A Critical Appraisal of DNA Transfer from Plants to Parasitic Cyst Nematodes.

Plant-parasitic nematodes are one of the most economically important pests of crops. It is widely accepted that horizontal gene transfer-the natural acquisition of foreign genes in parasitic nematodes-contributes to parasitism. However, an apparent paradox has emerged from horizontal gene transfer analyses: On the one hand, distantly related organisms with very dissimilar genetic structures (i.e. bacteria), and only transient interactions with nematodes as far as we know, dominate the list of putative donors, while on the other hand, considerably more closely related organisms (i.e. the host plant), with similar genetic structure (i.e. introns) and documented long-term associations with nematodes, are rare among the list of putative donors. Given that these nematodes ingest cytoplasm from a living plant cell for several weeks, there seems to be a conspicuous absence of plant-derived cases. Here, we used comparative genomic approaches to evaluate possible plant-derived horizontal gene transfer events in plant parasitic nematodes. Our evidence supports a cautionary message for plant-derived horizontal gene transfer cases in the sugar beet cyst nematode, Heterodera schachtii. We propose a 4-step model for horizontal gene transfer from plant to parasite in order to evaluate why the absence of plant-derived horizontal gene transfer cases is observed. We find that the plant genome is mobilized by the nematode during infection, but that uptake of the said "mobilome" is the first major barrier to horizontal gene transfer from host to nematode. These results provide new insight into our understanding of the prevalence/role of nucleic acid exchange in the arms race between plants and plant parasites.

Diversity and evolution of transposable elements in the plant-parasitic nematodes.

BACKGROUND: Transposable elements (TEs) are mobile DNA sequences that propagate within genomes, occupying a significant portion of eukaryotic genomes and serving as a source of genetic variation and innovation. TEs can impact genome dynamics through their repetitive nature and mobility. Nematodes are incredibly versatile organisms, capable of thriving in a wide range of environments. The plant-parasitic nematodes are able to infect nearly all vascular plants, leading to significant crop losses and management expenses worldwide. It is worth noting that plant parasitism has evolved independently at least three times within this nematode group. Furthermore, the genome size of plant-parasitic nematodes can vary substantially, spanning from 41.5 Mbp to 235 Mbp. To investigate genome size variation and evolution in plant-parasitic nematodes, TE composition, diversity, and evolution were analysed in 26 plant-parasitic nematodes from 9 distinct genera in Clade IV. RESULTS: Interestingly, despite certain species lacking specific types of DNA transposons or retrotransposon superfamilies, they still exhibit a diverse range of TE content. Identification of species-specific TE repertoire in nematode genomes provides a deeper understanding of genome evolution in plant-parasitic nematodes. An intriguing observation is that plant-parasitic nematodes possess extensive DNA transposons and retrotransposon insertions, including recent sightings of LTR/Gypsy and LTR/Pao superfamilies. Among them, the Gypsy superfamilies were found to encode Aspartic proteases in the plant-parasitic nematodes. CONCLUSIONS: The study of the transposable element (TE) composition in plant-parasitic nematodes has yielded insightful discoveries. The findings revealed that certain species exhibit lineage-specific variations in their TE makeup. Discovering the species-specific TE repertoire in nematode genomes is a crucial element in understanding the evolution of genomes in plant-parasitic nematodes. It allows us to gain a deeper insight into the intricate workings of these organisms and their genetic makeup. With this knowledge, we are gaining a fundamental piece in the puzzle of understanding the evolution of these parasites. Moreover, recent transpositions have led to the acquisition of new TE superfamilies, especially Gypsy and Pao retrotransposons, further expanding the diversity of TEs in these nematodes. Significantly, the widely distributed Gypsy superfamily possesses proteases that are exclusively associated with parasitism during nematode-host interactions. These discoveries provide a deeper understanding of the TE landscape within plant-parasitic nematodes.

VND Genes Redundantly Regulate Cell Wall Thickening during Parasitic Nematode Infection.

Plant parasitic root-knot nematodes are major agricultural pests worldwide, as they infect plant roots and cause substantial damages to crop plants. Root-knot nematodes induce specialized feeding cells known as giant cells (GCs) in the root vasculature, which serve as nutrient reservoirs for the infecting nematodes. Here, we show that the cell walls of GCs thicken to form pitted patterns that superficially resemble metaxylem cells. Interestingly, VASCULAR-RELATED NAC-DOMAIN1 (VND1) was found to be upregulated, while the xylem-type programmed cell death marker XYLEM CYSTEINE PEPTIDASE 1 was downregulated upon nematode infection. The vnd2 and vnd3 mutants showed reduced secondary cell wall pore size, while the vnd1 vnd2 vnd3 triple mutant produced significantly fewer nematode egg masses when compared with the wild type. These results suggest that the GC development pathway likely shares common signaling modules with the metaxylem differentiation pathway and VND1, VND2, and VND3 redundantly regulate plant-nematode interaction through secondary cell wall formation.

The other side of the coin: systemic effects of Serendipita indica root colonization on development of sedentary plant-parasitic nematodes in Arabidopsis thaliana.

MAIN CONCLUSION: Upon systemic S. indica colonization in split-root system cyst and root-knot nematodes benefit from endophyte-triggered carbon allocation and altered defense responses what significantly facilitates their development in A. thaliana. Serendipita indica is an endophytic fungus that establishes mutualistic relationships with different plants including Arabidopsis thaliana. It enhances host's growth and resistance to different abiotic and biotic stresses such as infestation by the cyst nematode Heterodera schachtii (CN). In this work, we show that S. indica also triggers similar direct reduction in development of the root-knot nematode Meloidogyne javanica (RKN) in A. thaliana. Further, to mimick the natural situation occurring frequently in soil where roots are unequally colonized by endophytes we used an in vitro split-root system with one half of A. thaliana root inoculated with S. indica and the other half infected with CN or RKN, respectively. Interestingly, in contrast to direct effects, systemic effects led to an increase in number of both nematodes. To elucidate this phenomenon, we focused on sugar metabolism and defense responses in systemic non-colonized roots of plants colonized by S. indica. We analyzed the expression of several SUSs and INVs as well as defense-related genes and measured sugar pools. The results show a significant downregulation of PDF1.2 as well as slightly increased sucrose levels in the non-colonized half of the root in three-chamber dish. Thus, we speculate that, in contrast to direct effects, both nematode species benefit from endophyte-triggered carbon allocation and altered defense responses in the systemic part of the root, which promotes their development. With this work, we highlight the complexity of this multilayered tripartite relationship and deliver new insights into sugar metabolism and plant defense responses during S. indica-nematode-plant interaction.

Silencing of the T-type voltage-gated calcium channel α1 subunit by fungus-mediated RNAi altered the structure of F-actin and caused defective behaviors in Ditylenchus destructor.

BACKGROUND: T-type calcium channels, characterized as low-voltage activated (LVA) calcium channels, play crucial physiological roles across a wide range of tissues, including both the neuronal and nonneuronal systems. Using in situ hybridization and RNA interference (RNAi) techniques in vitro, we previously identified the tissue distribution and physiological function of the T-type calcium channel α1 subunit (DdCα1G) in the plant-parasitic nematode Ditylenchus destructor. METHODS AND RESULTS: To further characterize the functional role of DdCα1G, we employed a combination of immunohistochemistry and fungus-mediated RNAi and found that DdCα1G was clearly distributed in stylet-related tissue, oesophageal gland-related tissue, secretory-excretory duct-related tissue and male spicule-related tissue. Silencing DdCα1G led to impairments in the locomotion, feeding, reproductive ability and protein secretion of nematodes. To confirm the defects in behavior, we used phalloidin staining to examine muscle changes in DdCα1G-RNAi nematodes. Our observations demonstrated that defective behaviors are associated with related muscular atrophy. CONCLUSION: Our findings provide a deeper understanding of the physiological functions of T-type calcium channels in plant-parasitic nematodes. The T-type calcium channel can be considered a promising target for sustainable nematode management practices.

Design, synthesis, and nematicidal activity of novel 1,2,4-oxadiazole derivatives containing amide fragments.

Plant-parasitic nematodes are seriously affecting agricultural production worldwide and there are few highly effective and low-risk nematicides to control nematode diseases. In order to discover new nematicides, a series of 1,2,4-oxadiazole derivatives containing amide fragments have been designed and synthesized with the principle of active substructure splicing. The nematicidal activity of the target compounds was evaluated in vitro and it indicated that compound C3 exhibited the most nematicidal activity against Bursaphelenchus xylophilus, Aphelenchoides besseyi, and Ditylenchus destructor with the LC50 values of 37.2, 36.6, and 43.4 µg/mL, respectively, which were superior to positive agent tioxazafen. The preliminary mechanism results revealed that compound C3 not only inhibited the reproduction of B. xylophilus populations, but also affected the production of ROS and the accumulation of lipofuscin and lipids. Furthermore, compound C3 showed good inhibition of succinate dehydrogenase (SDH) with the IC50 value of 45.5 µmol/L. Molecular docking indicated that compound C3 had excellent binding to amino acids around the SDH active pocket. This work indicated that 1,2,4-oxadiazole derivative containing amide fragment is a promising template for the discovery of new nematicides and compound C3 can be used as a potential nematicide candidate.

Clarifying the Functional Role of Serotonin in Meloidogyne graminicola Host Plant Parasitism by Immunolocalization and RNA Interference.

Serotonin (5-hydroxytryptamine) is an essential neurotransmitter involved in regulating various behaviors in plant-parasitic nematodes, including locomotion, egg laying, feeding, and mating. However, the functional role of serotonin in root-knot nematode invasion of host plants and the molecular mechanisms underlying feeding behavior remain poorly understood. In this study, we tested the effects of exogenous serotonin and the pharmacological compounds fluoxetine and methiothepin on the feeding behaviors of Meloidogyne graminicola. Our results suggested that M. graminicola possesses an endogenous serotonin signaling pathway and that serotonin plays a crucial role in modulating feeding behaviors in M. graminicola second-stage juveniles. We also identified and cloned the serotonin synthesis enzyme tryptophan hydroxylase (Mg-tph-1) in M. graminicola and investigated the role of endogenous serotonin by generating RNA interference nematodes in Mg-tph-1. Silencing Mg-tph-1 substantially reduced nematode invasion, development, and reproduction. According to the immunostaining results, we speculated that these serotonin immunoreactive cells near the nerve ring in M. graminicola are likely homologous to Caenorhabditis elegans ADFs, NSMs, and RIH serotonergic neurons. Furthermore, we investigated the impact of phytoserotonin on nematode invasion and development in rice by overexpressing OsTDC-3 or supplementing rice plants with tryptamine and found that an increase in phytoserotonin increases nematode pathogenicity. Overall, our study provides insights into the essential role of serotonin in M. graminicola host plant parasitism and proposes that the serotonergic signaling pathway could be a potential target for controlling plant-parasitic nematodes.

Advances in Migratory Plant Endoparasitic Nematode Effectors.

Unlike sedentary plant-parasitic nematodes, migratory plant endoparasitic nematodes (MPENs) are unable to establish permanent feeding sites, and all developmental stages (except eggs) can invade and feed on plant tissues and can be easily overlooked because of the unspecific symptoms. They cause numerous economic losses in agriculture, forestry, and horticulture. In order to understand the pathogenetic mechanism of MPENs, here we describe research on functions and host targets focused on currently identified effectors from six MPENs, namely Radopholus similis, Pratylenchus spp., Ditylenchus destructor, Bursaphelenchus xylophilus, Aphelenchoides besseyi, and Hirschmanniella oryzae. This information will provide valuable insights into understanding MPEN effectors and for future fostering advancements in plant protection.

Nematodes as suppressors and facilitators of plant performance.

Plant-nematode interactions are mainly considered from the negative aspect with a focus on plant-parasitic nematodes (PPNs), which is justified considering the agronomic losses caused by PPNs. Despite the fact that PPNs are outnumbered by nonparasitic free-living nematodes (FLNs), the functional importance of FLNs, especially with regard to plant performance, remains largely unknown. Here, we provide a comprehensive overview and most recent insights into soil nematodes by showing direct and indirect links of both PPNs and FLNs with plant performance. We especially emphasize the knowledge gaps and potential of FLNs as important indirect players in driving plant performance such as stimulating the resistance to pests via improving the disease suppressive activity of the rhizobiome. Together, we present a holistic view of soil nematodes as positive and negative contributors to plant performance, accentuating the positive but underexplored role of FLNs.

Host-delivered RNAi-mediated silencing using fusion cassettes of different functional groups of genes precludes Meloidogyne incognita multiplication in Nicotiana tabacum.

KEY MESSAGE: This study demonstrates multi-gene silencing approach for simultaneous silencing of several functional genes through a fusion gene strategy for protecting plants against root-knot nematode, Meloidogyne incognita. The ability of root-knot nematode (RKN), Meloidogyne incognita, to cause extensive yield decline in a wide range of cultivated crops is well-documented. Due to the inadequacies of current management approaches, the alternatively employed contemporary RNA interference (RNAi)-based host-delivered gene silencing (HD-RNAi) strategy targeting different functional effectors/genes has shown substantial potential to combat RKNs. In this direction, we have explored the possibility of simultaneous silencing of four esophageal gland genes, six plant cell-wall modifying enzymes (PCWMEs) and a serine protease gene of M. incognita using the fusion approach. In vitro RNAi showed that combinatorial gene silencing is the most effective in affecting nematode behavior in terms of reduced attraction, penetration, development, and reproduction in tomato and adzuki beans. In addition, qRT-PCR analysis of M. incognita J2s soaked in fusion-dsRNA showed perturbed expression of all the genes comprising the fusion construct confirming successful dsRNA processing which is also supported by increased mRNA abundance of five key-RNAi pathway genes. In addition, hairpin RNA expressing constructs of multi-gene fusion cassettes were developed and used for generation of Nicotiana tabacum transgenic plants. The integration of gene constructs and expression of siRNAs in transgenic events were confirmed by Southern and Northern blot analyses. Besides, bio-efficacy analyses of transgenic events, conferred up to 87% reduction in M. incognita multiplication. Correspondingly, reduced transcript accumulation of the target genes in the M. incognita females extracted from transgenic events confirmed successful gene silencing.

First Report of the Root-Knot Nematode Meloidogyne luci on Tomato in Serbia.

Root-knot nematode (RKN) Meloidogyne luci Carneiro, Correa, Almeida, Gomes, Deimi, Castagnone-Sereno, and Karssen, 2014 was described from Brazil, Chile and Iran, parasitizing in various crops (Carneiro et al. 2014). It was later also described from Slovenia, Italy, Greece, Portugal, Turkey and Guatemala (review in Geric Stare et al. 2017). It is considered an extremely damaging pest as it has a wide host range and infects numerous higher plants, including monocotyledons and dicotyledons as well as herbaceous and woody plants. This species was included in the European Plant Protection Organisation Alert List of harmful organisms. In Europe, M. luci has been detected in both greenhouse and field agricultural production (review in Geric Stare et al. 2017). Furthermore, M. luci has been shown to survive winter in the field under continental and sub-Mediterranean climatic conditions (Strajnar et al. 2011). In August 2021, an official survey for quarantine RKN in Serbia (Province Vojvodina) revealed in a greenhouse in the village of Lugovo (43043'32,562; 19008'55,168), near Sombor, yellowing, stunning and extensive root galls on tomato (Solanum lycopersicum L.) cultivar Diva F1 caused by an unknown Meloidogyne sp. (Fig. 1). As correct identification is essential for effective pest management program, the next step was to identify the nematode species. Morphological characterization performed on freshly isolated females revealed perineal patterns similar to M. incognita (Kofoid and White, 1919) Chitwood, 1949. The shape was oval to squarish with the dorsal arch rounded to moderately high and without shoulders. The dorsal striae were wavy and continuous. The ventral striae were smooth and the lateral lines were weakly demarcated. The perivulval region was without striae (Fig. 2). The female stylet was robust with well-developed knobs and the stylet cone slightly curved dorsally. Although morphological characters was very variable, the nematode was suspected as M. luci based on comparison with originally described M. luci and M. luci populations from Slovenia, Greece and Turkey. Identification was achieved with subsequent species-specific PCR and sequence analysis. The nematode was determined to belong to the tropical RKN group and the M. ethiopica group using two PCR reactions as described by Geric Stare et al. (2019) (Figs. 3 and 4). Identification was confirmed by species-specific PCR of M. luci as described by Maleita et al. (2021), and a band of approximately 770 bp was obtained (Fig. 5). In addition, the identification was confirmed by sequence analyses. The region of mtDNA was amplified with primers C2F3 and 1108 (Powers and Harris 1993), cloned, sequenced (acc. no. OQ211107), and compared to other Meloidogyne spp. sequences from the Genbank. The determined sequence is 100% identical to an unidentified Meloidogyne sp. from Serbia, while the next highest scores are sequences of M. luci from Slovenia, Greece and Iran, all of which have 99.94% sequence identity. In phylogenetic tree, all M. luci sequences including the sequence from Serbia belong to a single clade. Egg masses isolated from infected tomato roots were used to establish a nematode culture in greenhouse and they caused typical root galls on cultivar Maraton of tomato. The galling index assessed 110 days-post-inoculation was in the range 4-5 according to the scoring scheme (1-10) for field evaluation of RKN infestations (Zeck 1971). To our knowledge, this is the first report of M. luci in Serbia. The authors hypothesize that climate change and higher temperatures could lead to much greater spread and damage to various agricultural crops in the field by M. luci in the future. National surveillance program for RKN in Serbia continued in 2022 and 2023. A management program to control the spread and damage from M. luci will be implemented in Serbia in 2023. Acknowledgments: This work was financially supported by the Serbian Plant Protection Directorate of MAFWM in the frame of Program of Measures in Plant Health in 2021, the Slovenian Research Agency in the frame of Research Programme Agrobiodiversity (P4-0072) and the Ministry of Agriculture, Forestry and Food of the Republic of Slovenia in the frame of Expert work in the field of plant protection (C2337).

Distribution, Diversity, and Soil Associations of Wine Grape Plant-Parasitic Nematodes in Georgia, U.S.A., Vineyards.

Wine grape (Vitis vinifera and V. vinifera hybrids) production in Georgia occurs in three distinct regions (North, West, and South) which can be characterized by sandy, sandy-loam, or sandy clay-loam soils. We studied plant-parasitic nematode (PPN) communities in 15 wine grape vineyards from the three primary growing regions to understand which nematodes are a concern and what soil characteristics are associated with their occurrence and relative abundance. Twelve genera of PPNs were detected throughout the state: Belonolaimus, Helicotylenchus, Hemicycliophora, Heterodera, Hoplolaimus, Meloidogyne, Mesocriconema, Paratrichodorus, Paratylenchus, Pratylenchus, Tylenchorhynchus, and Xiphinema. Nonmetric multidimensional scaling ordination and multirank permutation procedure identified PPN community differences and soil characteristics that were associated by region. Indicator species analysis identified Helicotylenchus, Mesocriconema, Tylenchorhynchus, and Xiphinema as statistically associated with the West while Meloidogyne and Paratrichodorus were associated with the South. Our analyses further suggested that soil texture (percent sand, percent clay, and percent silt) and the lime buffer capacity at equilibrium (LBCEQ) were associated with PPN community structure while pH was not. When focused on a single vineyard in the North, multiple logistic regression analysis suggested a statistically significant association between Meloidogyne spp. and soil characteristics, including percentages of sand, pH, and LBCEQ. Our study supports the association between soil characteristics and specific nematode genera, as well as the emergence of LBCEQ, the soil measurement with the strongest statistical association with nematode community structure and Meloidogyne presence.

Plant-Parasitic Nematodes and their Effects on Ornamental Plants: A Review.

Worldwide, the ornamental plant industry is estimated to be valued at $70 billion, with the United States' ornamental plant industry valued at $4.8 billion in 2020. Ornamental plants are cultivated for numerous reasons worldwide, such as decorative, medicinal, social, and utility purposes, making the ornamental field a high growth industry. One of the main pathogen groups affecting the yield and growth of the ornamental plant industry is plant-parasitic nematodes, which are microscopic roundworms that feed on plant parts causing significant yield loss. There are many kinds of plant-parasitic nematodes that affect ornamental plants, with the main genera being Meloidogyne spp., Aphelenchoides spp., Paratylenchus spp., Pratylenchus spp., Helicotylenchus spp., Radopholus spp., Xiphinema spp., Trichodorus spp., Paratrichodorus spp., Rotylenchulus spp., and Longidorus spp. The aim of this review is to focus on the effects, hosts, and symptoms of these major plant-parasitic nematodes on ornamental plants and synthesize current management strategies in the ornamental plant industry.

First Reports and Morphological and Molecular Characterization of Pratylenchus delattrei and Quinisulcius capitatus Associated with Chickpea in Ethiopia.

Chickpea (Cicer arietinum L.) is classed among the most important leguminous crops of high economic value in Ethiopia. Two plant-parasitic nematode species, Pratylenchus delattrei and Quinisulcius capitatus, were recovered from chickpea-growing areas in Ethiopia and characterized using molecular and morphological data, including the first scanning electron microscopy data for P. delattrei. New sequences of D2-D3 of 28S, ITS rDNA and mtDNA COI genes have been obtained from these species, providing the first COI sequences for P. delattrei and Q. capitatus, with both species being found for the first time on chickpea in Ethiopia. Furthermore, Pratylenchus delattrei was recovered in Ethiopia for the first time. The information obtained about these nematodes will be crucial to developing effective nematode management plans for future chickpea production.

Disruption of carbon for nutrient exchange between potato and arbuscular mycorrhizal fungi enhanced cyst nematode fitness and host pest tolerance.

Plants simultaneously interact with a range of biotrophic symbionts, ranging from mutualists such as arbuscular mycorrhizal fungi (AMF), to parasites such as the potato cyst nematode (PCN). The exchange of mycorrhizal-acquired nutrients for plant-fixed carbon (C) is well studied; however, the impact of competing symbionts remains underexplored. In this study, we examined mycorrhizal nutrient and host resource allocation in potato with and without AMF and PCN using radioisotope tracing, whilst determining the consequences of such allocation. The presence of PCN disrupted C for nutrient exchange between plants and AMF, with plant C overwhelmingly obtained by the nematodes. Despite this, AMF maintained transfer of nutrients on PCN-infected potato, ultimately losing out in their C for nutrient exchange with the host. Whilst PCN exploited the greater nutrient reserves to drive population growth on AMF-potato, the fungus imparted tolerance to allow the host to bear the parasitic burden. Our findings provide important insights into the belowground dynamics of plant-AMF symbioses, where simultaneous nutritional and nonnutritional benefits conferred by AMF to hosts and their parasites are seldom considered in plant community dynamics. Our findings suggest this may be a critical oversight, particularly in the consideration of C and nutrient flows in plant and soil communities.

A real-time recombinase polymerase amplification assay for fast and accurate detection of Ditylenchus destructor.

Ditylenchus destructor is a plant-parasitic nematode that seriously infests sweet potato crop in China. Thus, fast and accurate detection of D. destructor in soil and plant tissue samples is of great significance. In this study, a real-time recombinase polymerase amplification (RPA) assay was developed for the rapid and accurate detection of D. destructor in various samples. The RPA assay could be easily operated and detected as low as 1/500 individual J4 nematode DNA per reaction in 20 min at 39 °C with high specificity. The assay meets the requirements of rapid detection prior to port quarantine as well as on-site real-time detection and can be applied to detect the parasite in soil and plant samples. The modified gDNA extraction method for a single nematode established in this study significantly reduced the time of detection and improved the applicability of the real-time RPA assay for on-site detection in different environments. The real-time RPA assay to detect D. destructor will be useful for epidemiological investigations in the field as well as for quarantine processes in the sweet potato and potato trade.

Nematicidal effects of a peptidase (NlpC/P60) from the phytopathogen Pseudomonas syringae MB03 on the model nematode Caenorhabditis elegans and plant-parasitic Meloidogyne incognita.

BACKGROUND: Plant-parasitic nematodes (PPNs) are severe threats to agricultural yields and continue to be challenging to treat in several crops worldwide. Microbial-based control has been suggested as a better alternative to chemical control. In this study, we aimed to identify and characterize nematicidal virulence factors of a common phytopathogenic bacterium, Pseudomonas syringae, mainly focusing on the nematicidal and suppressive activities of an NlpC/P60 family peptidase, namely, Peptidase03, against the model nematode Caenorhabditis elegans and an agriculturally important PPN, Meloidogyne incognita. METHODS AND RESULTS: Genome-wide virulence factor prediction of the P. syringae wild-type strain MB03 revealed numerous nematode pathogenic determinants. We selected 11 predicted nematicidal genes for cloning and induced expression in an Escherichia coli expression system and then performed comparative nematicidal bioassays on the model nematode C. elegans. The recombinant strain expressing Peptidase03 showed the highest level of toxicity against C. elegans, with 75.9% mortality, compared to the other tested strains. Purified Peptidase03 showed significant toxicity against C. elegans and M. incognita, with half lethal concentration (LC50) values of 147.9 µg/mL and 211.50 µg/mL, respectively. We also demonstrated that Peptidase03 could damage the intestinal tissues of C. elegans and exhibit detrimental effects on its growth, brood size, and locomotion. CONCLUSIONS: The Peptidase03 protein from P. syringae MB03 had significant nematicidal and suppressive activities against C. elegans and M. incognita, thereby showing potential for the development of an effective PPN-controlling agent for use in agricultural practice.

In planta RNAi approach targeting three M. incognita effector genes disturbed the process of infection and reduced plant susceptibility.

Meloidogyne incognita is the most economically important species of the root-knot nematode complex causing damage to several crops worldwide. During parasitism in host plants, M. incognita secretes several effector proteins to suppress the plant immune system, manipulate the plant cell cycle, and promote parasitism. Several effector proteins have been identified, but their relationship with plant parasitism by M. incognita has not been fully confirmed. Herein, the Minc01696, Minc00344, and Minc00801 putative effector genes were evaluated to assess their importance during soybean and Nicotiana tabacum parasitism by M. incognita. For this study, we used in planta RNAi technology to overexpress dsRNA molecules capable of producing siRNAs that target and downregulate these nematode effector genes. Soybean composite roots and N. tabacum lines were successfully generated, and susceptibility level to M. incognita was evaluated. Consistently, both transgenic soybean roots and transgenic N. tabacum lines carrying the RNAi strategy showed reduced susceptibility to M. incognita. The number of galls per plant and the number of egg masses per plant were reduced by up to 85% in transgenic soybean roots, supported by the downregulation of effector genes in M. incognita during parasitism. Similarly, the number of galls per plant, the number of egg masses per plant, and the nematode reproduction factor were reduced by up to 83% in transgenic N. tabacum lines, which was also supported by the downregulation of the Minc00801 effector gene during parasitism. Therefore, our data indicate that all three effector genes can be a target in the development of new biotechnological tools based on the RNAi strategy in economically important crops for M. incognita control.

First report of root knot nematodes Meloidogyne incognita and M. javanica parasitizing sweet potato, Ipomoea batatas L., in Portugal.

Sweet potato, Ipomoea batatas L., is a tuberous root vegetable rich in low glycemic sugars, vitamins and fibers (Galvão et al., 2021). Although it is widely cropped and consumed in tropical regions, in Europe consumer demand is growing exponentially (CBI, 2021). In Portugal, the production area of sweet potato increased from 588 ha in 2011 to 954 ha in 2017, and exports increased from 2404 tons in 2011 to 13412 tons in 2019 (FAOSTAT, 2021). During a survey carried out in August 2019, sweet potato plants were collected in Almada (38°39'40"N 9°10'54"W) and Belmonte (38°39'40"N 9°10'54"W), South and Centre regions of Portugal, respectively. No symptoms were observed on leaves, however, roots presented numerous galls and/or small spots (females and respective egg masses) were observed in the tuberous root flesh, suggestive of root knot nematodes (RKN, Meloidogyne spp.) infection. At least 8 individual females and respective egg masses were handpicked from roots of each sample and characterized biochemically by electrophoretic analysis of esterases (Pais & Abrantes, 1989). Phenotypes I2 and J3, attributed to M. incognita and M. javanica, respectively, were present in samples from Almada, whereas only phenotype I2 was found from Belmonte sample (Santos et al., 2019). Pure RKN cultures were established on tomato cv. Coração-de-Boi to obtain inoculum for molecular characterization and host suitability assays. Molecular characterization was performed by DNA amplification with M. incognita (Mi-F/Mi-R) and M. javanica (Fjav/Rjav) species-specific primers (Zijlstra et al., 2000; Meng et al., 2004). DNA amplification resulted in unique bands of ≈900 bp and ≈650 bp, respectively, confirming the RKN species identification. The host suitability of sweet potato cvs. Lira (local variety, purple skin, yellow flesh) and Murasaki (purple skin, white/pale to yellow flesh) to M. javanica (Almada) and M. incognita (Belmonte) isolates was assessed. Sweet potato slips with ≈10 cm roots were transplanted to 500 cm3 pots (one slip/pot) and after 2 weeks, each plant was inoculated with 5000 eggs + second-stage juveniles (Pi, initial population density) and maintained in a growth chamber (25±2°C; 12:12 h photoperiod). Tomato cv. Coração-de-Boi was included as a positive control. Each RKN species-plant germplasm combination was repeated 6 times. At 60 days after inoculation, host suitability was evaluated on the basis of root gall index (GI) and reproduction factor (Rf=final population density/Pi) (Sasser et al., 1984). Sweet potato cv. Lira was susceptible (GI=5; Rf=111.8) to M. incognita and resistant (GI=2; Rf=0.11) to M. javanica; while cv. Murasaki was hypersusceptible (GI=5; Rf=0.9) to M. incognita and susceptible (GI=5; Rf=5.5) to M. javanica. Although cultivars varied in their response to M. incognita and M. javanica isolates and variation in the final population density was high, both RKN isolates reproduced in these sweet potato cultivars. In previous studies, cv. Murasaki was considered resistant to M. enterolobii and to M. incognita (La Bonte et al. 2008; Schwarz et al., 2021). Depending on the RKN species, cultivation of cvs. Murasaki and Lira may thus benefit succeeding crops, but they should be combined with other management strategies to further reduce RKN populations in the field. In Portugal, M. incognita and M. javanica have been found associated with economically important horticultural crops, such as tomato and potato, trees and weeds (Santos et al., 2019; Maleita et al., 2021). To our knowledge, these species are reported for the first time parasitizing sweet potato in Portugal and this is the first report on the occurrence of M. incognita and M. javanica infecting sweet potato in Europe. Although findings were not totally unexpected due to the wide distribution and host range of these RKN species, they are of crucial importance since the sweet potato production in Europe has almost doubled from 50 (2011) to 97 thousand tons (2017), with Spain, Portugal, Italy and Greece being the largest producers (FAO, 2021). Our findings also reveal that sweet potato cropped in Portugal have different susceptibility levels to these common RKN species, reinforcing the importance of cultivar selection in RKN management.

Aphelenchoides besseyi Ab-FAR-1 Interacts with Arabidopsis thaliana AtADF3 to Interfere with Actin Cytoskeleton, and Promotes Nematode Parasitism and Pathogenicity.

Fatty acid and retinol binding proteins (FAR) are unique proteins found in nematodes and are considered potential targets for controlling these parasites. However, their functions in nematode parasitism and pathogenicity and interaction with hosts are still unclear. In this study, we investigated the specific roles of rice white tip nematodes (RWTNs), Aphelenchoides besseyi, and a protein, Ab-FAR-1, to elucidate the parasitic and pathogenic processes of nematodes. The results showed that the expression level of Ab-far-1 was significantly up-regulated after A. besseyi infection of the plant. The immunofluorescence and subcellular localisation showed that Ab-FAR-1 was secreted into plant tissues mainly through the body wall of nematodes and might act in the nucleus and cytoplasm of plant cells. The pathogenicity of RWTNs was enhanced in Arabidopsis thaliana overexpressing Ab-FAR-1 and inhibited in Ab-far-1 RNAi A. thaliana. Yeast two-hybrid, Co-IP, BiFC, and nematode inoculation experiments showed that Ab-FAR-1 could interact with the A. thaliana actin-depolymerizing factor protein AtADF3, and the A. thaliana adf3 mutant was more susceptible to nematodes. An in vitro actin filament depolymerisation assay demonstrated that Ab-FAR-1 could inhibit AtADF3-mediated depolymerisation of actin filaments, and the turnover process of cellular actin filaments was also affected in A. thaliana overexpressing Ab-FAR-1. In addition, flg22-mediated host defence responses were suppressed in A. thaliana overexpressing Ab-FAR-1 and adf3 mutants. Therefore, this study confirmed that RWTNs can affect the turnover of actin filament remodelling mediated by AtADF3 through Ab-FAR-1 secretion and thus inhibit plant PAMP-triggered immunity (PTI), promoting the parasitism and pathogenicity of nematodes.