Tomato Mi-gene Resistance-Breaking Populations of Meloidogyne Show Variable Reproduction on Susceptible and Resistant Crop Cultivars.

Sixteen Meloidogyne isolates from tomato fields in California grown with resistant cultivars were multiplied on resistant tomato in a greenhouse. Of these resistance-breaking isolates, one was identified as M. javanica, and all others as M. incognita. The reproduction of the M. javanica isolate and four M. incognita isolates on six resistant tomato cultivars and on susceptible and resistant cultivars of pepper, sweetpotato, green bean, cotton, and cowpea was evaluated and compared to an avirulent M. incognita population in greenhouse pot trials. On resistant tomato cultivars, there were minor but significant differences between the resistance-breaking Meloidogyne isolates and between the different tomato cultivars. Of the other resistant crop cultivars, pepper was resistant to all isolates and green bean to all M. incognita isolates, while cotton and cowpea allowed reproduction of one of the resistance-breaking M. incognita isolates. The resistant sweetpotato cv. Bonita behaved like resistant tomato, allowing reproduction of all five resistance-breaking isolates but not of the avirulent M. incognita. Our results showed that variability exists among resistance-breaking Meloidogyne isolates, and that isolates overcoming resistance in tomato may also be virulent on resistant sweetpotato.

Morphological and molecular characterization of two new species of the genus Aporcelinus Andrássy, 2009 (Nematoda, Dorylaimida, Aporcelaimidae) from the USA, with new insights on the phylogeny of the genus.

Two new species of the genus Aporcelinus from the USA are described and illustrated. Aporcelinus floridensis sp. n. is characterized by its 1.12-1.52 mm long body, lip region offset by marked constriction and 14.5-17.0 µm broad with perioral liplets, odontostyle 16.5-20.0 µm at its ventral side and 1.1-1.2 times the lip region diameter, neck 316-395 µm long, pharyngeal expansion occupying 43-48% of total neck length, uterus simple and 33-56 µm long or 0.8-1.2 times the corresponding body diameter, V = 48-54, female tail conical (36-49 µm long, c = 27-41, c' = 1.2-2.0) with finely rounded terminus and no hyaline portion, and male absent. Aporcelinus paolae sp. n. is characterized by its 1.29-1.80 mm long body, lip region offset by marked constriction and 14-16 µm broad, odontostyle 15-17 µm at its ventral side and 1.0-1.1 times the lip region diameter, neck 314-397 µm long, pharyngeal expansion occupying 43-53% of total neck length, uterus tripartite and 128-164 µm long or 2.6-3.6 times the corresponding body diameter, V = 53-57, female tail conical (30-39 µm long, c = 40-51, c' = 1.1-1.3) with finely rounded terminus and variably re-curved dorsad, male tail conical (27-36 µm, c = 39-59, c' = 0.9-1.2), ventrally straight and dorsally convex, spicules 48-54 µm long, and 7-9 irregularly spaced ventromedian supplements lacking hiatus. The analyses of the D2-D3 expansion segments of 28S rRNA (LSU) gene sequences of the two new species confirmed the monophyly of the genus, based upon currently available data, showing a close relationship between the genera Aporcelinus and Makatinus, and justified the placement of Aporcelaimellus, Makatinus and Aporcelinus under the subfamily Aporcelaimellinae.

The utility of mtDNA and rDNA for barcoding and phylogeny of plant-parasitic nematodes from Longidoridae (Nematoda, Enoplea).

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.

A new stem nematode, Ditylenchus oncogenus n. sp. (Nematoda: Tylenchida), parasitizing sowthistle from Adriatic coast dunes in southern Italy.

Morphological and molecular analyses of a stem nematode causing a severe disease on infected sowthistle (Sonchus bulbosus) plants, involving the formation of gall-like structures on infected leaves and stems, have led to the description of a new species named Ditylenchus oncogenus n. sp. Morphologically, the new species is characterized by a medium to large body size (all adults more than 1 mm in length); a delicate stylet (9.0-11.0 µm long) with minute, rounded knobs; a long post-vulval uterine sac (c. 65% of the vulva-anus distance); six incisures at the lateral fields and characteristic D. destructor-pattern of spicules (with pronounced ventral tumulus and anteriorly pointed, less sclerotized, cuticle parts present within the lamina). The results of molecular analysis of rRNA gene sequences, including the D2-D3 expansion regions of 28S rRNA, internal transcribed spacer (ITS) rRNA, partial 18S rRNA gene, the protein-coding mitochondrial gene, cytochrome oxidase c subunit I (COI), and the heat-shock protein 90 (hsp90) gene, support the new species status. The results of a host-suitability test indicated that the new species does not parasitize potato (Solanum tuberosum) tubers and broad bean (Vicia faba) seedlings. Histopathological observations on naturally infected sowthistle tissues revealed that D. oncogenus n. sp. causes floral stem neoplasia and midrib leaf gall formation on the type, and to date only known, host. The galls were characterized by extensive hyperplasia, where several necrotic cells in the neoplasic area were directly damaged by feeding of the nematode, whereas a number of adjacent cells showed typical cytological changes, such as granulated cytoplasm with hypertrophied nuclei and nucleoli.

Molecular Characterization of Meloidogyne christiei Golden and Kaplan, 1986 (Nematoda, Meloidogynidae) Topotype Population Infecting Turkey Oak (Quercus laevies) in Florida.

Meloidogyne christiei isolated from turkey oak, Quercus laevies, from the type locality in Florida was characterized using isozyme profiles and ribosomal and mitochondrial gene sequences. The phenotype N1a detected from a single egg-laying female of M. christiei showed one very strong band of malate dehydrogenase (MDH) activity; however, no esterase (EST) activity was identified from macerate of one or even 20 females per well. Phylogenetic relationships within the genus Meloidogyne as inferred from Bayesian analysis of partial 18S ribosomal RNA (rRNA), D2-D3 of 28S rRNA, internal transcribed spacer (ITS) rRNA, and cytochrome oxidase subunit II (COII)-16S rRNA of mitochondrial DNA (mtDNA) gene fragments showed that M. christiei formed a separate lineage within the crown group of Meloidogyne and its relationships with any of three Meloidogyne clades were not resolved.

First Report of the Giant Stem Nematode, Ditylenchus gigas, from Broad Bean in Iran.

The giant stem nematode, Ditylenchus gigas (Nematoda: Tylenchida) has been recorded from several European and African countries mainly bordering the Mediterranean Sea (2). This nematode causes considerable yield loss of broad bean, Vicia faba, and it may induce more severe damage than the typical faba bean race of D. dipsaci. Spread of infestation through seed limits export of broad bean and has made these nematodes quarantine pests in many countries (2). Broad bean is cultivated in the north, west, southwest, and central parts of Iran. Although D. dipsaci has been reported from different crops and regions in Iran, there is no record of broad bean infection by this nematode. A survey of broad bean fields was conducted in the north and west provinces in a continuation of a study on different populations of D. dipsaci in Iran in May to July of 2007 and 2008 and resampling from some farms in June 2012. The sampling was performed at flowering stage and after. The aboveground plant samples were collected and cut into pieces of 2 to 3 cm, then incubated for 5 to 6 h in Whitehead trays. Morphological and molecular analysis of isolated nematodes from Kermanshah and Lorestan provinces revealed the presence of D. gigas in the samples. Of the 23 plant samples of cv. Barekat collected from Mazandaran and Golestan provinces in the north, 47.8% were infected with stem nematode, mostly with high population density of over 20,000 nematodes per 5 plant stems. The percentage of infected samples of broad bean cv. Shakhbozy collected in Lorestan and Kermanshah provinces in the west was 76.5%. The symptoms of infection were observed as necrotic lesions on the stem surface and reduction of internode distances in severe infection. The giant stem nematode population from Kermanshah showed the following characters: females (n = 20), L = 1,650 ± 140 (1,270 to 1,875) µm; b' = 8.6 ± 0.6 (7.7 to 10.0), c = 19.0 ± 1.3 (19.2 to 21.2), c' = 4.7 ± 0.5 (1.1 to 5.3), stylet = 11.6 ± 0.5 (11 to 12) µm; post vulval sac = 96 ± 16 (58 to 140) µm; vulval-anus distance = 217.0 ± 21.0 (178 to 272) µm, tail = 86.4 ± 9.4 (66 to 102) µm; males (n = 10), L = 1,495 ± 148 (1,236 to 1,636) µm; b' = 7.7 ± 0.3 (7.3 to 8.1), c = 17.3 ± 0.7 (16.3 to 18.6), stylet = 11.3 ± 0.5 (11 to 12) µm, tail = 86.5 ± 8.5 (71 to 95) µm, spicules = 24.8 ± 1.7 (23 to 28) µm. The morphological and morpohometric features were generally in agreement with those published for D. gigas (2). The morphological identification of D. gigas from Iran was supported by the analyses of the ITS rRNA and the D2-D3 expansion segments of the 28S rRNA gene sequences. The rRNA gene of D. gigas from broad bean and D. dipsaci from garlic were amplified and sequenced using two primer sets: (i) the TW81 and AB28 for the ITS-rRNA and (ii) D2A and D3B for partly 28S rRNA gene, as described by (2). New sequences were deposited in the GenBank under accession numbers KC310732 through KC310735. The Iranian D. gigas sequences showed 100% similarity with those of the Italian D. gigas isolates (ITS rRNA: HQ219231, HQ219232; D2-D3 of 28S rRNA: HQ219217 and HQ219216). The identification was further supported by PCR with species specific SCAR (sequence characterized amplified region) primers for this species (1). The specimens from broad bean generated a specific fragment ∼200 bp for D. gigas, whereas the samples with D. dipsaci from garlic and alfalfa produced one fragment ∼250 bp specific for this species. To our knowledge, this is the first report of D. gigas from broad bean in Iran. References: (1) M. Esquibet et al. Genome 46:1077, 2003. (2) N. Vovlas et al. Plant Pathol. 60:762, 2011.

First Report of Ditylenchus dipsaci on Garlic in Minnesota.

In the summer of 2011, two independent garlic samples from Morrison and Dakota counties and in 2012 one garlic sample from Carver county in Minnesota were submitted by commercial growers to the University of Minnesota Plant Disease Clinic for disease analyses. Symptoms of the above-ground plant parts were stunting and chlorosis. Symptoms of bulbs were necrosis, underdevelopment, and distortion. Upon microscopic examination, phytonematodes exuded into the surrounding water droplet. Nematodes were present in the protective leaves, abscission zone, and cloves in all submitted bulbs (n = 18) for analyses. Morphometric examination of females, males, and juveniles determined that they were Ditylenchus dipsaci. Nematodes extracted from garlic cloves were fixed in TAF (97 ml formalin [40%], 2 ml triethanolamine, and 91 ml dH2O). Morphological observations and measurements were made under an Olympus BX51 microscope equipped with a Nomarski differential interference contrast. Female (n = 6) measurements were: L = 1.411 to 1.636 mm, a = 38 to 44, b = 5.8 to 8.0, c = 14 to 17, stylet = 11.5 to 12.3 µm, V = 79 to 81%, and tail = 95 to 105 µm. The body was almost straight, when heat relaxed, lip region flattened, median bulb oval, and isthmus elongate and slender. The basal pharyngeal bulb overlapped the intestine. The post-vulval uterine branch was about half of vulva-anus distance. The tail was conoid with a pointed terminus. Male (n = 9) measurements were: L = 1.372 to 1.558 mm, a = 40 to 50, b = 6.5 to 7.0, c = 14 to 16, stylet = 11.5 to 12.3 µm, spicules = 22 to 27 µm, and gubernaculum = 9 to 10 µm. The bursa was leptoderan and spicules were curved with simple gubernaculum. Morphology and morphometrics of females and males of D. dipsaci from Minnesota generally fit the descriptions provided for the type and other populations by Hopper (1) and other authors. Several specimens were also taken for molecular identification. DNA extraction, PCR, and sequencing protocols were as described by Subbotin et al. (2). The TW81 and AB28 primers were used for amplification of ITS-rRNA region and the D2A and D3B primers were used for amplification of the D2-D3 expansion segments of 28S rRNA gene. Comparison of the ITS and D2-D3 of 28 rRNA gene sequences showed 100 and 99% identity with corresponding gene sequences of D. dipsaci published in the GenBank (2). The sequences were submitted in the GenBank under accession numbers JX123258 and X123259. This nematode problem has not been known to occur in either of these locations previously. The most likely source of introduction of D. dipsaci are imported garlic seed bulbs. To our knowledge, this is the first report of D. dipsaci affecting garlic or any other crops in Minnesota. The garlic produced in these locations was considered unmarketable and complete loss to the farmers. The presence of D. dipsaci could have a significant economic impact in the emerging multi-million dollar garlic industry in Minnesota. References: (1) D. J. Hooper. Ditylenchus dipsaci. CIH Descriptions of Plant-Parasitic Nematodes Set 1, No. 14, 1972. (2) S. A. Subbotin et al. Phytopathology 95:1308, 2005.

Characterization of the Cystoid Nematode Meloidoderita kirjanovae (Nemata: Sphaeronematidae) from Southern Italy.

A population of the cystoid nematode Meloidoderita kirjanovae was detected parasitizing water mint (Mentha aquatica) in southern Italy. The morphological identification of this species was confirmed by molecular analysis using the internal transcribed spacer 1 (ITS1) and 5.8S gene sequences of nuclear ribosomal DNA (rDNA), which clearly separated it from the closely related species Meloidoderita polygoni. A phylogenetic analysis of M. kirjanovae with species of related genera was conducted using sequences of the D2-D3 expansion segments of the 28S nuclear ribosomal RNA gene. The resulting phylogenetic tree was congruent with trees from an extended dataset for Criconematina and Tylenchida. The basal position of the genus Meloidoderita together with Sphaeronema within the Criconematina clade in this tree may indicate their close relationships. The anatomical changes induced by M. kirjanovae population from Italy in water mint were similar to those reported for a nematode population infecting roots of M. longifolia in Israel. Nematode feeding caused the formation of a stellar syncytium that disorganized the pericycle and vascular root tissues.

Morphological and Molecular Characterization of Longidorus americanum n. sp. (Nematoda: Longidoridae), a Needle Nematode Parasitizing Pine in Georgia.

We describe and illustrate a new needle nematode, Longidorus americanum n. sp., associated with patches of severely stunted and chlorotic loblolly pine, (Pinus taeda L.) seedlings in seedbeds at the Flint River Nursery (Byromville, GA). It is characterized by having females with a body length of 5.4-9.0 mm; lip region slightly swollen, anteriorly flattened, giving the anterior end a truncate appearance; long odontostyle (124-165 microm); vulva at 44%-52% of body length; and tail conoid, bluntly rounded to almost hemispherical. Males are rare but present, and in general shorter than females. The new species is morphologically similar to L. biformis, L. paravineacola, L. saginus, and L. tarjani but differs from these species either by the body, odontostyle and total stylet length, or by head and tail shape. Sequence data from the D2-D3 region of the 28S rDNA distinguishes this new species from other Longidorus species. Phylogenetic relationships of Longidorus americanum n. sp. with other longidorids based on analysis of this DNA fragment are presented. Additional information regarding the distribution of this species within the region is required.

Molecular Characterization of Aldolase from Heterodera glycines and Globodera rostochiensis.

Fructose-bisphosphate aldolase (EC 4.1.2.13) is a key enzyme in glycolysis. We have characterized full-length coding sequences for aldolase genes from the cyst nematodes Heterodera glycines and Globodera rostochiensis, the first for any plant-parasitic nematode. Nucleotide homology is high (83% identity), and the respective sequences encode 40 kDa proteins with 89% amino acid identity. Genomic sequences contain six introns located at identical positions in both genes. Intron 4 in the H. glycines gene is >500 bp. Partial genomic sequences determined for seven other cyst nematode species reveal that the large fourth intron is characteristic of Heterodera but not Globodera aldolase genes. Total aldolase-like specific activity in homogenates from H. glycines was 2-fold lower than in either Caenorhabditis elegans or Panagrellus redivivus (P = 0.001). Activity in H. glycines samples was higher in juvenile stages than in adults (P = 0.003). Heterodera glycines aldolase has Km = 41 microM and is inhibited by treatment with carboxypeptidase A or sodium borohydride.

Phylogenetic relationships within the cyst-forming nematodes (Nematoda, Heteroderidae) based on analysis of sequences from the ITS regions of ribosomal DNA.

The ITS1, ITS2, and 5.8S gene sequences of nuclear ribosomal DNA from 40 taxa of the family Heteroderidae (including the genera Afenestrata, Cactodera, Heterodera, Globodera, Punctodera, Meloidodera, Cryphodera, and Thecavermiculatus) were sequenced and analyzed. The ITS regions displayed high levels of sequence divergence within Heteroderinae and compared to outgroup taxa. Unlike recent findings in root knot nematodes, ITS sequence polymorphism does not appear to complicate phylogenetic analysis of cyst nematodes. Phylogenetic analyses with maximum-parsimony, minimum-evolution, and maximum-likelihood methods were performed with a range of computer alignments, including elision and culled alignments. All multiple alignments and phylogenetic methods yielded similar basic structure for phylogenetic relationships of Heteroderidae. The cyst-forming nematodes are represented by six main clades corresponding to morphological characters and host specialization, with certain clades assuming different positions depending on alignment procedure and/or method of phylogenetic inference. Hypotheses of monophyly of Punctoderinae and Heteroderinae are, respectively, strongly and moderately supported by the ITS data across most alignments. Close relationships were revealed between the Avenae and the Sacchari groups and between the Humuli group and the species H. salixophila within Heteroderinae. The Goettingiana group occupies a basal position within this subfamily. The validity of the genera Afenestrata and Bidera was tested and is discussed based on molecular data. We conclude that ITS sequence data are appropriate for studies of relationships within the different species groups and less so for recovery of more ancient speciations within Heteroderidae.

Agreia bicolorata gen. nov., sp. nov., to accommodate actinobacteria isolated from narrow reed grass infected by the nematode Heteroanguina graminophila.

Agreia bicolorata gen. nov., sp. nov. (type strain VKM Ac-1804T=UCM Ac-620T) is proposed to accommodate aerobic, oxidase- and catalase-positive, weakly motile, coryneform actinobacteria isolated from leaf galls induced by the plant-parasitic nematode Heteroanguina graminophila in narrow reed grass, Calamagrostis neglecta. Bacteria assigned to Agreia bicolorata gen. nov., sp. nov. form a distinct lineage within the phylogenetic branch of the family Microbacteriaceae and possess the following chemotaxonomic characteristics: B-type peptidoglycan containing 2,4-diaminobutyric acid, ornithine, alanine, glycine, glutamate and hydroxyglutamate; cell wall sugars rhamnose, fucose and mannose; MK-10 as major menaquinone; phosphatidylglycerol and diphosphatidylglycerol as principal phospholipids; and 12-methyltetradecanoic acid (anteiso-15:0), 14-methyl-pentadecanoic acid (iso-16:0) and 14-methyl-hexadecanoic acid (anteiso-17:0) as predominant fatty acids. The DNA G+C content of Agreia bicolorata is about 67.0 mol %.

Leifsonia poae gen. nov., sp. nov., isolated from nematode galls on Poa annua, and reclassification of 'Corynebacterium aquaticum' Leifson 1962 as Leifsonia aquatica (ex Leifson 1962) gen. nov., nom. rev., comb. nov. and Clavibacter xyli Davis et al. 1984 with two subspecies as Leifsonia xyli (Davis et al. 1984) gen. nov., comb. nov.

The new genus Leifsonia gen. nov. with two new species, Leifsonia poae sp. nov. (type strain VKM Ac-1401T) and Leifsonia aquatica (ex Leifson 1962) nom. rev., comb. nov. (the type species, with VKM Ac-1400T = DSM 20146T = JCM 1368T as type strain), is proposed to accommodate bacteria found in Poa annua root gall, induced by the nematode Subanguina radicicola, and 'Corynebacterium aquaticum' Leifson 1962. Further, it is proposed to reclassify Clavibacter xyli Davis et al. 1984 with two subspecies in the new genus as Leifsonia xyli (Davis et al. 1984) comb. nov., Leifsonia xyli subsp. xyli (Davis et al. 1984) comb. nov. and Leifsonia xyli subsp. cynodontis (Davis et al. 1984) comb. nov. Members of the proposed genus are characterized by coryneform morphology, peptidoglycans based upon 2,4-diaminobutyric acid, the major menaquinone MK-11, phosphatidylglycerol and diphosphatidylglycerol as principal phospholipids, the high content of anteiso- and iso-branched saturated fatty acids, and a DNA G+C base composition of 66-73 mol%. They form a distinct phylogenetic branch attached to the line of descent of Agromyces spp. The new and reclassified species of the new genus clearly differ from each other phylogenetically and phenetically and can be recognized by their morphologies, the cell wall sugar composition, the requirement of complex media for growth, and numerous physiological characteristics, including the oxidase reaction.