First reports of Hemicycliophora poranga, Helicotylenchus dihystera and Tylenchorhynchus zeae (Tylenchomorpha) from Greece and further records of four other nematode species.

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.

First Report of Longidorus Leptocephalus Hooper, 1961 (Nematoda: Longidoridae) from Greece.

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.

Morphological and molecular characterisation of Longidorus pauli (Nematoda: Longidoridae), first report from Greece.

Sampling for needle nematodes was carried out in a grapevine area in Thessaloniki, North Greece and two nematode species of Longidorus (L. pauli and L. pisi) were collected. Nematodes were extracted from 500 cm3 of soil by modified sieving and decanting method, processed to glycerol and mounted on permanent slides, and subsequently identified morphologically and molecularly. Nematode DNA was extracted from single individuals and PCR assays were conducted to amplify D2-D3 expansion segments of 28S rRNA, ITS1 rRNA, and partial mitochondrial coxI regions. Morphology and morphometry data obtained from these populations were consistent with L. pauli and L. pisi identifications. To our knowledge, this is the first report of L. pauli for Greece, and the second world report after the original description from Idleb, Syria, extending the geographical distribution of this species in the Mediterranean Basin.

New Distribution and Molecular Diversity of the Reniform Nematode Rotylenchulus macrosoma (Nematoda: Rotylenchulinae) in Europe.

Reniform nematodes of the genus Rotylenchulus are semi-endoparasites of numerous herbaceous and woody plant species roots and occur largely in regions with temperate, subtropical, and tropical climates. In this study, we provide new records of the nematode Rotylenchulus macrosoma in eight European countries (Czech Republic, France, Germany, Hungary, Italy, Romania, Serbia, and Portugal), in addition to the six Mediterranean countries (Greece, Israel, Jordan, Spain, Syria, and Turkey) where the nematode was previously reported. Four new host species (corn, pea, wheat, and an almond-peach hybrid rootstock) are added to the recorded host species (bean, chickpea, hazelnut, peanut, soybean, and wild and cultivated olive). Molecular analyses based on the cytochrome c oxidase subunit coxI and D2-D3 segments of 28S RNA markers showed high diversity and pronounced genetic structure among populations of Rotylenchulus macrosoma. However, the complexity of phylogeographic patterns in plant-parasitic nematodes may be related to the intrinsic heterogeneity in the distribution of soil organisms, a rare occurrence of a species, or the potential human impact associated with agricultural practices.

Structural Diversity and Highly Specific Host-Pathogen Transcriptional Regulation of Defensin Genes Is Revealed in Tomato.

Defensins are small and rather ubiquitous cysteine-rich anti-microbial peptides. These proteins may act against pathogenic microorganisms either directly (by binding and disrupting membranes) or indirectly (as signaling molecules that participate in the organization of the cellular defense). Even though defensins are widespread across eukaryotes, still, extensive nucleotide and amino acid dissimilarities hamper the elucidation of their response to stimuli and mode of function. In the current study, we screened the Solanum lycopersicum genome for the identification of defensin genes, predicted the relating protein structures, and further studied their transcriptional responses to biotic (Verticillium dahliae, Meloidogyne javanica, Cucumber Mosaic Virus, and Potato Virus Y infections) and abiotic (cold stress) stimuli. Tomato defensin sequences were classified into two groups (C8 and C12). Our data indicate that the transcription of defensin coding genes primarily depends on the specific pathogen recognition patterns of V. dahliae and M. javanica. The immunodetection of plant defensin 1 protein was achieved only in the roots of plants inoculated with V. dahliae. In contrast, the almost null effects of viral infections and cold stress, and the failure to substantially induce the gene transcription suggest that these factors are probably not primarily targeted by the tomato defensin network.

Compatibility of fluazaindolizine and oxamyl with Pasteuria penetrans on spore attachment to juveniles of Meloidogyne javanica and M. incognita.

Fluazaindolizine is a novel sulfonamide nematicide that is the active ingredient (a.i.) of Salibro™, a.i. Reklemel™. Its compatibility with Pasteuria penetrans, a bacterial parasite of root-knot nematodes (Meloidogyne spp.), was investigated in populations of M. javanica and M. incognita. Spores of a single P. penetrans isolate (Pp 3) or a blend of six isolates were incubated in the suspensions of fluazaindolizine (SalibroTM 500SC, at 5, 50, and 250 ppm a.i.) and oxamyl (Vydate™ 10 L, 10% (a.i.) at 25 and 50 ppm a.i.) for 1, 7, and 21 days; controls were incubated in water. Thereafter, the suspensions were washed through a cellulose filter (3 µm) so as to remove the nematicide, and the spores retained on the filter were suspended in water. Juveniles (J2) were exposed in these spore suspensions in Petri dishes and the number of attached spores was recorded. Neither fluazaindolizine nor oxamyl, at all the tested dosages, had any negative effect on the rate of spore attachment. The spore encumbered J2 from some experiments were used to infect tomatoes. Females without egg masses were extracted from the roots after 50 days and checked for eggs in ovaries and mature spores of P. penetrans. Despite no mature spores present in the females, there was evidence of a low percentage of infection in a few treatments. A possible explanation is that since the bacterium had been kept stored in the form of dried roots for a long period, its ability to infect nematodes was decreased.Fluazaindolizine is a novel sulfonamide nematicide that is the active ingredient (a.i.) of Salibro™, a.i. Reklemel™. Its compatibility with Pasteuria penetrans, a bacterial parasite of root-knot nematodes (Meloidogyne spp.), was investigated in populations of M. javanica and M. incognita. Spores of a single P. penetrans isolate (Pp 3) or a blend of six isolates were incubated in the suspensions of fluazaindolizine (SalibroTM 500SC, at 5, 50, and 250 ppm a.i.) and oxamyl (Vydate™ 10 L, 10% (a.i.) at 25 and 50 ppm a.i.) for 1, 7, and 21 days; controls were incubated in water. Thereafter, the suspensions were washed through a cellulose filter (3 µm) so as to remove the nematicide, and the spores retained on the filter were suspended in water. Juveniles (J2) were exposed in these spore suspensions in Petri dishes and the number of attached spores was recorded. Neither fluazaindolizine nor oxamyl, at all the tested dosages, had any negative effect on the rate of spore attachment. The spore encumbered J2 from some experiments were used to infect tomatoes. Females without egg masses were extracted from the roots after 50 days and checked for eggs in ovaries and mature spores of P. penetrans. Despite no mature spores present in the females, there was evidence of a low percentage of infection in a few treatments. A possible explanation is that since the bacterium had been kept stored in the form of dried roots for a long period, its ability to infect nematodes was decreased.

First report of cultivated Cretan mountain tea (Sideritis syriaca) as a host of Meloidogyne hapla and M. javanica in Crete, with some additional records on the occurrence of Meloidogyne species in Greece.

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.

First Report of Bitylenchus hispaniensis, Pratylenchoides alkani, and Helicotylenchus vulgaris in Association with Cultivated and Wild Olives in Crete, Greece and Molecular Identification of Helicotylenchus microlobus and Merlinius brevidens.

Nematode samplings in cultivated and wild olive in Crete, Greece, yielded the presence of Bitylenchus hispaniensis , Helicotylenchus microlobus , Helicotylenchus vulgaris , Merlinius brevidens , and Pratylenchoides alkani . With the exception of H. microlobus and M. brevidens , reports of these plant-parasitic nematode species constitute new records for Greece. Bitylenchus hispaniensis is also reported for first time in a country outside of Spain, where it was originally described. Pratylenchoides alkani is herein reported for the second time in the Mediterranean area and for the first time in association with olive. Two further populations of H. microlobus and H. vulgaris , from walnut and goji berry from Greece, were identified. Molecular data for all of these nematode species are provided, resulting in the first integrative identification of these Greek populations.

Morphological and Molecular Identification of Longidorus euonymus and Helicotylenchus multicinctus from the Rhizosphere of Grapevine and Banana in Greece.

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.

Distribution and function of carbamate hydrolase genes cehA and mcd in soils: the distinct role of soil pH.

Synthetic carbamates constitute a significant pesticide group with oxamyl being a leading compound in the nematicide market. Oxamyl degradation in soil is mainly microbially mediated. However, the distribution and function of carbamate hydrolase genes (cehA, mcd, cahA) associated with the soil biodegradation of carbamates is not yet clear. We studied oxamyl degradation in 16 soils from a potato monoculture area in Greece where oxamyl is regularly used. Oxamyl showed low persistence (DT50 2.4-26.7 days). q-PCR detected the cehA and mcd genes in 10 and three soils, respectively. The abundance of the cehA gene was positively correlated with pH, while both cehA abundance and pH were negatively correlated with oxamyl DT50. Amongst the carbamates used in the study region, oxamyl stimulated the abundance and expression only of the cehA gene, while carbofuran stimulated the abundance and expression of both genes. The cehA gene was also detected in pristine soils upon repeated treatments with oxamyl and carbofuran and only in soils with pH ≥7.2, where the most rapid degradation of oxamyl was observed. These results have major implications regarding the maintenance of carbamate hydrolase genes in soils, have practical implications regarding the agricultural use of carbamates, and provide insights into the evolution of cehA.

First Report of Longidorus kuiperi and Rotylenchus eximius from Coastal Sand Dunes in Crete, Greece.

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 cm3 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.

First Report of the Carolina Spiral Nematode, Scutellonema brachyurus, from Soil of a Garden in Crete, Greece.

Members of the genus Scutellonema can cause substantial crop losses to ornamental and cultivated plants directly by feeding ectoparasitically on plant roots (Bridge et al., 2005; Coyne et al., 2006). In May 2015, a soil sample from a house garden from Heraklion city in Crete, Greece, was sent for diagnosis of plant-parasitic nematodes. In this place, there had been cactus (Opuntia sp.) plants (probably imported), which were uprooted 3 to 4 years earlier. After that, the area was cropped with cucumber (Cucumis sativus L.) in spring-summer and leaf vegetables such as spinach (Spinacia oleracea L.) and chicory (Cichorium intybus L.) in autumn-winter. The soil was collected 1 mon after the end of chicory crop. A population density (ca. 30 individuals/100 cm(3) of soil) of spiral nematodes (Scutellonema sp.) was found by extracting soil with the wet sieving and decanting method (Cobb, 1918). Morphological and molecular analyses of females identified the species as Scutellonema brachyurus (Steiner, 1938) Andrássy, 1958. The morphology of females was characterized by a hemispherical lip region with four to six annuli, morphometric data for 12 females were L, 640 to 760 µm; a, 24.6 to 30.6; b, 5.8 to 7.4; c, 69.1 to 99.3; c´ 0.5 to 0.6; stylet, 24.5 to 27.5 µm with anterior part shorter than posterior; and spermatheca nonfunctional and male absent. The morphology agreed with the description of S. brachyurus (van den Berg et al., 2013). Alignment indicated that the D2-D3 and ITS sequences (KU059494 and KU059495, respectively) showed 99% and 100% to 99% similarity, respectively, to other sequences of S. brachyurus (type A) deposited in GenBank from the United States, Italy, and Korea (JX472037-JX472046, DQ328753, FJ485643; and JX472069, JX472070, JX472071, respectively), differing from one to six nucleotides. Phylogenetic analyses using Bayesian inference of these sequences placed the Scutellonema sp. in a highly supported (100%) clade that included all S. brachyurus (type A) sequences deposited in GenBank (van den Berg et al., 2013). All identification methods were consistent with S. brachyurus. To our knowledge, this is the first report of S. brachyurus for Greece. As the cucumbers and the leaf vegetables cultivated in the area were seed planted, we consider that the nematode originated most probably from the cactus plants which had been previously root ball planted. Scutellonema brachyurus may represent a threat for ornamental and cultivated plants production in Crete, Greece. The nematode has been already reported, mainly in greenhouses of six European countries (CABI and EPPO, 2006). In most of these cases, it is hypothesized that the nematode was introduced by imported plant material.

Isolation of Oxamyl-degrading Bacteria and Identification of cehA as a Novel Oxamyl Hydrolase Gene.

Microbial degradation is the main process controlling the environmental dissipation of the nematicide oxamyl. Despite that, little is known regarding the microorganisms involved in its biotransformation. We report the isolation of four oxamyl-degrading bacterial strains from an agricultural soil exhibiting enhanced biodegradation of oxamyl. Multilocus sequence analysis (MLSA) assigned the isolated bacteria to different subgroups of the genus Pseudomonas. The isolated bacteria hydrolyzed oxamyl to oxamyl oxime, which was not further transformed, and utilized methylamine as a C and N source. This was further supported by the detection of methylamine dehydrogenase in three of the four isolates. All oxamyl-degrading strains carried a gene highly homologous to a carbamate-hydrolase gene cehA previously identified in carbaryl- and carbofuran-degrading strains. Transcription analysis verified its direct involvement in the hydrolysis of oxamyl. Selected isolates exhibited relaxed degrading specificity and transformed all carbamates tested including the oximino carbamates aldicarb and methomyl (structurally related to oxamyl) and the aryl-methyl carbamates carbofuran and carbaryl which share with oxamyl only the carbamate moiety.

Isolation of soil bacteria able to hydrolyze both organophosphate and carbamate pesticides.

Two bacteria identified as Pseudomonas putida and Acinetobacter rhizosphaerae able to rapidly degrade the organophosphate (OP) fenamiphos (FEN) were isolated. Denaturating gradient gel electrophoresis analysis revealed that the two isolates were dominant members of the enrichment culture. Clone libraries further showed that bacteria belonging to α-, ß-, γ-proteobacteria and Bacteroidetes were also present in the final enrichment but were not isolated. Both strains hydrolyzed FEN to fenamiphos phenol which was further transformed, only by P. putida. The two strains were using FEN as C and N source. Cross-feeding studies with other pesticides showed that P. putida degraded OPs with a P-O-C linkage and unexpectedly degraded the carbamates oxamyl and carbofuran being the first wild-type bacterial strain able to degrade both OPs and carbamates. The same isolate exhibited high bioremediation potential against spillage-level concentrations of aged residues of FEN and its oxidized derivatives.

Investigation of alternatives to methyl bromide for management of Meloidogyne javanica on greenhouse grown tomato.

The root galling index and the densities of eggs in roots and juveniles in soil of the root-knot nematode Meloidogyne javanica (Treub) Chitwood on tomato, and the effect of these on crop yield were assessed in greenhouse experiments applying various treatments at two different sites in Crete, Greece. Tomato crops were grown for four cycles by rotating nematode-resistant (first and third spring crops) with susceptible (second and fourth autumn crops) cultivars and receiving the following treatments: (a) untreated control; (b) methyl bromide application before the first and third crops; (c) application of the fungus Pochonia chlamydosporia (Goddard) Zare, Gams & Evans before planting the first, third and fourth crops with a supplementary application three weeks after the beginning of the fourth crop; (d) application of oxamyl in both sites and fenamiphos in site 1 only at the second and fourth crops; (e) combination of treatments (c) and (d). The fungus density in soil was monitored three weeks after application and at the end of each crop, when roots were lifted. Pochonia chlamydosporia had a variable establishment and did not control the nematode. Its pathogenicity on eggs was not demonstrated, as in all cases galls were big, with all egg masses inside and protected from infection. The methyl bromide treatment significantly reduced root galling and egg production compared to other treatments in all crop cycles and the yield of the fourth crop was significantly greater. Nematicides reduced nematode densities compared with untreated controls and the fungus treatment, but they were less effective than methyl bromide and resulted in increased yield in one site only.