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Cyst-forming nematodes of the genus Heterodera are highly derived and economically important plant parasites. The Schachtii group of this genus is one of the largest ones with a total of 18 species parasitising dicotyledons. In this study, we provided comprehensive phylogenetic analyses of several hundred COI and ITS rRNA gene sequences of selected species from the Schachtii group, including H. betae, H. cajani, H. ciceri, H. galeopsidis, H. glycines, H. medicaginis, H. mediterranea, H. schachtii, H. sonchophila and H. trifolii, using Bayesian inference, maximum likelihood, and statistical parsimony. One hundred and twenty four new COI, 57 ITS rRNA and eight hsp90 gene sequences from 81 nematode populations collected in 19 countries were obtained in this study. Our study showed that the ITS rRNA gene has limited discrimination power compared to the COI gene. However, our analysis also revealed that partial COI gene sequences were identical for H. trifolii, H. betae and H. galeopsidis. Based on the results of phylogeographical analysis and age estimation of clades with a molecular clock approach, it was hypothesised that the majority of the Schachtii group species originated and diversified in the Mediterranean Basin biodiversity hotspot during the Pleistocene and then dispersed from this region across the world. The Sino-Japanese Floristic Region is likely one of the centres of diversification for the soybean cyst nematode, which showed distinct population structure. The possible role of hybridisation and polyploidisation in the evolution of species of the Schachtii group is discussed.

The ring nematode genus Xenocriconemella De Grisse and Loof, 1965 comprises only one nominal species, Xenocriconemella macrodora (Taylor, 1936) De Grisse and Loof, 1965. The initial objective of the present study was to investigate the morphological-morphometric and molecular diversity of 28 X. macrodora populations in the Iberian Peninsula associated with tree forests (mainly Quercus spp.). However, a detailed integrative taxonomic analysis (morphological-morphometric and molecular data) from each population and analysis of this data using principal component analysis (PCA) for morphometric data (including these 28 populations and other 25 X. macrodora populations around the world) and molecular and phylogenetic species delimitation methods revealed that X. macrodora forms a species complex. This species complex is composed by species that are morphometricly and morphologically similar, but clearly different at the molecular level. Three new species are described applying integrative taxonomy, namely as Xenocriconemella iberica sp. nov., Xenocriconemella paraiberica sp. nov. and Xenocriconemella pradense sp. nov. However, the molecular diversity of this species in USA and Italy confirmed that additional species are likely present in this species complex, and the diversity of this group may be higher than expected. The study of X. macrodora topotypes can clarify the position of this species using molecular markers under an integrative approach.

Ring nematodes are obligate ectoparasites on crops and natural herbaceous and woody plants, and some species are of economic importance and cause damage to roots of several crops. Recent integrative taxonomical analyses recognized the existence of two cryptic species within the Criconema annuliferum morphotype in Spain. In this study, we corroborated that morphometric, morphological and a multi-locus analysis (including the ribosomal markers D2-D3 expansion segments of 28S rRNA, ITS rRNA, 18S RNA, and the mitochondrial DNA cytochrome oxidase I gene) identified a new lineage clearly separated from C. annuliferum, C. paraannuliferum and C. plesioannuliferum. The new lineage was described herein as Criconema pseudoannuliferum sp. nov., confirming that C. annuliferum species complex species complex comprises a hyper-cryptic species complex. This research analysed soil samples from the rhizosphere of maritime pine (Pinus pinaster Ait.) forests in Bermeja-Crestellina Mountain, located at the western part of Málaga province, southern Spain. The integrative taxonomical analyses revealed the occurrence of a new cryptic species identified using females, males and juveniles with detailed morphology, morphometry and molecular markers, described herein as Criconema pseudoannuliferum sp. nov. All molecular markers (D2-D3, ITS, 18S and COI) were obtained from the same individual that was also used for morphological and morphometric analyses. This research demonstrated the hidden diversity within the C. annuliferum species complex species complex can reach to four lineages under ribosomal and mitochondrial gene markers for one morphospecies group, which includes four species, viz. C. annuliferum, C. paraannuliferum, C. plesioannuliferum, and C. pseudoannuliferum sp. nov. Criconema pseudoannuliferum sp. nov. was detected in moderate soil density in two maritime pine forests (5 and 25 nematodes/500 cm3 of soil) suggesting that does not cause damage to maritime pine.

Ring nematodes are obligate ectoparasites on cultivated and wild herbaceous and woody plants, inhabiting many types of soil, but particularly sandy soils. This study explored the morphometrical and molecular diversity of ring nematodes resembling Criconema annuliferum in 222 soil samples from fruit crops in Spain, including almond, apricot, peach and plum, as well as populations from cultivated and wild olives, and common yew. Ring nematodes of the genus Criconema were detected in 12 samples from under Prunus spp. (5.5%), showing a low to moderate nematode soil densities in several localities from southeastern and northeastern Spain. The soil population densities of Criconema associated with Prunus spp. ranged from 1 nematode/500 cm3 of soil in apricot at Sástago (Zaragoza province) to 7950 and 42,491 nematodes/500 cm3 of soil in peach at Ricla and Calasparra (Murcia province), respectively. The integrative taxonomical analyses reveal the presence of two cryptic species identified using females, males (when available), and juveniles with detailed morphology, morphometry, and molecular markers (D2-D3, ITS, 18S, and COI), described herein as Criconema paraannuliferum sp. nov. and Criconema plesioannuliferum sp. nov. All molecular markers from each species were obtained from the same individuals, and these individuals were also used for morphological and morphometric analyses. Criconema paraannuliferum sp. nov. was found in a high soil density in two peach fields (7950 and 42,491 nematodes/500 cm3 of soil) showing the possibility of being pathogenic in some circumstances.

Parasitic nematodes of millipedes from Nigeria are molecularly characterized for the first time. During nematode surveys on live giant African millipedes from several localities in Nigeria, 4 species of rhigonematids were identified by application of integrative taxonomical approaches (morpho-anatomy and molecular markers), including Brumptaemilius sp., Gilsonema gabonensis, Obainia pachnephorus, and Rhigonema disparovis. The results of morphometric and molecular analyses of D2-D3 28S, ITS, partial 18S rRNA, and cytochrome oxidase c subunit 1 (COI) gene sequences further characterized the rhigonematid species, and clearly separated them from other related species. Phylogenetic relationships based on 28S and 18S rRNA genes suggest that genera within Ransomnematoidea (Ransomnema, Heth, Carnoya, Brumptaemilius, Cattiena, Insulanema, Gilsonema) and Rhigonematoidea (Rhigonema, Obainia, Xystrognathus, Trachyglossoides, Ichthyocephaloides) clustered rather closer than could be expected in view of their morphological differences. Phylogenetic relationships based on ITS and COI are congruent with those of other ribosomal genes; however, they are not conclusive due to the scarcity of available sequences of these genes for these genera in NCBI.

Nematode samplings in various areas and crops of Greece were carried out and the recovered nematode species were characterized using morphological and molecular data. Seven species of plant-parasitic nematodes were recovered, three of which are reported for the first time in Greece, including Hemicycliophora poranga, Helicotylenchus dihystera and Tylenchorhynchus zeae. Four other recovered species had already been reported in Greece, including Bitylenchus hispaniensis, Helicotylenchus microlobus, Nanidorus minor and Scutellonema brachyurus. D2-D3 segments of 28S rRNA gene for all of these nematode species are provided.

Meloidogyne enterolobii Yang & Eisenback, 1983 (guava root-knot nematode) is an important disease in subtropical to tropical climate in several areas of the world (Subbotin et al., 2021). It is a highly polyphagous root-knot nematode species causing major damage to a range of economically important crops. The expansion of this species is increasing worldwide creating a potential problem to the maintenance of resistance genes to other major Meloidogyne species (Castagnone-Sereno and Castillo, 2020). Additionally, the diagnosis of M. enterolobii can be challenging due to morphological similarities with other root-knot nematode species (Castagnone-Sereno, 2012). In the African continent, it has been cited in several countries of Equatorial and South Africa (Subbotin et al., 2021), but not in North Africa. Two guava groves (at Bany Salama, Natrn vally, El Beheira governorate, 30.322043N, 30.518529E; and Izbat Al Halawijah, Monshaah Alaweyah, Abu El Matamir, El Beheira governorate 30.9398050N, 30.1484430E), in Egypt, were found with significant symptoms of tree decline and root galling damage. The presence of egg masses and females of root-knot nematodes were found inside the galls (Figure 1A, B). Nematodes were extracted from soil samples with levels of 12300 and 12600 second-stage juveniles (J2s)/250 g of soil using a modified Baerman method (Hooper, 1986), respectively. Nematode root density was 24367 eggs/g of root, using the protocol described in Hussey and Barker (1973) for Izbat Al Halawijah population. For morphological and morphometrical identification, J2s and females were fixed using a hot formalin solution (4% v/v). DNA was isolated from single J2s specimen for: i) testing multiplex specific-PCR assay for M. incognita, M. javanica and M. arenaria (Kiewnick et al., 2013), and ii) amplifying and sequencing of cytochrome oxidase subunit II (COII) and the 16S rRNA mitochondrial region using the primer pair C2F3 (5'-GGTCAATGTTCAGAAATTTGTGG-3') (Powers and Harris

Heterodera zeae Koshy, Swarup & Sethi, 1971 (corn cyst nematode) is an important disease of corn in several areas of the world, including India, Nepal, Pakistan, Egypt, USA, Greece and Portugal (Subbotin et al., 2010). It is a sedentary semi-endoparasite feeding on corn roots and other Poaceae plants and has been associated with significant yield losses in corn (Subbotin et al., 2010). During autumn 2022 a plant-parasitic nematode survey performed in corn at central-western area of Spain (Talavera de la Reina, Toledo), revealed a commercial field with stunted plants. Nematodes were extracted from soil by centrifugal-flotation method (Coolen, 1979). Corn roots inspection detected infections by immature and mature cysts, and soil revealed also mature live cysts and second-stage juveniles (J2s) with a population density of 1010 eggs and J2s/500 cm3 soil (including eggs from cysts). J2s and cysts were processed to pure glycerine using De Grisse's (1969) method. DNA was isolated from single live fresh J2s specimens for amplifying and sequencing of cytochrome c oxidase subunit II (COII) mitochondrial region using the primer pair species-specific H.Gly-COIIF_inFOR/P116F-1R (Riepsamen et al., 2011); D2 and D3 expansion domains of the 28S rRNA were amplified using the D2A/D3B primers (De Ley et al. 1999); internal transcribed spacer (ITS) region using primers TW81/AB28 (Subbotin et al., 2001); and cytochrome c oxidase subunit 1 (COI) gene was amplified using the primers JB3/JB5 (Bowles et al., 1992). Brown cysts were lemon-shaped with a protruding vulval cone with fenestra ambifenestrate, bullae prominent below underbridge and characteristically arranged in finger-like bullae (Fig. 1). J2 with slightly offset lip region (3-5 annuli), stylet strong with rounded stylet knobs, lateral field with four lines, and tail short and tapering conically. Measurements of cysts (n=10) included body length 559 (432-688) µm, body width 450 (340-522) µm, fenestral length 40 (36-43) µm, semif

Los pasados 35 años han sido testigos de importantes avances en el conocimiento sobre los organismos fitopatógenos, demasiado numerosos y diversos en naturaleza para poder resumirlos en unas páginas. Muchos de dichos avances han estado mediados por el desarrollo de nuevas metodologías, instrumentos y protocolos de estudio, en particular los concernientes a las tecnologías de análisis y secuenciación del ADN que comenzaron con su amplificación mediada por una ADN polimerasa termoestable (PCR) –y sus posteriores derivados: PCR cuantitativa, en tiempo real, digital, etc. Estos avances continuaron con el uso de las plataformas de secuenciación masiva para el análisis de los genomas de estos organismos, incluso a partir de la matriz vegetal que infectaban sin necesidad de su aislamiento, así como el uso de diversas tecnologías -ómicas para el análisis masivo de la expresión diferencial de genes (genómica), proteínas (proteómica) y metabolitos (metabolómica). Todo ello ha tenido profundas repercusiones, por ejemplo, sobre la taxonomía y relaciones filogenéticas de estos organismos fitopatógenos, la comprensión de la regulación genética de la patogenicidad y de los factores (efectores) de virulencia, la resistencia a la infección en la planta. Asimismo, las tecnologías de observación microscópica y el uso de genes que codifican proteínas fluorescentes de diferentes propiedades espectrales han propiciado una mejor compresión de los procesos de infección (Deal, 2011). En las siguientes secciones del artículo se presentan algunos avances ilustrativos seleccionados por expertos para cada uno de los grandes grupos de organismos fitopatógenos: hongos, oomicetos, bacterias, virus y nematodos. Cabe decir que muchos de los avances presentados para cada organismo fitopatógeno son aplicables a todos los demás. Si bien, con objeto de no incurrir en reiteraciones, y extendernos en exceso, se ha intentado, en la medida de lo posible, seleccionar avances con aspectos diferenciadores.

Los cultivos de Prunus (principalmente almendro, melocotón-nectarino, ciruelo y cerezo) están ampliamente extendidos por las zonas mediterráneas del territorio español. Las nuevas condiciones climáticas provocadas por el cambio climático (mayores temperaturas y sequía), la introducción de nuevos patógenos y la intensificación de los cultivos de Prunus pueden incrementar la incidencia y patogenicidad de algunas enfermedades en el futuro con estos nuevos condicionantes. Este artículo pretende abordar una revisión de las principales enfermedades causadas por patógenos de suelo en los cultivos de Prunus, tanto tradicionales como emergentes, así como medidas de control actuales y sus posibles estrategias de prevención y control.

The Mediterranean olive cyst nematode, Heterodera mediterranea, is a pest of olive trees that is found in Spain, Italy, and Tunisia. In this paper, conventional and real-time PCR assays for the identification of H. mediterranea was developed. The hsp90 gene fragment was used to design a species-specific primer and a TaqMan probe. The specificity of the primer/probe set was tested in singleplex or multiplex reactions against Mediterranean olive cyst nematode and non-target nematodes. In multiplex real-time PCR experiments with the specific and universal primer/probe sets, fluorescent signals were simultaneously monitored for hsp90 and D3 of 28S rRNA target genes. The results of the experiments showed that the assays with species-specific primer/probe were sensitive enough to detect the DNA from 0.03 of a second-stage juvenile (J2) H. mediterranea in a reaction tube.

This study evaluates plant growth promotion and the suppressive effect of the burrowing nematode (Radopholus similis) in banana Musa AAA cv Grande Naine, by two ensilaged biostimulants (EBSs) in two greenhouse trials and in two different commercial farms. Conductive (CS) and suppressive (SS) soils to plant parasitic nematodes were used for EBSs production. The EBSs were incorporated in the growth substrate at 10% w/w before planting the in vitro banana plants and before R. similis inoculation in the greenhouse trials. In commercial banana plantations, the treatments were applied every four months by incorporating 500 g of the EBS into the soil in front of the successional sucker of each banana plant. The results showed that both EBSs were effective, stimulating the root growth and reducing R. similis. The EBS with CS reduced R. similis consistently in greenhouse and field evaluations. The data suggests the potential of EBSs to promote unfavorable conditions for the burrowing nematode reproduction and more favorable conditions to the development and production of the crop, which could contribute to promote more sustainable banana production.

Paratylenchus species are obligate ectoparasitic nematodes on cultivated and wild herbaceous and woody plants occupying numerous soil categories. Several species may cause damage to several crops (viz. P. dianthus, P. enigmaticus, P. microdorus, P. hamatus and P. epacris on carnation, lettuce, rose and walnut, respectively). This investigation proves and emphasizes the relevance of applying integrative taxonomy for the accurate detection of Paratylenchus species in mountainous wild environments in the Malaga province, Southern Spain. This research analyzed 45 soil samples of maritimus pine and one of green heather in southern Spain and identified fourteen Paratylenchus species, two of them are described herein as new species (P. paraaonli sp. nov., P. plesiostraeleni sp. nov.), six of them were first reports for Spain (P. canchicus, P. nainianus, P. neonanus, P. salubris, Paratylenchus sp. 2 SAS, and P. wuae), and six species (P. caravaquenus, P. microdorus, P. nanus, P. neoamblycephalus, P. sheri, and P. variabilis) have been already reported in Spain. Accordingly, these data increase the biodiversity of pin nematodes in Spain comprising a total of 47 species (33.1% out of 142 total species of this genus). Phylogenetic analyses based on ribosomal and mitochondrial markers (D2-D3, ITS, and partial COI) resulted in a consistent position for the newly described Paratylenchus species in this study (P. plesiostraeleni sp. nov., P. paraaonli sp. nov.). Paratylenchus plesiostraeleni sp. nov. grouped in a separated subclade as unequivocal species from the P. straeleni-complex species (including P. straeleni and P. parastraeleni), and P. paraaonli sp. nov. clustered with P. vitecus, but clearly separate from this species. This study indicates that Paratylenchus species diversity in natural environments may be higher than expected, and this study may help in accurate identifications.

This study aimed to evaluate the prevalence and distribution of Paratylenchus species in the main areas of Prunus spp. production in Spain, their ecological constraints, and new molecular tools for the specific identification of major species. Pin nematodes are recognized as plant-parasitic nematodes with a wide host range and global distribution. Therefore, understanding the environmental and agronomic factors involved in their distribution is critical to design environmental and sustainable management strategies to reduce plant damage. A total of 219 sampling sites were surveyed and 12 Paratylenchus species were identified based on an integrative taxonomic approach (P. baldaccii, P. enigmaticus, P. goodeyi, P. hamatus, P. holdemani, P. indalus, P. israelensis, P. pedrami, P. tateae, P. tenuicaudatus, P. veruculatus, and P. zurgenerus). The most common pin nematode was P. hamatus, followed by P. tenuicaudatus. Nematode abundance was influenced by climatic characteristics, soil chemical properties, and agronomic management practices. Nine explanatory variables were selected as the most strongly associated with Paratylenchus distribution. Specifically, P. tenuicaudatus was significantly correlated with soil chemical characteristics, such as pH and carbon, sulfur, and sodium content, whereas P. goodeyi was closely related to fields with <10 years of almond cultivation. Species-specific PCRs were developed for P. hamatus and P. tenuicaudatus and their validity was evaluated studying the molecular variability of these species and against other Paratylenchus species.

Pin nematodes of the genus Paratylenchus comprise 140 species. This group of nematodes is characterized by a quite homogeneous morphology and cosmopolitan distribution and is prevalent in cultivated and natural soil ecosystems. The present study describes the first interactive and illustrated web-assisted polytomous identification key for the genus Paratylenchus. The updated Paratylenchus species polytomous key was based on a wide list of 24 diagnostic characters generated for the 140 species comprising this genus. Here we developed a web-assisted method to achieve an easy and accurate Paratylenchus species characterization that will greatly improve the identification of these plant-parasitic nematodes for many diagnostic laboratories and researchers. However, this identification needs to be completed with the use of molecular markers available for the species due to the existence of species complexes studied in former researches. This idea is pointed in the polytomous key in the specific species complexes up-to-know. In some cases, the presence in the soil as survival stage of few individuals in the fourth-stage juvenile (J4) required the use of molecular markers for species identification. We suggest the use of at least a fragment of mitochondrial COI gene for species identification or the combination of nuclear D2-D3 regions of the 28S rRNA and the COI to complement each marker. However, for some species complexes, the use of the D2-D3 regions alone has not enough resolution to separate the putative species inside the species complex. Web-based polytomous key was constructed using the free software Xper3, for computers and mobile devices (smartphones, tablets, and pocket PCS).

Radopholus similis is one of the most important diseases in banana in tropical/subtropical regions worldwide. The knowledge of its molecular diversity in specific areas could help to understand its distribution, ways of expansion, control methods, differential pathogenicity, and sustainable plant breeding programs. This study explores the molecular diversity of R. similis in an extensive survey in banana plantations in Costa Rica using two molecular markers, one genomic (ITS region) and another mitochondrial (COI). The results showed a low molecular variability, being dominated by a prevalent haplotype in both markers. Three haplotypes have been detected for each molecular marker respectively. The haplotypes present in Costa Rica are similar to others found in other areas of Central America (Colombia, Panama) and these haplotypes are also distributed worldwide. These results suggested a unique or limited number of introductions of this pathogen in Central America and a possible expansion within the continent by infected plant material.

Longidorid nematodes comprise more than 500 species, and Longidorus and Xiphinema are the most diversified, prevalent, and cosmopolitan genera within plant-parasitic nematodes. The genus Longidorus comprise a group of species, some of which are vectors of plant viruses. New sampling for needle nematodes was carried out in a grapevine area in Thessaloniki, northern Greece, and one nematode species of Longidorus (L. leptocephalus) was recovered. Nematodes were extracted from soil samples by modified sieving and a decanting method. Extracted specimens were processed using glycerol, mounted on permanent slides, and subsequently identified morphologically. Nematode DNA was extracted from individual, live specimens, and PCR (Polymerase Chain Reaction) assays were performed for D2-D3 expansion segments of 28S rRNA, ITS1 rRNA, and partial mitochondrial COI regions. Morphology and morphometric data obtained from this population were consistent with the original description and reported populations of L. leptocephalus. To our knowledge, this is the first report of L. leptocephalus in Greece and the second in the Mediterranean Basin after the record of the species from Slovenia, extending the geographical distribution of this species in Europe.

Entomopathogenic nematodes (EPNs) are biological control agents distributed worldwide in natural and agricultural soils. Recent evidence supports the hypothesis that stable rhizospheres of perennial crops can favor the occurrence of EPNs. However, different agricultural management practices can modulate their natural distribution in agroecosystems, hence should be considered in conservation biological control programs to support as high as possible native EPN populations. Cultivated olive (Olea europaea subsp. europaea), originally from the Mediterranean Basin, is a key perennial crop susceptible to a diverse range of arthropod pests that account for their life with at least one soil inhabitant stage. In this study, we investigated, for the first time, the occurrence of EPNs and associated soil organisms in olive groves managed with different practices. We surveyed 89 commercial olive groves located in Andalusia (Southern Spain) and differently handled for pest management (conventional vs. organic), soil management (tilling vs. cover cropping), and irrigation regime (rain feed vs. drip-irrigation). By using species-specific primers-probe sets and quantitative real-time PCR approaches, we screened the occurrence of nine species of EPNs, five free-living nematodes (FLNs), and six nematophagous fungi (NF). We found EPNs in 23.6% of the sites, identifying only two EPN species: Steinernema feltiae (15.7%) and Heterorhabditis bacteriophora (7.9%). Pest and soil management did not affect EPN abundance, but drip irrigation increased their occurrence (P < 0.025). On the contrary, we recorded lower FLN numbers in irrigated groves (P = 0.003), particularly for Oscheius tipulae and Pristionchus maupasi. Regarding NF, both organic management (P < 0.001) and drip irrigation (P < 0.035) enhanced their abundance. Four abiotic explanatory variables (extractable P, clay content, elevation, and precipitation) explained 85.6% of the EPN, FLN, and NF species variation in multivariate an

Grapevines are challenged by a range of diseases and pests, causing economic losses and requiring often costly approaches to mitigate damage. Public interest in reducing the use of chemicals is a related challenge, along with climate change. Yet, the Vitis gene pool provides vast resources for the development of genetic resistance in rootstock and scion cultivars. Traditional breeding approaches have made great strides in the development of adaptive traits, and recent access to ‘omic technologies has further facilitated the identification of useful loci along with rapid trait introgression from wild species. Moreover, marker technologies are now used to stack multiple genes for the same trait into a single genotype, a heretofore barely accessible technology. Genomic technologies are also impacting germplasm characterization, and thereby facilitating “Breeding by Design” approaches. Genetic transformation and gene-editing technologies are also applicable for both cultivar improvement as well as functional studies of genes. The landscape for acceptance of new resistant cultivars is complex and with wine grapes, subject to high degrees of regulation especially in the European Union. With rootstocks, as well as table/raisin grapes, gaining acceptance in the marketplace for new cultivars developed through either traditional or marker-assisted approaches is routine. Yet even in the highly regulated EU environment, the adoption of new wine cultivars of interspecific origins is beginning to take place in both traditional wine growing regions as well as non-traditional regions nearby.