PESTOLIVE: an historical and ecological approach for understanding and managing soil-borne parasite communities on olive in the Mediterranean basin.

Cultivated plants usually differ from their wild progenitors in several morphological and/or physiological traits. Their microbe communities might also differ because of adaptation to new conditions related to cultivation. To test this hypothesis, we investigated morphological traits in a parthenogenetic root-knot nematode (Meloidogyne javanica) from natural and agricultural environments. Seventeen populations of M. javanica were sampled on cultivated and wild olives in Morocco, then maintained in controlled conditions for a ‘common garden’ experiment. We estimated the genetic variation based on three traits (stylet size, neck width and body width) by a quantitative genetic design (ten families per population and nine individuals per family were measured), and molecular variation was investigated with a mitochondrial marker to identify the genetic lineages of nematode isolates sampled from wild and cultivated olives. Significant morphological differences were detected between individuals from wild vs. cultivated hosts for the three traits, whereas no phylogenetic clustering was observed among isolates collected on those two hosts. Our results thus suggest an adaptive response of the asexual parasite, possibly related to the deep modification of soil nematode communities between natural olive stands and orchards.

The present study is part of a survey for the identification of plant-parasitic nematodes in the rhizosphere of cultivated and wild olive trees in Crete, Greece. Sixteen species corresponding to 13 genera are added to 20 species belonging to 8 genera, previously reported in the survey. Seven nematode species, Filenchus ditissimus , Filenchus vulgaris , Ogma civellae , Pratylenchoides crenicauda , Psilenchus hilarulus , Tylenchus elegans , and Zygotylenchus guevarai , are recorded for the first time in Greece.

Description of the subject. In Morocco, expanding agricultural sector under “Morocco Green Plan” will also introduce plant diseases and plant-parasitic nematodes (PPN). Soil substrate used in most of the olive nurseries is potentially inhabited by PPN and natural enemies. Objectives. This work assessed the co-occurrence of PPN and nematophagous fungi (NF) in substrates and potential of NF to suppress PPN. Method: The diversity of PPN and NF genera was characterized in 305 substrate samples, isolated from 25 olive nurseries and functional activity (nematicidal and enzymatic) of NF was evaluated. Results. High PPN diversity was detected in the isolated samples and substrates were differentiated by NF activity. Global partition between PPN and NF diversity patterns confirmed prey-predator antagonism. Moreover, PPN-NF patterns depend on the fungal survival (saprophytes vs. obligate parasites) and antagonism (predation vs. toxicity) processes. However, even the NF strains with maximum fitness (growth, sporulation, enzymatic activity) were not inevitably the most effective against PPN. Consequently, it was hypothesized that predation and competition could be an adaptation process of NF to overcome environmental stress and exploit alternative or supplementary nutrient sources. Conclusions. The use of diverse native NF from cultivated and wild soils offer future prospects for the development of PPN management strategies in olive nurseries. Key words: Biological control, Communities, Soil Ecology, Nursery, Invasion, Predation

Datasets presented here were employed in the main work "Spatial structure and soil properties shape local community structure of plant-parasitic nematodes in cultivated olive trees in southern Spain" Archidona-Yuste et al., 2020. In this research, we aimed to unravel the diversity of plant-parasitic nematodes (PPN) associated with cultivated olive (Olea europaea subsp. europaea var. europaea) in southern Spain, Andalusia. The olive growing area of Andalusia is of high agriculture and socioeconomic importance with an extensive distribution of this crop. To this end, we conducted a systematic survey comprising 376 commercial olive orchards covering the diversity of cropping systems applied. Data showed 128 species of PPN belonging to 38 genera and to 13 families. In addition, an extensive data set regarding to potential factors in structuring the community patterns of PPN found in the 376 commercial olive orchards sampled is provided. Three variables data set were compiled including above-ground environment , soil and agronomic management. Overall, 48 explanatory variables were selected as determinist processes on shaping the diversity of PPN. Finally, data also showed the values regarding to the partition of beta diversity into contributions of single sites to DOI of original article: https://doi.

Datasets presented here were employed in the main work "Spatial structure and soil properties shape local community structure of plant-parasitic nematodes in cultivated olive trees in southern Spain" Archidona-Yuste et al., 2020. In this research, we aimed to unravel the diversity of plant-parasitic nematodes (PPN) associated with cultivated olive (Olea europaea subsp. europaea var. europaea) in southern Spain, Andalusia. The olive growing area of Andalusia is of high agriculture and socio-economic importance with an extensive distribution of this crop. To this end, we conducted a systematic survey comprising 376 commercial olive orchards covering the diversity of cropping systems applied. Data showed 128 species of PPN belonging to 38 genera and to 13 families. In addition, an extensive data set regarding to potential factors in structuring the community patterns of PPN found in the 376 commercial olive orchards sampled is provided. Three variables data set were compiled including above-ground environment, soil and agronomic management. Overall, 48 explanatory variables were selected as determinist processes on shaping the diversity of PPN. Finally, data also showed the values regarding to the partition of beta diversity into contributions of single sites to overall beta diversity (LCBD) and intro contributions of individual species to overall beta diversity (SCBD). Data may serve as benchmarks for other groups working in the field of PPN diversity associated with crops and of belowground communities and ecosystems.

Human activities increase in ecosystems, and cropping systems are more and more intensified. In Morocco, the development of an intensive agriculture in the "Morocco Green Plan" will necessarily induce the emergence of plant diseases, including plant-parasitic nematodes (PPN). Their management is a major challenge in agriculture. On the olive tree, they imperil production, mainly in nurseries and in high-density orchards. Researches were led to better understand (i) the response of PPN and of associated nematophagous fungus (NF) communities to the habitat origin of the breeding substrates and climate in nurseries, and (ii) the response of PPN communities to olive domestication and crop intensification. The goal was to evaluate the PPN risk in order to give recommendations to stakeholders, and to consider biocontrol strategies. Morphological, biochemical and molecular diagnosis of the total PPN fauna detected in the 25 nurseries (i.e., 305 olive cuttings sampled) and in the 49 orchards (i.e., 213 soil samples) revealed a wide species diversity (47 genera and 117 species), many species being described for the first time on olive tree, and a new species (Meloidogyne spartelensis) has been discovered. A focus on root-knot nematodes (RKN - Meloidogyne spp.) indicated the dominance of M. javanica. It was established that M. javanica disperses from nurseries to orchards first by the way of contaminated substrates introduced in nurseries, especially from cultivated soils, and then by the wide spread of infested olive trees in orchards. Consequently, olive production systems in Morocco are exposed to strong RKN invasion risks. PPN invasive processes are enhanced in orchards by the intensification of the cropping systems. Indeed, the anthropogenic gradient (oleaster vs feral olive vs traditional cultivation vs high-density cultivation) is the main variable that drives the diversity, the species / trophic / life strategy structure of the communities, the dominance of the taxa, and the community patterns by mostly reducing the species richness and increasing the abundance of PPN. The olive varieties multiplied in nurseries and used in orchards had no effect on the PPN diversity. In order to develop biocontrol strategies against PPN, nematophagous fungi (NF) were looked for in nurseries. Thus, 70 strains were isolated, including a large diversity of Orbiliaceae with trapping organs and of toxic Hypocreaceae. Egg-parasitic and endoparasitic species were also observed. In vitro predation tests revealed that Talaromyces assiutensis and Orbiliaceae strains were able to kill 100% and 50 to 80% of RKN juveniles respectively. It was established that the climate (rainfall and minimum temperatures) and the habitat origin of the substrates were the main drivers for both PPN and NF communities. Co-structure analyses between PPN and NF communities indicated significant correlations between Orbiliaceae and Meloidogyne spp., which confirms the interest of NF as biocontrol agents for the management of PPN. These studies validate that the diversity of PPN communities may be relevant indicators (i) to assess the health of substrates in olive nurseries for certification purposes and (ii) to rethink olive cropping strategies. They offer future prospects for the development of microbiological management strategies of these parasites respectful of the environment. This 5-years research involved 5 Moroccan and 2 French research teams and was funded by two projects (ARIMNet and TOUBKAL). It involved 2 MSc and 2 PhD students granted by UIZ-FSA, Tishreen University (Syria) and IRD (France). It produced 8 WOS publications and 11 international congress presentations.

Cultivated Cretan mountain tea or Malotira (Sideritis syriaca L.) was found to be infected by Meloidogyne hapla and Meloidogyne javanica in the island of Crete. The authors provide the first molecular characterization of M. hapla in Greece and the first report of Cretan mountain tea or Malotira as a host of Meloidogyne species worldwide. In addition, Meloidogyne hispanica was found infecting aloe (Andros island) and corn (Drama, North Greece) consisting the first reports of natural infection of these plants by M. hispanica in Europe. Furthermore, infection of corn by M. incognita and soybean by M. javanica (Drama, North Greece) are reported for the first time in Greece. Integrative taxonomical approach based on perineal pattern and EP/st ratio, as well as the region of the mitochondrial genome between the cytochrome oxidase subunit II (coxII) and 16S rRNA mitochondrial DNA (mtDNA) genes was used to differentiate Meloidogyne species. Cultivated Cretan mountain tea or Malotira (Sideritis syriaca L.) was found to be infected by Meloidogyne hapla and Meloidogyne javanica in the island of Crete. The authors provide the first molecular characterization of M. hapla in Greece and the first report of Cretan mountain tea or Malotira as a host of Meloidogyne species worldwide. In addition, Meloidogyne hispanica was found infecting aloe (Andros island) and corn (Drama, North Greece) consisting the first reports of natural infection of these plants by M. hispanica in Europe. Furthermore, infection of corn by M. incognita and soybean by M. javanica (Drama, North Greece) are reported for the first time in Greece. Integrative taxonomical approach based on perineal pattern and EP/st ratio, as well as the region of the mitochondrial genome between the cytochrome oxidase subunit II (coxII) and 16S rRNA mitochondrial DNA (mtDNA) genes was used to differentiate Meloidogyne species.

Root-knot nematodes of the genus Meloidogyne are recognised worldwide as a major production constraint in crops of primary economic importance. Knowledge of their diversity and prevalence, as well as the major environmental and agronomical cues for understanding their distribution in specific areas is of vital importance for designing control measures to reduce significant damage. We provide the first detailed information on the diversity, distribution and levels of Meloidogyne species infecting wild and cultivated olive soils in a wide region in southern Spain that included 499 sampling sites. Overall Meloidogyne spp. were found in 6.6% of sampled olive plants, with 6.6% and 6.5% for cultivated and wild olive, respectively. We identified five previously described Meloidogyne spp. Meloidogyne arenaria, M. baetica, M. hapla, M. incognita, M. javanica) and one new species (Meloidogyne oleae sp. nov.) which, characterized using integrative taxonomy, increases the known biodiversity of Meloidogyne spp. affecting olive. Meloidogyne arenaria and M. incognita were only found infecting cultivated olive varieties, while, M. baetica was only found infecting wild olive. Three major parameters drive the distribution of Meloidogyne spp. in cultivated olives in southern Spain, cover vegetation on alley, irrigation and soil texture, but different species respond differently to them. In particular the presence of M. incognita is highly correlated with sandy loamy soils, the presence of M. javanica with irrigated soils and cover vegetation, while the presence of M. arenaria is correlated with the absence of cover vegetation on alley and absence of irrigation. These parameters likely influence the selection of each particular Meloidogyne species from a major dispersal source, such as the rooted plantlets used to establish the orchards.

Background Root-knot nematodes (RKN) are major pest of olive tree (Olea europaea ssp. europaea), especially in nurseries and high-density orchards. Soil samples were collected from main olive growing areas of Morocco, to characterize Meloidogyne species and to discuss the contribution of biotic and abiotic factors in their spatial distribution. ResultsRKN were found in 159 soil samples out of 305 from nurseries (52.1% occurrence) and in 11 out of 49 soil samples from orchards (23.2% occurrence). Biochemical and molecular characterisation (PAGE esterase and SCAR) revealed the dominance of M. javanica both in nurseries and orchards with minor presence of M. incognita only in nurseries, and M. arenaria in only one nursery. RKN were distributed on aggregated basis. Frequent presence of M. javanica in orchards might have come from nurseries. In contrast, the detection of M. incognita in nurseries alone suggests that this species could not reproduce in orchards because of either the competition with other plant-parasitic nematodes or unfit local habitats. The impact of environmental variables (climate, habitat origin and physicochemical characteristics of the substrates) on the distribution of Meloidogyne species is also discussed. Conclusion Olive nurseries in Morocco are not able to guarantee the safety of rooted plants. As a result, olive production systems are exposed to strong RKN invasion risks. Consequently, the use of healthy substrates in nurseries may prevent plant-parasitic nematode induction in orchards.

Plant-parasitic nematodes are major pests in agriculture. Microbial antagonists such as nematophagous fungi are of great interest for developing alternative biocontrol strategies to replace nematicide use. This study aims to isolate, identify and assess the diversity and the in vitro predatory activity of nematophagous fungi from various olive nurseries in Morocco. A total of 305 soil samples were collected in 25 nurseries in Moroccan olive-producing areas (Jbala, Guerouane, Haouz and Souss). Seventy-three pure fungal strains from nematophagous fungi were recovered from nursery substrates infested with root-knot nematodes. Diversity indices showed that fungi were more numerous in southern regions, whereas fungal communities were more numerically alike in northern regions, partly depending on the occurrence of root-knot nematodes. One fungal strain, Talaromyces assiutensis, killed all Meloidogyne javanica juveniles during in vitro predation tests. Orbiliaceae (Arthrobotrys spp., Drechslerella brochopaga, Monacrosporium spp.) trapped 50 to 80% of juveniles. Purpureocillium and Trichoderma species killed 30 to 50% of juveniles. The diversity of nematophagous communities could be affected by the north-south climate gradient of Morocco, by their nematode prey expansion, and, to a lesser extent, by the habitat origin of the substrates used. Talaromyces assiutensis was the most efficient nematophagous species against M. javanica. The use of native nematophagous fungi is a promising alternative strategy for nematode control in olive nurseries and to prevent nematode introduction into olive orchards.

Plant-parasitic nematodes (PPN) are key impediments to efficient global crop production and impair the quality of susceptible plants in nurseries as well. In this context, nematode communities were determined in 305 solid substrate samples collected from 25 olive (Olea. europaea. subsp. europaea) nurseries in Morocco. Taxonomical and functional diversity as well as the structures of PPN communities were described and then compared between regions, cultivars as well as according to biotic and abiotic factors. A high diversity of PPN was observed, with the detection of 63 species and 26 genera. The most dominant taxa detected were spiral nematodes (Helicotylenchus spp. and Rotylenchus spp.), stunt nematodes (Tylenchorhynchus spp.), grazer nematodes (Tylenchus spp.) and root-knot nematodes (Meloidogyne spp.). Hoplolaimidae nematodes (Helicotylenchus spp. and Rotylenchus spp.) and Tylenchus spp. were better adapted to rainy conditions that prevailed in the northern regions of Morocco. Multiblock analyses demonstrated that functional diversity (cp and trophic groups) was more affected by the environment than taxonomical diversity (total number, species richness, locale diversity and evenness). They also indicated that PPN communities were more impacted by climatic variables (rainfall and minimum temperature) and nursery substrate origins than by soil physic-chemical factors. Nevertheless, a co-inertia analysis showed that N, P and K amendments in olive nurseries enhanced the development of harmful PPN, especially root-knot nematodes.

Plant-parasitic nematodes such as Longidorus euonymus and Helicotylenchus multicintctus are species widely distributed in central Europe as well as in Mediterranean area. In Greece, both species have been previously reported but no morphometrics or molecular data were available for these species. Nematode surveys in the rhizosphere of grapevines in Athens carried out in 2016 and 2017, yielded a Longidorus species identified as Longidorus euonymus. Similarly, a population of Helicotylenchus multicinctus was detected infecting banana roots from an outdoor crop in Tertsa, Crete. For both species, morphometrics and molecular data of Greek populations were provided, resulting in the first integrative identification of both nematode species based on morphometric and molecular markers, confirming the occurrence of these two nematodes in Greece as had been stated in earlier reports.

• Background. Unraveling domestication processes is crucial for understanding how species respond to anthropogenic pressures, forecasting crop responses to future global changes, and improving breeding programs. Domestication processes for clonally propagated perennials differ markedly from those for seed-propagated annual crops, mostly due to long generation times, clonal propagation and recurrent admixture with local forms, leading to a limited number of generations of selection from wild ancestors. However, additional case studies are required to document this process more fully. • Scope. The olive is an iconic species in Mediterranean cultural history. Its multiple uses and omnipresence in traditional agrosystems have made this species an economic pillar and cornerstone of Mediterranean agriculture. However, major questions about the domestication history of the olive remain unanswered. New paleobotanical, archeological, historical and molecular data have recently accumulated for olive, making it timely to carry out a critical re-evaluation of the biogeography of wild olives and the history of their cultivation. We review here the chronological history of wild olives and discuss the questions that remain unanswered, or even unasked, about their domestication history in the Mediterranean Basin. We argue that more detailed ecological genomics studies of wild and cultivated olives are crucial to improve our understanding of olive domestication. Multidisciplinary research integrating genomics, metagenomics and community ecology will make it possible to decipher the evolutionary ecology of one of the most iconic domesticated fruit trees worldwide. • Conclusion: The olive is a relevant model for improving our knowledge of domestication processes in clonally propagated perennial crops, particularly those of the Mediterranean Basin. Future studies on the ecological and genomic shifts associated with domestication in olive and its associated community will provide insight into the phenotypic and molecular bases of crop adaptation to human uses.

The traditional identification of plant-parasitic nematode species by morphology and morphometric studies is very difficult because of high morphological variability that can lead to considerable overlap of many characteristics and their ambiguous interpretation. For this reason, it is essential to implement approaches to ensure accurate species identification. DNA barcoding aids in identification and advances species discovery. This study sought to unravel the use of the mitochondrial marker cytochrome c oxidase subunit 1 (coxI) as barcode for Longidoridae species identification, and as a phylogenetic marker. The results showed that mitochondrial and ribosomal markers could be used as barcoding markers, except for some species from the Xiphinema americanum group. The ITS1 region showed a promising role in barcoding for species identification because of the clear molecular variability among species. Some species presented important molecular variability in coxI. The analysis of the newly provided sequences and the sequences deposited in GenBank showed plausible misidentifications, and the use of voucher species and topotype specimens is a priority for this group of nematodes. The use of coxI and D2 and D3 expansion segments of the 28S rRNA gene did not clarify the phylogeny at the genus level.

The spiral nematode Helicotylenchus oleae is an ectoparasite of olive roots and is distributed in some countries in the Mediterranean Basin. In this study, we provided morphological and molecular characterisation of topotypes from southern Italy, as well as of several other populations from Crete (Greece) and Spain. Correct identification of plant-parasitic nematode species is essential to establish appropriate control strategies and for preventing their spread to other areas. Helicotylenchus oleae is reported for the first time in Greece (Crete). Integrative morphometric and molecular data for H. oleae populations using D2-D3 expansion segments of 28S rDNA, ITS-rDNA, and the cytochrome c oxidase subunit 1 (coxI), were in agreement with the original descriptions of the species, except for some minor differences, which may be a result of intraspecific variability. The phylogenetic relationships of this species with other representatives of Helicotylenchus spp. using D2-D3 expansion segments and the ITS-rRNA region was studied showing a low intraspecific diversity for H. oleae species. For the first time, coxI molecular data is obtained for the genus Helicotylenchus.

Root-knot nematodes (Meloidogyne incognita and M. javanica) were found infecting cultivated aloe (Aloe vera) plants on the island of Crete, Greece. The infected plants showed reddish brownish leaf discolouration associated with poor plant growth, and roots with dark necrotic areas and irregular thickening but without the typical root-knot nematode galls. Females and egg masses were embedded deeply inside the root. Nematodes were identified using PCR assays either of females, juveniles and males directly obtained from aloe roots and surrounding soil, or of females generated by culturing the nematodes from the aloe on potted tomato plants. Histopathological studies showed female and juvenile bodies and feeding sites surrounded by darkened cells, suggesting the presence of phenolic contents at high levels. Transverse and longitudinal sections confirmed that this reaction was not related to a hypersensitive response because the feeding site was established, but that giant cell formation was accompanied by phenolic compound accumulation. Egg masses isolated from aloe roots were placed in water but juveniles did not hatch. Inspection under the light microscope showed different levels of darkening, with the majority of eggs undeveloped or degenerated. Additionally, PCR reactions which were successful for females failed in these egg masses, suggesting DNA degradation or presence of inhibitory compounds. These results indicate a possible plant defence mechanism impairing viability and development of eggs which are laid inside the aloe roots. It is suggested that the females either produced detoxification enzymes or expressed higher tolerance levels to toxic compounds and thus their development was not impaired.

A new bisexual species of Rotylenchus from North-western Iran is described and illustrated based on morphological, morphometric and molecular studies. Rotylenchus urmiaensis n. sp. is characterised by having a truncate lip region with irregular longitudinal striation, lateral field areolated only in pharynx region, stylet length 34-40 μm, vulva positioned at 53-61%, and female tail conoid-rounded to dorsally convex-conoid with 5-10 annuli. Rotylenchus urmiaensis n. sp. appears to be similar to R. striaticeps, from which it may be differentiated morphologically by a slightly shorter body length (870-1269 vs 1000-1723 μm), shorter stylet (34-40 vs 39-50 μm), female tail shape (conoid-rounded to dorsally convex-conoid vs rounded), frequency of males (rare vs common as abundant as females), shorter spicules (39-43 vs 41-50 μm) and phasmid position (varying from three annuli anterior or three annuli posterior to anus vs at level to seven annuli anterior to anus), and molecularly. The results of phylogenetic analyses based on sequences of D2-D3 expansion region of 28S, ITS-rDNA, 18S rDNA and partial cytochrome c oxidase subunit 1 (coxI) mtDNA, confirmed the species differentiation and the close molecular relationship between R. urmiaensis n. sp. and R. striaticeps.

A new longidorid nematode, Longidorus asiaticus n. sp., is described and illustrated from a population extracted from soil associated with the movement of crape myrtle (Lagerstroemia indica) flowering bonsai trees imported from China into Italy. The new needle nematode is characterised by a small body size (2.74–3.52mm), a bluntly-rounded lip region, ca 12 μm wide, continuous with body contour, amphidial fovea pocket-shape with posterior end rounded not bilobed, a moderately long and flexible odontostyle ca 85 μm long, stylet guiding ring located at ca 37 μm from anterior end, vulva almost equatorial (48–54 %), tail short, about 2/3 of its width, dorsally convex-conoid, with rounded terminus, with a c’ ratio ca 0.7, bearing two pairs of caudal pores and male absent. Integrative diagnosis was completed with molecular data obtained using D2-D3 expansion segments of 28S rDNA, ITS-rDNA, and partial 18S-rDNA. The phylogenetic relationships of this species with other Longidorus spp. using D2-D3 expansion segments, ITS and partial 18S-rDNA indicated that L. asiaticus n. sp. clustered together with L. hangzhouensis, Longidorus sp. JH-2014, and L. camelliae: all of them sharing a common Asiatic geographic origin.

The reniform nematodes of the genus Rotylenchulus are semi-endoparasites of numerous herbaceous and woody plant roots and distributed in regions with Mediterranean, subtropical and tropical climates. In this study, we provide morphological and molecular characterisation of three out of 11 valid species of the genus Rotylenchulus: R. macrodoratus, R. macrosoma, and R. reniformis from Greece (Crete), Italy and Spain. The overall prevalence of reniform nematodes in wild and cultivated olives in Greece, Italy, and Spain was 11.5%, 19.0% and 0.6%, respectively. In Greece, R. macrodoratus and R. macrosoma were detected in cultivated olive with a prevalence of 8.2% and 6.2%, respectively, but none of them were found in wild olive. This is the first report of R. macrosoma in Greece. Only one reniform nematode species was detected in olive from Italy and Spain, viz. R. macrodoratus and R. macrosoma, respectively. The parasitism of R. macrosoma on hazelnut in northern Spain was also confirmed for the first time. This study demonstrates that R. macrodoratus and R. macrosoma have two distinct rRNA gene types in their genomes, specifically the two types of D2-D3 for R. macrosoma and R. macrodoratus, the two types of ITS for R. macrodoratus and the testing of the ITS variability in other R. macrosoma populations in different countries. Rotylenchulus macrosoma from Greece and Spain showed differences in nucleotide sequences in the ITS region and D2-D3 of 28S rRNA gene.

Plant-parasitic nematode species have been reported on several occasions from coastal sand dunes, including Longidorus and Rotylenchus species (Vovlas et al., 2008; De Luca et al., 2009; Mateille et al., 2014). In April 2016, 10 soil samples of 3 to 4 kg from the rhizosphere of Tamarix smyrnensis with different vegetation around (viz. Elymus farctus, Lycium schweinfurthii, Crithmum maritimum, and Arthrocnemum sp.) were collected for diagnosis of plant-parasitic nematodes. The area of sampling was a coastal sand dune near the archeological site of Komos, southwest of Crete, Greece. Low soil populations of a needle and a spiral nematode were detected (3 and 8 individuals/1,000 cm(3) of soil, respectively), which prompted us to undertake a detailed morphological and molecular comparative study with previous reported data. Nematodes were extracted from soil with the wet sieving and decanting method (Cobb, 1918). Morphological and molecular analyses of females identified these species as Longidorus kuiperi Brinkman, Loof and Barbez, 1987, and Rotylenchus eximius Siddiqi, 1964. The morphology of L. kuiperi females (six specimens studied) was characterized by having a slender body; very broad lip region (27 ± 1.5 [25 to 30] μm in width); short, hemispherical tail; body length of (7.1 ± 0.8 [6.5 to 8.5] mm); vulva position at 47% to 55% of body length; odontostyle length of (105 ± 6.5 [90 to 115] μm); males very common (but less frequent than females [45% vs 55%]); tail region with 15 to 20 supplements and bulged terminal cuticle. The morphology of R. eximius females (four specimens studied) was characterized by having a hemispherical lip region clearly set off; with four annuli; body without longitudinal striations; lateral fields areolated in the pharyngeal region only; stylet 36 to 38 μm; and broadly rounded tail. The morphology of the isolated nematodes agreed with previous descriptions of L. kuiperi (Brinkman et al., 1987; De Luca et al., 2009), and R. eximius (Siddiqi, 1964; Castillo and Vovlas, 2005). A single individual was used for DNA extraction. Primers and polymerase chain reaction conditions used in this research were specified in Cantalapiedra et al. (2013) and Archidona-Yuste et al. (2016), and a single amplicon of 800 and 1,100 bp was obtained and sequenced, respectively. D2-D3 (KX398055-KX398056) and ITS sequence alignments (751 and 648 bp, respectively) from L. kuiperi (KX398057) showed 98% to 99% similarity, differing in 4, and from 6 to 12 nucleotides, respectively, to other sequences of L. kuiperi deposited in GenBank from Italy and Spain (AM911623, AM905267-AM905270, respectively), with a query coverage of 99%. Similarly, D2-D3 sequence alignment from R. eximius (KX398058) showed 100% to 99% to another sequence of R. eximius deposited in GenBank from Italy and Spain (EU280794, DQ328741), differing in zero to three nucleotides, respectively, and a query coverage of 81%. Phylogenetic analyses using Bayesian Inference placed L. kuiperi in a highly supported (100%) clade that included all L. kuiperi sequences deposited in GenBank (Archidona-Yuste et al., 2016), and R. eximius in a highly supported (100%) clade that included all R. eximius sequences deposited in GenBank (Cantalapiedra-Navarrete et al., 2013). All identification methods were consistent with L. kuiperi and R. eximius. To our knowledge, this is the first report of L. kuiperi and R. eximius in Greece. Consequently, all these data suggest that coastal sand dunes in Europe constitute environmental conditions optimal for colonization and development of L. kuiperi, as previously reported (De Luca et al., 2009). Similarly, R. eximius has been reported in several Mediterranean countries, including Italy, Morocco, Spain, and Tunisia (Castillo and Vovlas, 2005), and this report extend the geographical distribution of this species.

Plant-parasitic nematodes (PPN) are major crop pests. In olive (Olea europaea) orchards and nurseries, the damages they induce are poorly documented all over the world. However, these parasites significantly contribute to economic losses in the ten-top olive producing countries in the world, especially in the Mediterranean basin (Spain, Italy, Greece, Tunisia, and Morocco). Moreover, their damages increase with cropping intensification. PPN are everywhere found in communities (i.e. species population mixtures). As for all life organisms, diversity and structures of PPN communities respond to evolutionary, environmental and anthropogenic forces. Instead of controlling the main pathogenic nematode species as usual, one of the innovative strategies to control PPN would be to manage diversity in communities in order to lead them to be less pathogenic. The present study is conducted under the PESTOLIVE project (“Contribution of olive history for the management of soil-borne pests in the Mediterranean basin”) funded by ARIMNET (www.arimnet.fr) and supported by the 7th EU-FP7. It aims at understanding the contribution of olive domestication and human impacts on the PPN communities by analyzing the diversity of PPN in cultivated olive compared to wild olive in Morocco. Thus, 220 samples were collected in 2012 in several sites with cultivated and feral olive trees (i.e. wild olive resulting from cultivated olive) in the olive production areas located all along the Atlas foothills, as well as on wild olive (i.e. O. europaea subsp. europaea in the Rif and O. europaea subsp. maroccana in the Western High Atlas). Morphobiometric observations revealed a significant diversity of PPN, belonging to 12 families and 28 genera. Our results showed the presence of genera Pratylenchus and Meloidogyne in all sampled systems that are known harmful taxa for agriculture especially in nurseries. These two genera were more abundant in the cropped systems. Principal component analysis revealed no significant effect of olive systems on the diversity of PPN in communities but on the PPN community patterns (Favored presence of Heteroderidae and Longidoridae in nature systems and others as Meloidogyne spp. were favoured in irrigated conditions). Other factors such as olive genotype, soil physic-chemical characteristics, geo-climatic characteristics, associated plants with olive trees were also discussed as major factors affecting the plant-parasitic nematode community patterns.

Plant-parasitic nematodes significantly contribute to economic losses in the ten top olive producing countries in the world, especially in the Mediterranean basin (Spain, Italy, Greece, Tunisia, and Morocco). Diversity and structures of plant-parasitic nematode communities respond to evolutionary, environmental and anthropogenic forces. Instead of controlling the main pathogenic nematode species as usual, one of the innovative strategies to control plant-parasitic nematodes would be to manage diversity in communities in order to lead them to be less pathogenic. The present study aims at understanding the contribution of olive domestication and human impacts on the plant-parasitic nematode communities by analyzing the diversity of plant-parasitic nematodes in cultivated olive compared to wild olive in Morocco. Thus, 220 samples were collected in 2012 in several sites with cultivated and feral olive trees (i.e. wild olive resulting from cultivated olive) in the olive production areas located all along the Atlas foothills, as well as on wild olive Morphobiometric observations revealed a significant diversity of plant-parasitic nematodes, belonging to 12 families and 28 genera. Our results showed the presence of genera Pratylenchus and Meloidogyne in all sampled systems that are known harmful taxa for agriculture especially in nurseries. These two genera were more abundant in the cropped systems. Principal component analysis revealed no significant effect of olive systems on the diversity of PPN in communities but on the PPN community patterns Other factors such as olive genotype, soil physic-chemical characteristics, geo-climatic characteristics, associated plants with olive trees will also be discussed as major factors affecting the plant-parasitic nematode community patterns.

Plant-parasitic nematode communities associated to wild and cultivated olive (Olea europaea) in South Italy

Objectives Plant-parasitic nematodes (PPN) are everywhere present in communities. Community patterns are used as bio-indicators for plant-production. But at the same time, plant-parasitic nematodes are one of the main biotic stresses on crops. Then, considering the new cultivation programs pushing olive production with high technologies in Morocco ('Green Morocco program') and the enhancement of rooted cutting production, nurseries established in southern Morocco were surveyed in order to analyse the associated PPN communities and the contribution of olive varieties and soil substrates on their diversity. Materials and methods Twenty-two commercial olive nurseries (Olea europaea) located in the Haouz and Souss-Massa-Draa regions in Morocco were selected for nematode surveys conducted in 2013 (Fig. 1). In each nursery surveyed, five rooted cuttings (as replicates) of each variety were randomly sampled (Fig. 2) , and then brought and kept under controlled greenhouse conditions. The 210 rooted cuttings collected belonged to the following olive varieties: 'Picholine marocaine' (65 samples), 'Haouzia' (60 samples), 'Menara' (45 samples), 'Picholine du Languedoc' (25 samples), 'Arbequina' (10 samples) and 'Manzanille' (5 samples). The first three varieties sampled are the most commonly produced in Morocco. PPN (Fig. 3) were extracted using NF ISO 23611-4 soil elutriation method. They were observed at genus rank and enumerated under a stereomicroscope (x60 magnification). Population levels were expressed per dm 3 of fresh soil. Plant-parasitic nematode communities in southern Morocco olive nurseries as affected by olive diversity and substrates

Ecology and genetics of nematode communities

Ecology and genetics of nematode communities

The olive tree (Olea europaea ssp. europaea.) is one of the most ancient cultivated trees. It is an emblematic species owing to its ecological, economic and cultural importance, especially in the Mediterranean Basin. Plant-parasitic nematodes are major damaging pests on olive trees, mainly in nurseries. They significantly contribute to economic losses in the top-ten olive-producing countries in the world. However, the damages they induce in orchards and nurseries are specifically documented only in a few countries. This review aims to update knowledge about the olive-nematode pathosystem by: (1) updating the list of plant-parasitic nematodes associated with olive trees; (2) analysing their diversity (taxonomic level, trophic groups, dominance of taxa), which allowed us (i) to assess the richness observed in each country, and (ii) to exhibit and describe the most important taxa able to induce damages on olive trees such as: Meloidogyne, Pratylenchus, Helicotylenchus, Xiphinema, Tylenchulus, Rotylenchulus, Heterodera (distribution especially in the Mediterranean Basin, pathogenicity and reactions of olive trees); (3) describing some management strategies focusing on alternative control methods; (4) suggesting new approaches for controlling plant-parasitic nematodes based on the management of the diversity of their communities, which are structured by several environmental factors such as olive diversity (due to domestication of wild olive in the past, and to breeding now), cropping systems (from traditional to high-density orchards), irrigation, and terroirs.

Mousadik, 2015. Plant-parasitic nematodes associated with olive tree in Southern Morocco. Int. J. Agric. Biol., Abstract Plant-parasitic nematodes affect significantly the production of susceptible plants, including olive trees. In this context, nematode communities were determined in soil samples collected from 23 olive growing sites in the Haouz and Souss regions (southern Morocco). These sites corresponded to various modalities: wild (Olea europaea L. ssp. sylvestris) or cropped olive (Olea. europaea L. ssp. europaea), traditional or high-density cropping, rainfed or irrigated. Even free-living nematodes prevailed in most of the sites, high population levels of plant-parasitic nematodes were observed in rainfed cropping systems than in irrigated systems. Ten nematode families and 14 genera were identified. The most important plant-parasitic nematodes detected, in order decreasing frequency of infestation (percentage of samples), were spiral nematodes Helicotylenchus spp. (100%) and Rotylenchus spp. (87%), lesion nematodes Pratylenchus spp. (100%) and root-knot nematodes Meloidogyne spp. (40%). Most of the nematode species were assigned to more or less colonizer nematodes, whereas only one family (Longidoridae) was assigned to persistent nematodes. Considering the two producing areas, plant-parasitic nematodes were significantly more abundant in the Souss region than in the Haouz region, but nematode diversity was higher in the second one. The prevalence of Meloidogyne spp. in the Haouz region would be related to the predominance of irrigated cropping systems in this region. Hoplolaimidae nematodes (Helicotylenchus spp. and Rotylenchus spp.) are better adapted to rainfed conditions that prevail in the Souss region. Co-inertia analysis showed the importance of soil physic-chemical characteristics (e.g., pH, texture and nutrients) on the structure of the plant-parasitic nematode community patterns. The high occurrence of Helicotylenchus spp. in olive orchards may be induced by fertilization. Aphelenchoides spp., Gracilacus spp., Pratylenchus spp., Rotylenchidae and Tylenchidae were supported by coarse textures as it was observed in the Souss region.

A new mono-sexual root-lesion nematode species, Pratylenchus oleae n. sp., parasitizing roots of olive plants cv. Koroneiki in commercial fields at Ouled Chamekh (central Tunisia), and wild and cultivated olive (cv. Picual) plants in Agua Amarga (southern Spain) is described. The new species is characterised by the female having a lip region slightly offset and bearing three annuli, stylet 16.5 (14.5-17.0) μm long, with prominent rounded knobs, pharyngeal overlapping rather long (22–36) μm, lateral fields areolated and with four incisures and diagonal lines in middle band, spermatheca rounded but nonfunctional, tail short, conoid-rounded to subcylindrical, usually annulated terminus,males unknown, and a specific D2-D3, ITS1, 18S-rRNA, hsp90 and COI sequences. Morphologically this species is related to P. cruciferus, P. delattrei, and P. kumamotoensis. The results of the phylogenetic analysis based on sequences of the D2-D3 expansion regions of 28S, partial 18S and ITS rRNA genes confirmed the close relationship of P. oleae n. sp. with P. dunensis, P. penetrans, P. pinguicaudatus, from which was clearly separated. A PCR-based diagnostic assay was also developed for identification of P. oleae n. sp. using the species-specific primers Poleae_fw1_4 and Poleae_rv1 that amplify a 547-bp fragment in the internal transcribed spacer (ITS1) region of ribosomal DNA, which clearly separate from other root-lesion nematodes damaging olive such as P. penetrans and P. vulnus. Keywords Bayesian inference . Cytochrome c oxidase subunit 1 (COI) . Heat shock protein 90 (hsp90) . Maximumlikelihood . Olea europaea subsp. europaea . Olea europaea subsp. sylvestris . Phylogeny. rDNA. Root-lesion . Taxonomy

Stunt nematodes are characterized by phenotypic plasticity, with overlapping morphology and morphometry leading to potential misidentification. Consequently, the application of integrative taxonomic approaches is useful to species delimitation based on a combination of different perspectives, e.g. morphology and DNA sequences. We conducted nematode surveys in cultivated and natural environments in Spain and the USA, from which we identified 18 known species of the family Telotylenchidae and two new taxa within the studied samples. These species were morphologically, morphometrically, and molecularly characterized. The results of light and scanning electron microscopic observations, and molecular and phylogenetic analysis also allowed two new species to be distinguished, described herein as Bitylenchus hispaniensis sp. nov. and Tylenchorhynchus mediterraneus sp. nov. The phylogenetic analysis was carried out using molecular data from nuclear ribosomal DNA genes [D2–D3 expansion segments of the large ribosomal subunit (28S), internal transcribed spacer (ITS), and partial small ribosomal subunit (18S)]. We also provide here a test of alternative hypotheses that confirms the monophyly of both Tylenchorhynchus and Bitylenchus sensu Siddiqi’s classification but does not support Fortuner & Luc’s conceptual view of Tylenchorhynchus as a large genus. Ancestral state reconstructions of several diagnostic morphological characters using a maximum parsimony approach showed congruence in morphological and molecular evolution for stylet knob inclination and tail tip annulation. Our analysis emphasizes some of the problems related to the taxonomy and phylogeny of nematodes of Telotylenchinae.

A population of Xiphinema barense from wild olive trees in Torre Pozzella, Brindisi province, southern Italy, is described using both morphological and molecular studies and compared with the description of the type specimens. The wild olive nematode population agrees very well with all morphometrics provided in the original description. However, detailed observations of the lumen of the tubular portion of the uterus in paratypes and specimens of the new population revealed a clear pseudo-Z-organ with small granules mixed with crystalloid bodies which were previously undetected. Photomicrographs of adult paratypes, which were lacking in the original description, and of specimens of the new population from wild olive trees are provided. The results of the phylogenetic analyses based on the sequences of the D2-D3 expansion regions of the 28S rRNA gene and ITS rRNA genes confirm the species differentiation and indicate the phylogenetic position of X. barense and its relationship with closely related species.

PESTOLIVE (Contribution of olive history for the management of soil-borne parasites in the Mediterranean Basin) is a project funded by ARIMNet, an ERANET action supported by the 7th European Framework Programme and by non-European Mediterranean countries. PESTOLIVE aims at producing knowledge and tools for a new and efficient management of plant-parasitic nematodes (PPN) and plant-pathogenic fungi (PPF) in olive (Olea europaea L.) cropping systems and nurseries, while reducing the use of pesticides. Because of the anthropic continuum from Olea post-glacial refuges to Oleasters (domestication) and then to olive-trees (breeding and cropping), the fragmentation of the PPN and PPF communities and of their natural enemies could explain the scattered diversity of the control techniques (especially resistance rootstocks, biocontrol, cropping strategies) developed and applied all around the Mediterranean Basin. The novelty of PESTOLIVE is based on: i) the analysis and the management of the parasite diversity (ecology of communities) instead of controlling emblematic species (population approach), and ii) the involvement of knowledge about the historical co-adaptation of soil-borne parasite and natural enemies communities to olive-tree domestication (origins and past assemblages) and breeding that follows the history of O. europaea around the Mediterranean Basin. Hypothesis about soil-borne pest control by managing their diversity Following objectives for restoration of disturbed agro-systems, it is becoming urgent to understand the contribution of parasitism within all degradation functions, in particular that of PPN and PPF, and especially in the context of fast programmed elimination of pesticides. On the other hand, the control of plant parasites leads to the decrease of their biodiversity whose risk should be assessed if biodiversity is considered as the driving force of ecological Proceedings of the 5 th Int. Conf. Olivebioteq 2014 332 resilience. Then, evaluating the ecological sustainability of plant resistance and of biocontrol strategies by assessing their long-term effects on parasite communities appears as a new research alternative for crop protection. The diversity of parasite communities can thus inform on the aptitude of environment to either facilitate or not the epidemic phenomena, on resistance and the resilience capacities of soils. The comparative studies of environment with contrasted anthropic levels (Mateille et al., 2008) will seek for information and knowledge about biotic trade-offs in ecosystems, in order to introduce them in agro-systems (resilience). Considering olive protection, PPN and PPF control strategies appear to be developed in each Mediterranean producing country according to their own constraints, depending above all on the parasite diversity encountered in each of them (Lockwood, 1988). Despite their high quality, researches conducted on plant resistance and biocontrol would lead to empirical results and practices if they are not based on knowledge about the origins of the parasite communities and about their past assemblages. Because of the anthropic continuum from Olea post-glacial refuges to Oleasters (domestication) and then to olive-trees (breeding and cropping), the fragmentation of the PPN and PPF communities and of their associated antagonists could be hypothesized and could explain their present distribution. Aim of PESTOLIVE The aim of PESTOLIVE (www1.montpellier.inra.fr/CBGP/pestolive) is to help the development of new sustainable management strategies by involving enlightenment about historical co-adaptation of soil-borne parasite communities to olive-tree domestication, breeding and cropping. In order to provide adequate answers to questions emerging from the weakening of Mediterranean eco-anthroposystems due to soil-borne parasites, PESTOLIVE would develop an integrated network of pan-Mediterranean research focused on two soil-borne pest groups, PPN and PPF, and on their common antagonists, especially mycorrhizae.