pinea tree with wilting symptoms in Coimbra, Portugal. Nematodes were transferred to Botrytis cinerea cultures to establish a nematode isolate which was characterised using morphological, morphometrical and molecular methods. Restriction fragment length polymorphism (RFLP) analysis carried out with five endonucleases of amplified internal transcribed spacer (ITS) of the rDNA region, revealed the characteristic restriction pattern described for Bursaphelenchus arthuri. Sequencing of the D2-D3 expansion region of the large subunit (LSU) of rDNA confirmed this identification. Phylogenetic analysis based on multiple sequence alignment of selected D2-D3 sequences showed that the Portuguese B. arthuri isolate formed a separate group, along with the other B. arthuri isolate from Taiwan available in GenBank, from those of other isolates of closely related nematodes species belonging to the Fungivorus group. This study is the first report of B. arthuri in Europe and from P. pinea.

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease and is considered an A2 quarantine organism by the European Plant Protection Organisation. In Europe, this nematode has been reported in Pinus pinaster, P. radiata, and P. nigra. In May 2024, severe wilting symptoms were observed in P. sylvestris trees at Serra da Lousã (Coimbra, the central area of continental Portugal). Wood samples were collected from six wilted trees, and the presence of PWN was investigated. From these, B. xylophilus specimens were detected in five out of the six trees. Species identification was performed based on species-specific morphological diagnostic characters, and this was confirmed by real-time PCR using species-specific primers targeting the B. xylophilus satellite DNA region. This study presents the first detection of B. xylophilus in P. sylvestris in Portugal and in Europe.

Pine wilt disease (PWD) is a devastating forest disease caused by the pinewood nematode (PWN), Bursaphelenchus xylophilus, a migratory endoparasite that infects several coniferous species. During the last 20 years, advances have been made for understanding the molecular bases of PWN-host trees interactions. Major advances emerged from transcriptomic and genomic studies, which revealed some unique features related to PWN pathogenicity and constituted fundamental data that allowed the development of postgenomic studies. Here we review the proteomic approaches that were applied to study PWD and integrated the current knowledge on the molecular basis of the PWN pathogenicity. Proteomics has been useful for understanding cellular activities and protein functions involved in PWN-host trees interactions, shedding light into the mechanisms associated with PWN pathogenicity and being promising tools to better clarify host trees PWN resistance/susceptibility.

Meloidogyne chitwoodi, M. enterolobii, and M. luci are present in some EU countries, with restricted distributions, and plant resistance can be used to manage these nematodes. Two pot experiments were conducted under controlled conditions for 56 d to assess the host suitability of two potential rootstocks, Cucumis metuliferus BGV11135 and Citrullus amarus BGV5167, to one isolate of each nematode. The susceptible cucumber (Cucumis sativus) ‘Dasher II’, watermelon (Citrullus lanatus) ‘Sugar Baby’ and tomato (Solanum lycopersicum) ‘Coração-de-Boi’ were included for comparisons. A histopathological study using confocal-laser microscopy was also conducted 15 d after nematode inoculations. In the pot test, the rootstocks showed lower numbers of galls, egg masses, and eggs per plant than their susceptible ones. Reproduction indices of the rootstocks varied from immune to moderately resistant, depending on the isolate-rootstock combination. In the histopathological study, M. enterolobii and M. luci induced similar numbers of giant cells (GC) per feeding site in all germplasms. However, GC volumes and numbers of nuclei in rootstocks were lower than in the susceptible germplasms. GCs induced by M. chitwoodi were only detected in susceptible cucumber. These results emphasize the potential of C. metuliferus and C. amarus as effective, eco-friendly strategies for managing root-knot nematodes, and show the complex these host-pathogen interactions.

To demonstrate the efficacy of the vacuum pressure impregnation (VPI) with commercial wood preservative products to eliminate the quarantine organism, pinewood nematode (PWN), Bursaphelenchus xylophilus , and other nematodes from maritime pine ( Pinus pinaster ) wood, in vitro assays and industrial assays in horizontal industrial autoclave tanks were conducted. In vitro nematicidal activity assays through direct exposure of the PWN 3rd-stage dispersal juveniles, the resistance juvenile stage, extracted from naturally infected P. pinaster revealed 100% nematode mortality with three commercial wood preservatives. Nematode mortality was also assessed in VPI industrial assays with the three commercial wood preservatives using naturally PWN infected P. pinaster experimental units, with various diameters and sizes. After VPI treatment, the nematode mortality ranged from 99.9761 to 100%. After incubation, the mortality of the total number of nematodes increased and, in all sections, the nematode mortality was higher than 99.9981% and in some it was 100% indicating that wood impregnated with preservative products does not constitute an environment favorable to the reproduction and development of nematodes. Overall, our findings demonstrated that the efficiency of the VPI process results from the joint action of the physical effect of pressure and vacuum and of the nematicidal effect of the preservative product. VPI treatment can be considered a valuable approach to eliminate PWN and other nematodes from maritime pine wood avoiding the subsequent application of the heat treatment.

In a survey for Burspahelenchus species in a declining centennial stone pine, Pinus pinea, in Portugal, few specimens of Potensaphelenchus stammeri were extracted from wood samples, and an isolate was established in fungus cultures. The Portuguese P. stammeri isolate was characterised and identified based on morphological and morphometric diagnostic characters of females and males and by sequencing the D2-D3 expansion region of a large subunit (LSU) ribosomal DNA. Phylogenetic analysis by the multiple sequence alignment of selected relevant D2-D3 sequences including sequences of different isolates of P. stammeri revealed that this Portuguese P. stammeri isolate forms a clade with other P. stammeri isolates. Potensaphelenchus stammeri is reported in Portugal, and it is associated with Pinus pinea; moreover, the morphological and morphometric data of a Portuguese isolate were presented for the first time.

The root-knot nematode (RKN) Meloidogyne luci presents a threat to the production of several important crops. This nematode species was added to the European Plant Protection Organization Alert list in 2017. The scarce availability of efficient nematicides to control RKN and the phasing out of nematicides from the market have intensified the search for alternatives, such as phytochemicals with bionematicidal properties. The nematicidal activity of 1,4-naphthoquinone (1,4-NTQ) against M. luci has been demonstrated; however, knowledge of the potential mode(s) of action of this compound is still scarce. In this study, the transcriptome profile of M. luci second-stage juveniles (J2), the infective stage, in response to 1,4-NTQ exposure was determined by RNA-seq to identify genes and pathways that might be involved in 1,4-NTQ's mode(s) of action. Control treatments, consisting of nematodes exposed to Tween® 80 (1,4-NTQ solvent) and to water, were included in the analysis. A large set of differentially expressed genes (DEGs) was found among the three tested conditions, and a high number of downregulated genes were found between 1,4-NTQ treatment and water control, reflecting the inhibitory effect of this compound on M. luci, with a great impact on processes related to translation (ribosome pathway). Several other nematode gene networks and metabolic pathways affected by 1,4-NTQ were also identified, clarifying the possible mode of action of this promising bionematicide.

Plant-parasitic nematodes (PPN), “the unseen enemies” of plants are a threat to the potato (Solanum tuberosum) production affecting their quality and yield. The most destructive species belong to the genera Globodera (Heteroderidae), Meloidogyne (Meloidogynidae), Pratylenchus/Nacobbus (Pratylenchidae), Ditylenchus (Anguinidae), and Nanidorus/Paratrichodorus/Trichodorus (Trichodoridae). Other PPN have been reported associated with the potato crop but the data on their economic importance is limited. Some of these nematodes are considered invasive species and, in a survey based on scientific and economic importance, Meloidogyne spp., Globodera spp., Pratylenchus spp., Radopholus similis, Ditylenchus dipsaci, Rotylenchus reniformis, and Nacobbus aberrans are in the top 10 list. Relevant information on the biology, host range, symptoms and diagnosis, survival and dissemination, environmental factors, disease complexes, economic importance, and management of the most aggressive species is given in this chapter.

The root-knot nematode (RKN) Meloidogyne luci is included in the Alert List of the European Plant Protection Organization, because it has potential negative impacts on economically important crops. Identification of plant species/cultivars resistant to M. luci is important for its management. Susceptibility of 35 commercial plant species/cultivars, from nine families to a M. luci isolate from Portugal was evaluated in pot assays, assessing root gall index (GI) and reproduction factor (Rf) 60 d after inoculation, with tomato ‘Coração-de-Boi’ used as the positive susceptible experimental control. Presence/absence of RKN resistance genes was also determined in the tomato and pepper cultivars. One cultivar of cabbage, three of lettuce, ten of pepper, one of sugar beet, and all the cultivars of Cucurbitaceae (five), Fabaceae (two) and Poaceae (one) were susceptible to M. luci (GI = 4-5; Rf = 2.1-152.3). One cultivar each of carrot, passion fruit, lettuce ‘Cocktail’, cabbage ‘Bacalan’, ‘Coração’ and ‘Lombarda’, and spinach ‘Tayto’ were resistant/hypersensitive (Rf < 1; GI > 2). The tomato ‘Actimino’, ‘Briomino’, ‘Veinal’ and ‘Vimeiro’, which carried at least one copy of the Mi-1.2 gene, were resistant to the nematode (GI = 1-2; 0.0 < Rf < 0.1). These results indicate that the tomato cultivars have potential to contribute to reduction of M. luci populations in agro-ecosystems and improve the crop yields.

The root lesion nematode Pratylenchus penetrans is an important plant-parasitic nematode of potato. In this study, the susceptibility of commercial potato cultivars to P. penetrans was assessed. Nematode penetration was evaluated in cultivars Agria, Camel, Kennebec, Laura, Royata, and Stemster at 1, 3, 7, and 15 days after inoculation (DAI) with 750 nematodes/plant, and an egression assay at 3 DAI with 1000 nematodes/plant. Reproduction assays of cultivars Agata, Agria, Camel, Désirée, Dirosso, Kennebec, Laura, Picasso, Royata, and Stemster were performed in 2 L pots inoculated with four P. penetrans/g soil and quantified at 60 DAI. Tenue or moderate root cell browning to advanced necrotic areas were observed after nematode penetration, and the number of nematodes/g of root gradually increased with time of infection. A lower number of deposited eggs and nematodes were observed within the roots of cultivar Laura in all assays comparatively to other cultivars. The susceptibility index (SI) was significantly lower in cultivar Laura (0.4–0.6), followed by cultivars Camel and Picasso (0.8–0.9). All remaining cultivars showed SI values above 1. Although the potato susceptibility to the nematode varied among cultivars, no differences on the average number or weight of tubers produced by each plant of inoculated versus non-inoculated plants were detected. Our data reveals that these cultivars have a distinct ability to support the reproduction of P. penetrans.

The worldwide demand on bionematicides is in a growing trend, fueled by environmental concerns with potential negative impacts of synthetic products on ecosystems and non-target organisms, as well as by the need to provide solutions to organic farming management. As such, the main goal of the present study was to evaluate the impact of 1,4-naphthoquinone (NTQ), a nematicidal compound found in natural products such as walnut husk, on soil microbial community (non-target organisms), and determine ecotoxicological indicators in order to follow and quantify the effect of this compound. The effects on diversity and metabolic state of the microbial community were evaluated using a Phospholipid-derived Fatty Acid analysis (PLFA) method. Tests were conducted on a natural uncontaminated soil spiked with a range of NTQ concentrations (up to 768 mg/kg), as well as comparable control solutions containing solubilization enhancer Triton X-100 (TX100). NTQ impacted the soil microbial community, causing significant changes on global PLFA profile at 12 mg/kg, and significant changes on taxonomic biomarker balance at 96 mg/kg. TX100 also caused a significant effect on the global profile, but only at 192 mg/kg NTQ-equivalent. The modifications brought by either compounds were distinct, as different indicators were affected. The effects of NTQ on microbial community were quantifiable for several indicators by calculating half maximal effective concentrations (EC50). The method used proved to be suitable for the assessment of NTQ ecotoxicity in soils.

The scarce availability of efficient and eco-friendly nematicides to control root-knot nematodes (RKN), Meloidogyne spp., has encouraged research toward the development of bionematicides. Naphthoquinones, juglone (JUG) and 1,4-naphthoquinone (1,4-NTQ), are being explored as alternatives to synthetic nematicides to control RKN. This study expands the knowledge on the effects of these natural compounds toward M. luci life cycle (mortality, hatching, penetration, reproduction). M. luci second-stage juveniles (J2)/eggs were exposed to each compound (250, 150, 100, 50, and 20 ppm) to monitor nematode mortality and hatching during 72 h and 15 days, respectively. Tomato seedlings were then inoculated with 200 J2, which had been exposed to JUG/1,4-NTQ for 3 days. The number of nematodes inside the roots was determined at 3 days after inoculation, and the final population density was assessed at 45 days after inoculation. Moreover, the potential mode of action of JUG/1,4-NTQ was investigated for the first time on RKN, through the assessment of reactive oxygen species (ROS) generation, acetylcholinesterase (AChE) in vitro inhibitory activity and expression analysis of ache and glutathione-S-transferase (gst) genes. 1,4-NTQ was the most active compound, causing ≥50% J2 mortality at 250 ppm, within 24 h. At 20 and 50 ppm, hatching was reduced by ≈50% for both compounds. JUG showed a greater effect on M. luci penetration and reproduction, decreasing infection by ≈80% (50 ppm) on tomato plants. However, 1,4-NTQ-induced generation of ROS and nematode vacuolization was observed. Our study confirms that JUG/1,4-NTQ are promising nematicidal compounds, and new knowledge on their physiological impacts on Meloidogyne was provided to open new avenues for the development of innovative sustainable nematicides.

The pinewood nematode (PWN), Bursaphelenchus xylophilus, one of the most serious forest pests worldwide, is considered the causal agent of the pine wilt disease (PWD). The main host species belong to the genus Pinus, and a variation in the susceptibility of several pine species to PWN infection is well-known. It is also recognized that there is variation in the virulence among B. xylophilus isolates. In the present study, we applied a quantitative mass spectrometry-based proteomics approach to perform a deep characterization of proteomic changes across two B. xylophilus isolates with different virulence from different hosts and geographical origins. A total of 1,456 proteins were quantified and compared in the two isolates secretomes, and a total of 2,741 proteins were quantified and compared in the nematode proteomes in pine tree extract and fungus stimuli conditions. From the proteomic analyses, a group of proteins was selected and identified as potential virulence biomarkers and shed light on putative most pathogenic proteins of this plant-parasitic nematode. Proteomic data are available via ProteomeXchange with identifier PXD029377.

Summary Potato cyst nematodes (PCN), Globodera spp., cause damage to potatoes in more than 60 countries and several management strategies, including the application of chemical nematicides, are commonly used for their control. However, due to stringent regulations in Europe several nematicides have been, or are being, removed from the market due to their potential toxic effects on the environment and human health. New solutions and nematode management strategies are being sought to control these challenging and economically important nematodes. In this study, the effects of Salibro™, a novel sulfonamide nematicide based on the active ingredient fluazaindolizine (Reklemel™ active), were evaluated on the hatching, motility, infectivity and reproduction of PCN in the laboratory. Depending upon the duration of nematode pre-exposure, Salibro™ at concentrations of 5-250 mg fluazaindolizine (active substance) (a.s.) kg −1 (equivalent to 5-250 ppm a.s.) affected hatching, motility and infectivity of second-stage juveniles of G. pallida and G. rostochiensis , whereas the reproduction of G. pallida was only influenced at 5-50 mg a.s. kg −1 . Salibro™, under laboratory conditions, has intrinsic activity against both PCN species and could be a promising additional tool for the integrated management of PCN. Further studies are needed to demonstrate Salibro™ efficacy under field conditions.