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1.  First Report of Longidorus kuiperi and Rotylenchus eximius from Coastal Sand Dunes in Crete, Greece 
Journal of Nematology  2016;48(3):135.
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.
PMCID: PMC5070924  PMID: 27765985
Crete; detection; Longidorus kuiperi; needle nematode; Rotylenchus eximius; spiral nematode
2.  First Report of the Spiral Nematode Rotylenchus incultus (Nematoda: Hoplolaimidae) from Cultivated Olive in Tunisia, with Additional Molecular Data on Rotylenchus eximius 
Journal of Nematology  2016;48(3):136-138.
Spiral nematode species of the genus Rotylenchus have been reported on olive (Olea europaea L.) in several Mediterranean countries (Castillo et al., 2010; Ali et al., 2014). Nematological surveys for plant-parasitic nematodes on olive trees were carried out in Tunisia between 2013 and 2014, and two nematode species of Rotylenchus were collected from the rhizosphere of olive cv. Chemlali in several localities of Tunisia (Tables 1,2Table 1Rotylenchus eximius Siddiqi, 1964 and R. incultus Sher, 1965 sampled in olive cv. Chemlali in Tunisia and sequences used in this study.Table 2Morphometrics of Rotylenchus incultus Sher, 1965 from olive cv. Chemlali in Tunisia.a). Twenty-two soil samples of 3 to 4 kg were collected with a shovel from the upper 50 cm of soil from arbitrarily chosen olive trees. Nematodes were extracted from 500 cm3 of soil by centrifugal flotation method (Coolen, 1979). Specimens were heat killed by adding hot 4% formaldehyde solution and processed to pure glycerin using the De Grisse’s (1969) method. Measurements were done using a drawing tube attached to a Zeiss III compound microscope. Nematode DNA was extracted from single individuals and PCR assays were conducted as described by Castillo et al. (2003). Moderate-to-low soil populations of these spiral nematodes were detected (5.5–11.5, 1.5–5.0 individuals/500 cm3 of soil, respectively). This prompted us to undertake a detailed morphological and molecular comparative study with previous reported data. Morphological and molecular analyses of females identified these species as Rotylenchus eximius Siddiqi, 1964, and Rotylenchus incultus Sher, 1965. The morphology of R. eximius females (five specimens studied) was characterized by having a hemispherical lip region clearly off set, with four to five annuli, body without longitudinal striations, lateral fields areolated in the pharyngeal region only, stylet 32 to 36 μm long, and broadly rounded tail. The morphology of R. incultus females (51 females and 16 males; Table 2) was characterized by a hemispherical lip region with the basal annulus subdivided by irregular longitudinal striations, with three, rarely four annuli; stylet 21.5 to 27.5 μm long, female tail hemispherical with terminus regularly annulated; phasmids anterior to anus level (3–6 annuli above). The morphology of the isolated nematodes agreed with previous descriptions of R. eximius (Siddiqi, 1964; Castillo and Vovlas, 2005) and R. incultus (Sher, 1965; Castillo and Vovlas, 2005; Vovlas et al., 2008), respectively. A single individual was used for DNA extraction. Primers and PCR conditions used in this research were specified in Cantalapiedra-Navarrete et al. (2013), and a single amplicon of 800, 1,100, and 450 bp was obtained and sequenced for D2 to D3, ITS1, and cytochrome c oxidase subunit 1 (coxI), respectively. Sequence alignments for D2 to D3 (KX669231–KX669233), ITS1 (KX669238–KX669240), and coxI (KX669244–KX669245) from R. eximius, showed 99% to 97%, 98% to 94%, 93% similarity to other sequences of R. eximius deposited in GenBank (EU280794–DQ328741, EU373663–EU373664, JX015401–JX015402, respectively). Similarly, D2 to D3 (KX669234–KX669237), ITS1 (KX669241–KX669243), and coxI (KX669246–KX669249) sequence alignments from R. incultus, showed 99%, 99% to 95%, 99% to 90% similarity, respectively, to other sequences of R. incultus deposited in GenBank (EU280797, EU373672–EU373673, JX015403, respectively). The best fitted model of DNA evolution was obtained using jModelTest v. 2.1.7 (Darriba et al. 2012) with the Akaike information criterion. BI analyses were performed under the general time reversible (GTR) with invariable sites and a gamma-shaped distribution of substitution rates (GTR + I + G) model for ITS1 and coxI. Phylogenetic analyses of ITS1 and coxI using Bayesian inference (BI) placed R. eximius and R. incultus from Tunisia in subclades that included all R. eximius and R. incultus sequences deposited in GenBank (Fig. 1Fig. 1Phylogenetic relationships within Rotylenchus species found in Tunisia and other species from GenBank. Bayesian 50% majority rule consensus trees as inferred from ITS1 and coxI sequences alignments under the GTR + I + G model. Posterior probabilities more than 0.70 are given for appropriate clades. Newly obtained sequences in this study are in bold. Scale bar = expected changes per site.), which agrees with previous results (Cantalapiedra-Navarrete et al., 2013). Morphology, morphometry, and molecular and phylogenetic data obtained from these samples were consistent with R. eximius and R. incultus identification. To our knowledge, this is the first report of R. incultus in Tunisia. Consequently, all these data suggest that spiral nematode species of the genus Rotylenchus are predominant in olive as previously reported in other Mediterranean areas (Ali et al., 2014).
PMCID: PMC5070925  PMID: 27765986
Bayesian inference; detection; new geographic record; phylogeny; spiral nematodes
3.  First Report of Heterorhabditis amazonensis from Venezuela and Characterization of Three Populations 
Journal of Nematology  2016;48(3):139-147.
During a survey in western Venezuela in 2011, three new populations of Heterorhabditis amazonensis (LPV081, LPV156, and LPV498) were isolated. Some differences were found in terms of morphometry compared with the original description; however, the distance from the anterior end to the excretory pore is the most variable character; significantly shorter in all infective juveniles and in other developmental stages depending on the population. According to a Principal Component Analysis, LPV498 possesses more differences in morphometric characteristics and can be separated from the other two. Those intraspecific differences could be attributed to the geographic origin of the nematode. Molecular studies of ITS regions demonstrated that the sequences of the Venezuelan strains were identical to those of the type species originally isolated in the Brazilian Amazonian forest. This is an interesting fact because in several studies on heterorhabditids, intraspecific variability has been recorded. Herein, we present the first report of H. amazonensis in Venezuela and the characterization of three populations of this species.
PMCID: PMC5070926  PMID: 27765987
biogeography; entomopathogenic nematode; Heterorhabditis amazonensis; morphology; Venezuela
4.  Steinernema biddulphi n. sp., a New Entomopathogenic Nematode (Nematoda: Steinernematidae) from South Africa 
Journal of Nematology  2016;48(3):148-158.
A new species of entomopathogenic nematode (EPN), Steinernema biddulphi n. sp., was isolated from a maize field in Senekal, Free State Province of South Africa. Morphological and molecular studies indicated the distinctness of S. biddulphi n. sp. from other Steinernema species. Steinernema biddulphi n. sp. is characterized IJs with average body length of 663 μm (606–778 μm), lateral fields with six ridges in mid-body region forming the formula 2,6,2. Excretory pore located anterior to mid-pharynx (D% = 46). Hyaline layer occupies approximately half of tail length. Male spicules slightly to moderately curved, with a sharp tip and golden brown in color. The first generation of males lacking a mucron on the tail tip while the second generation males with a short filamentous mucron. Genital papillae with 11 pairs and one unpaired preanal papilla. The new species is further characterized by sequences of the internal transcribed spacer (ITS) and partial 28S regions (D2-D3) of the ribosomal DNA (rDNA). Phylogenetic data show that S. biddulphi n. sp. belongs to the “bicornutum” clade within the Steinernematidae family.
PMCID: PMC5070927  PMID: 27765988
D2-D3; entomopathogenic nematodes; ITS; new species; phylogeny; South Africa; Steinernema biddulphi; taxonomy
5.  Coronostoma claireae n. sp. (Nematoda: Rhabditida: Oxyuridomorpha: Coronostomatidae) from the Indigenous Milliped Narceus gordanus (Chamberlain, 1943) (Diplopoda: Spirobolida) in Ocala National Forest, Florida 
Journal of Nematology  2016;48(3):159-169.
Twenty-four individuals of Narceus gordanus (Diplopoda: Spirobolidae) were collected in Ocala National Forest, FL, between November 2013 and July 2014. Each specimen was dissected to extract the intestine, which was removed and examined for parasitic nematodes. Coronostoma claireae n. sp. was collected from the hindgut and midgut of 10 specimens, and its morphology was examined with brightfield, differential interference contrast, phase contrast, and scanning electron microscopy. This species is separated from other Coronostoma spp. by the following characteristics: body length less than 3 mm; head sense organs pit-like; first annule long, extending past middle of corpus, width similar to that of second annule; basal bulb pyriform; eggs larger than 60 × 50 µm. This species is the first North American record for the genus Coronostoma, which is removed from Thelastomatoidea: Thelastomatidae and reassigned to Coronostomatidae on the basis of presumed apomorphies. A key is provided for known Coronostoma spp. The superfamily Coronostomatoidea is re-established for Coronostomatidae and Traklosiidae.
PMCID: PMC5070928  PMID: 27765989
host-parasitic relationship; key; morphology; predator; nematophagy; scanning electron microscopy (SEM); Spirobolida; taxonomy; Thelastomatoidea; United States
6.  Curative Control of the Peachtree Borer Using Entomopathogenic Nematodes 
Journal of Nematology  2016;48(3):170-176.
The peachtree borer, Synanthedon exitiosa (Say 1823), is a major pest of stone fruit trees in North America. Current management relies upon preventative control using broad-spectrum chemical insecticides, primarily chlorpyrifos, applied in the late summer or early fall. However, due to missed applications, poor application timing, or other factors, high levels of S. exitiosa infestation may still occur and persist through the following spring. Curative treatments applied in the spring to established infestations would limit damage to the tree and prevent the next generation of S. exitiosa from emerging within the orchard. However, such curative measures for control of S. exitiosa do not exist. Our objective was to measure the efficacy of the entomopathogenic nematode, Steinernema carpocapsae, as a curative control for existing infestations of S. exitiosa. In peach orchards, spring applications of S. carpocapsae (obtained from a commercial source) were made to infested trees and compared with chlorpyrifos and a water-only control in 2014 and 2015. Additionally, types of spray equipment were compared: nematodes were applied via boom sprayer, handgun, or trunk sprayer. To control for effects of application method or nematode source, in vivo laboratory-grown S. carpocapsae, applied using a watering can, was also included. Treatment effects were assessed 39 d (2014) or 19 d (2015) later by measuring percentage of trees still infested, and also number of surviving S. exitiosa larvae per tree. Results indicated that S. carpocapsae provided significant curative control (e.g., >80% corrected control for the handgun application). In contrast, chlorpyrifos failed to reduce S. exitiosa infestations or number of surviving larvae. In most comparisons, no effect of nematode application method was detected; in one assessment, only the handgun and watering can methods reduced infestation. In conclusion, our study indicates that S. carpocapsae may be used as an effective curative measure for S. exitiosa infestations.
PMCID: PMC5070929  PMID: 27765990
application method; curative; entomopathogenic nematode; peachtree borer; Steinernema carpocapsae; Synanthedon exitiosa
7.  Effect of Application Timing of Oxamyl in Nonbearing Raspberry for Pratylenchus penetrans Management 
Journal of Nematology  2016;48(3):177-182.
In 2012, the Washington raspberry (Rubus idaeus) industry received a special local needs (SLN) 24(c) label to apply Vydate L® (active ingredient oxamyl) to nonbearing raspberry for the management of Pratylenchus penetrans. This is a new use pattern of this nematicide for raspberry growers; therefore, research was conducted to identify the optimum spring application timing of oxamyl for the suppression of P. penetrans. Three on-farm trials in each of 2012 and 2013 were established in Washington in newly planted raspberry trials on a range of varieties. Oxamyl was applied twice in April (2013 only), May, and June, and these treatments were compared to each other as well as a nontreated control. Population densities of P. penetrans were determined in the fall and spring postoxamyl applications for at least 1.5 years. Plant vigor was also evaluated in the trials. Combined results from 2012 and 2013 trials indicated that application timing in the spring was not critical. Oxamyl application reduced root P. penetrans population densities in all six trials. Reductions in P. penetrans population densities in roots of oxamyl-treated plants, regardless of application timing, ranged from 62% to 99% of densities in nontreated controls. Phytotoxicity to newly planted raspberry was never observed in any of the trials. A nonbearing application of oxamyl is an important addition to current control methods used to manage P. penetrans in raspberry in Washington.
PMCID: PMC5070930  PMID: 27765991
Praytlenchus penetrans; postplant nematicide; root lesion nematode; Rubus idaeus; vydate
8.  Evaluation of Steam and Soil Solarization for Meloidogyne arenaria Control in Florida Floriculture Crops 
Journal of Nematology  2016;48(3):183-192.
Steam and soil solarization were investigated for control of the root-knot nematode Meloidogyne arenaria in 2 yr of field trials on a commercial flower farm in Florida. The objective was to determine if preplant steam treatments in combination with solarization, or solarization alone effectively controlled nematodes compared to methyl bromide (MeBr). Trials were conducted in a field with naturally occurring populations of M. arenaria. Treatments were solarization alone, steam treatment after solarization using standard 7.6-cm-diameter perforated plastic drain tile (steam 1), steam treatment following solarization using custom-drilled plastic drain tile with 1.6-mm holes spaced every 3.8 cm (steam 2), and MeBr applied at 392 kg/ha 80:20 MeBr:chloropicrin. Drain tiles were buried approximately 35 cm deep with four tiles per 1.8 by 30 m plot. Steam application followed a 4-wk solarization period concluding in mid-October. All steam was generated using a Sioux propane boiler system. Plots were steamed for sufficient time to reach the target temperature of 70°C for 20 min. Solarization plastic was retained on the plots during steaming and plots were covered with a single layer of carpet padding to provide additional insulation. The floriculture crops larkspur (Delphinium elatum and Delphinium × belladonna), snapdragon (Antirrhinum majus), and sunflower (Helianthus annuus) were produced according to standard commercial practices. One month after treatment in both years of the study, soil populations of M. arenaria were lower in both steam treatments and in MeBr compared to solarization alone. At the end of the season in both years, galling on larkspur, snapdragon, and sunflowers was lower in both steam treatments than in solarization. Both steam treatments also provided control of M. arenaria in soil at the end of the season comparable to, or exceeding that provided by MeBr. Both steam treatments also reduced M. arenaria in snapdragon roots comparable to, or exceeding control with MeBr. Meloidogyne arenaria in soil increased in solarization alone. Solarization alone also had higher gall ratings on larkspur, snapdragon, and sunflower than all other treatments. Steam provided excellent control of M. arenaria in this study.
PMCID: PMC5070931  PMID: 27765992
Antirrhinum majus; Delphinium × belladonna; Delphinium elatum; fumigation; Helianthus annuus; methyl bromide alternatives; root-knot nematodes
9.  Mitochondrial Haplotype-based Identification of Root-knot Nematodes (Meloidogyne spp.) on Cut Foliage Crops in Florida 
Journal of Nematology  2016;48(3):193-202.
Florida accounts for more than 75% of the national cut foliage production. Unfortunately, root-knot nematodes (RKN) (Meloidogyne spp.) are a serious problem on these crops, rendering many farms unproductive. Currently, information on the Meloidogyne spp. occurring on most commonly cultivated cut foliage crops in Florida, and tools for their rapid identification are lacking. The objectives of this study were to (i) identify specific RKN infecting common ornamental cut foliage crops in Florida and (ii) evaluate the feasibility of using the mtDNA haplotype as a molecular diagnostic tool for rapid identification of large samples of RKN. A total of 200 Meloidogyne females were collected from cut foliage plant roots. Meloidogyne spp. were identified by PCR and RFLP of mitochondrial DNA. PCR and RFLP of mitochondrial DNA were effective in discriminating the Meloidogyne spp. present. Meloidogyne incognita is the most dominant RKN on cut foliage crops in Florida and must be a high target for making management decisions. Other Meloidogyne spp. identified include M. javanica, M. hapla, Meloidogyne sp. 1, and Meloidogyne sp. 2. The results for this study demonstrate the usefulness of the mtDNA haplotype-based designation as a valuable molecular tool for identification of Meloidogyne spp.
PMCID: PMC5070932  PMID: 27765993
cut foliage; endonuclease; identification; mitochondrial haplotype; MORF/MTHIS; mtDNA; PCR-RFLP; restriction enzymes; TRNAH/MRH106
10.  Morphological and Molecular Characterization of Gracilacus wuae n. sp. (Nematoda: Criconematoidea) Associated with Cow Parsnip (Heracleum maximum) in Ontario, Canada 
Journal of Nematology  2016;48(3):203-213.
Gracilacus wuae n. sp. from soil associated with cow parsnip in Ontario, Canada is described and illustrated. Morphologically, females have a long stylet ranging from 80 to 93 µm long, the lip region not offset from the body contour, without lateral lips but with large and flat submedian lobes, the mouth opening slit-like elongated laterally and surrounded by lateral flaps, the excretory pore is anterior to the knobs of the stylet; males without stylet and the pharynx degenerated. The fourth-stage juveniles lack a stylet, the pharynx degenerated, and can be differentiated into preadult females and males based on the position of the genital primordia. The third-stage juveniles are similar to females but smaller. Phylogenetic studies using the rDNA small subunit 18S, large subunit 28S D2/D3, and internal transcribed spacer (ITS) sequences collectively provide evidence of a grouping with other Gracilacus and some species of Paratylenchus with stylet length of females longer than 41 µm deposited in GenBank.
PMCID: PMC5070933  PMID: 27765994
Gracilacus; molecular; morphology; morphometric; new species; Paratylenchus; taxonomy
11.  Discopersicus n. gen., a New Member of the Family Tylenchidae Örley, 1880 with Detailed SEM Study on Two Known Species of the Genus Discotylenchus Siddiqi, 1980 (Nematoda; Tylenchidae) from Iran 
Journal of Nematology  2016;48(3):214-221.
Discopersicus iranicus n. gen., n. comb., previously described from Iran as a new species under the genus Discotylenchus, is illustrated using light microscope and scanning electron microscope (SEM) observations and further studied using molecular characters. SEM studies revealed the newly proposed genus has oblique amphidial apertures on the lateral sides of the lip region. SEM images are also provided for two species of Discotylenchus, namely D. discretus and D. brevicaudatus, as the first SEM study of the genus. These results confirmed longitudinal amphidial aperture type on lateral sides of the lip region in genus Discotylenchus, as noted by Siddiqi while erecting the genus with D. discretus as the type species. Molecular phylogenetic analyses using partial small subunit (SSU) and large subunit (LSU) rDNA sequences revealed the affinity of the genus Discopersicus n. gen. with members of the subfamily Boleodorinae, as supported by morphological characters (mainly, the oblique amphidial opening).
PMCID: PMC5070934  PMID: 27765995
18S rDNA; 28S rDNA; Boleodorinae; new genus; phylogeny; taxonomy
12.  A Third New Species of Aporcelinus Andrássy, 2009 (Dorylaimida, Aporcelaimidae) from Vietnam, with the First SEM Study of a Representative of the Genus 
Journal of Nematology  2016;48(2):104-108.
A new species, the third one from Vietnam, of the genus Aporcelinus is described from natural areas. Aporcelinus falcicaudatus sp. n. is characterized by its 1.28 to 1.63 mm long body, lip region offset by weak constriction and 16 to 18 µm broad, odontostyle 18 to 21 µm at its ventral side, neck 354 to 406 µm long, uterus tripartite and 61 to 95 µm long, V = 50 to 55, tail strongly recurved dorsad and conical (23–31 µm, c = 43–58, c′ = 0.7–0.9) with finely rounded tip, and male absent. Scanning electron microscope (SEM) study, the first of a representative of the genus, shows a lip region pattern significantly different from that observed in the typical aporcelaimid taxa.
PMCID: PMC4930313  PMID: 27418703
aporcelaims; description; free-living nematodes; southeast Asia; taxonomy
13.  Paurodontella parapitica n. sp. (Nematoda: Hexatylina, Sphaerularioidea) from Kermanshah Province, Western Iran 
Journal of Nematology  2016;48(2):109-115.
Paurodontella parapitica n. sp., collected from the rhizosphere of an apple tree in Kermanshah province, western Iran, is described. The new species is characterized by a body length of 505 to 723 µm (females) and 480 to 600 µm (males), lip region continuous by depression; 7 to 8 μm broad, 3 to 4 µm high, stylet length 7 to 9 µm or 1 to 1.3 times the lip region diameter, short postuterine sac of 4 to 6 μm long, lateral fields with five to six incisures; outer incisures crenated and inner incisures weakly crenated, excretory pore situated 90 to 100 µm from anterior end; functional males common in the population, with spicules 24 to 26 μm long. Tail of both sexes similar, almost straight and elongate-conoid. The new species resembles in morphology and morphometrics to four known species of the genus, namely P. apitica, P. minuta, P. myceliophaga, and P. sohailai. The results of phylogenetic analyses based on sequences of D2/D3 expansion region of 28S rRNA gene revealed this genus is polyphyletic in four different clades in Tylenchid.
PMCID: PMC4930314  PMID: 27418704
28S D2/D3; new species; molecular phylogeny; morphology; taxonomy
14.  Nematode Fauna of Tropical Rainforest in Brazil: A Descriptive and Seasonal Approach 
Journal of Nematology  2016;48(2):116-125.
Studies of nematode assemblages in natural ecosystems can contribute to better understanding of the occurrence, relevance, and ecology of plant-parasitic and other soil nematodes. Nematode assemblages and environmental parameters (organic matter, water content (WC), bulk density (BD), total porosity (Po), soil respiration, and soil texture) were investigated in two seasons (rainy and dry) in two forest areas of the Zona da Mata, Pernambuco State. The aim of our research was to evaluate the heterogeneity between two locations and seasons in the Brazilian Atlantic Forest. Structure and composition of the nematode assemblages differed between areas and across time. Rhabditidae dominated the rainy season in both forest soils. Rarefaction curves (RC) suggest that sampling to detect more nematode taxa should be more intensive in the rainy season. The forest soils have complex, stable soil food webs with high connectance and decomposition channels dominated by bacteria. The predator–prey relationships were not affected by changes in soil properties that fluctuate with time.
PMCID: PMC4930315  PMID: 27418705
ecological indices; ecology; metabolic footprints; predator–prey interactions; soil physical properties
15.  Efficacy of Various Application Methods of Fluensulfone for Managing Root-knot Nematodes in Vegetables 
Journal of Nematology  2016;48(2):65-71.
Fluensulfone is a new nematicide in the flouroalkenyl chemical group. A field experiment was conducted in 2012 and 2013 to evaluate the efficacy of various application methods of fluensulfone for control of Meloidogyne spp. in cucumber (Cucumis sativus). Treatments of fluensulfone (3.0 kg a.i./ha) were applied either as preplant incorporation (PPI) or via different drip irrigation methods: drip without pulse irrigation (Drip NP), pulse irrigation 1 hr after treatment (Drip +1P), and treatment at the same time as pulse irrigation (Drip =P). The experiment had eight replications per treatment and also included a PPI treatment of oxamyl (22.5 kg a.i./ha) and a nontreated control. Compared to the control, neither the oxamyl nor the fluensulfone PPI treatments reduced root galling by Meloidogyne spp. in cucumber. Among the drip treatments, Drip NP and Drip +1P reduced root galling compared to the control. Cucumber yield was greater in all fluensulfone treatments than in the control. In a growth-chamber experiment, the systemic activity and phytotoxicity of fluensulfone were also evaluated on tomato (Solanum lycopersicum), eggplant (Solanum melongena), cucumber, and squash (Curcurbita pepo). At the seedling stage, foliage of each crop was sprayed with fluensulfone at 3, 6, and 12 g a.i./liter, oxamyl at 4.8 g a.i./liter, or water (nontreated control). Each plant was inoculated with Meloidogyne incognita juveniles 2 d after treatment. There were six replications per treatment and the experiment was conducted twice. Foliar applications of fluensulfone reduced plant vigor and dry weight of eggplant and tomato, but not cucumber or squash; application of oxamyl had no effect on the vigor or weight of any of the crops. Typically, only the highest rate of fluensulfone was phytotoxic to eggplant and tomato. Tomato was the only crop tested in which there was a reduction in the number of nematodes or galls when fluensulfone or oxamyl was applied to the foliage compared to the nontreated control. This study demonstrates that control of Meloidogyne spp. may be obtained by drip and foliar applications of fluensulfone; however, the systemic activity of fluensulfone is crop specific and there is a risk of phytotoxicity with foliar applications.
PMCID: PMC4930317  PMID: 27418698
cucumber; fluensulfone; management; Meloidogyne spp.; nematicide; oxamyl; tomato; vegetable crops
16.  Reproduction of Meloidogyne incognita Race 3 on Flue-cured Tobacco Homozygous for Rk1 and/or Rk2 Resistance Genes 
Journal of Nematology  2016;48(2):79-86.
Most commercial tobacco cultivars possess the Rk1 resistance gene to races 1 and 3 of Meloidogyne incognita and race 1 of Meloidogyne arenaria, which has caused a shift in population prevalence in Virginia tobacco fields toward other species and races. A number of cultivars now also possess the Rk2 gene for root-knot resistance. Experiments were conducted in 2013 to 2014 to examine whether possessing both Rk1 and Rk2 increases resistance to a variant of M. incognita race 3 compared to either gene alone. Greenhouse trials were arranged in a completely randomized design with Coker 371-Gold (C371G; susceptible), NC 95 and SC 72 (Rk1Rk1), T-15-1-1 (Rk2Rk2), and STNCB-2-28 and NOD 8 (Rk1Rk1 and Rk2Rk2). Each plant was inoculated with 5,000 root-knot nematode eggs; data were collected 60 d postinoculation. Percent galling and numbers of egg masses and eggs were counted, the latter being used to calculate the reproductive index on each host. Despite variability, entries with both Rk1 and Rk2 conferred greater resistance to a variant of M. incognita race 3 than plants with Rk1 or Rk2 alone. Entries with Rk1 alone were successful in reducing root galling and nematode reproduction compared to the susceptible control. Entry T-15-1-1 did not reduce galling compared to the susceptible control but often suppressed reproduction.
PMCID: PMC4930319  PMID: 27418700
genetics; Meloidogyne incognita; Nicotiana tabacum; reproductive index; Virginia
17.  Sectonema caobangense sp. n. from Vietnam (Nematoda, Dorylaimida, Aporcelaimidae) 
Journal of Nematology  2016;48(2):95-103.
Sectonema caobangense sp. n. from evergreen forest soil in Vietnam is described, including scanning electron micrograph (SEM) observations and D2-D3 LSU rDNA analysis. The new species is characterized by its 3.12 to 5.80 mm long body, lip region offset by deep constriction and 21 to 23 μm broad, mural tooth 13 to 14 μm long at its ventral side, 940 to 1,112 μm long neck, pharyngeal expansion occupying 61% to 69% of total neck length, uterus a long simple tube-like structure 292 to 363 μm long or 2.7 to 2.9 times the corresponding body diameter, pars refringens vaginae well developed, V = 48 to 56, short (36–51 μm, c = 77–132, c′ = 0.5–0.8) and rounded tail, 87 to 99 μm long spicules, and four or five irregularly spaced ventromedian supplements bearing hiatus. Sectonema caobangense sp. n. differs from the typical pattern of Sectonema in the nature of the stomatal protrusible structure, bearing a mural tooth attached to the ventral side of the stoma. Molecular data obtained and the derived evolutionary trees support a close phylogenetic relationship with other Sectonema species.
PMCID: PMC4930321  PMID: 27418702
Bayesian inference; description; LSU ribosomal DNA; maximum likelihood; morphology; morphometrics; SEM; taxonomy
18.  The Effects of Nutrient Concentration, Addition of Thickeners, and Agitation Speed on Liquid Fermentation of Steinernema feltiae 
Journal of Nematology  2016;48(2):126-133.
Entomopathogenic nematode production in liquid fermentation still requires improvements to maximize efficiency, yield, and nematode quality. Therefore, this study was aimed at developing a more suitable liquid medium for mass production of Steinernema feltiae, by assessing the effects of nutrient concentration, thickeners (primarily agar), and agitation speed on infective juvenile (IJ) yield. Base medium (BM) contained yeast extract (2.3%), egg yolk (1.25%), NaCl (0.5%), and corn oil (4%). All media were inoculated with Xenorhabdus bovienii, and 2 d later, with 2-d-old S. feltiae juveniles. For the nutrient concentration experiment, we evaluated the base medium versus a modified base medium containing all the components, but with 3× concentrations of yeast extract (6.9%), egg yolk (3.75%), and corn oil (12%). The nematodes and bacteria were cultured in 150-ml Erlenmeyer flasks containing 50 ml of liquid medium at (25°C) and 180 rpm on a rotary shaker incubator. To assess the effect of thickeners, IJs were inoculated in BM with agar (0.2%), carrageen (0.2%), and carboxymethyl cellulose (0.2% and 0.5%). The addition of 3× more nutrients relative to the BM resulted in a significantly lower yield of nematodes. For agar and agitation speed experiments, five levels of agar in the BM (0%, 0.2%, 0.4%, 0.6%, and 0.8% agar) and two agitation speeds (180 and 280 rpm) were evaluated for production. Increasing agitation speed from 180 to 280 rpm and higher levels of agar in the medium (> 0.2%) significantly increased the yield of bacteria. At the lower agitation speed, media amended with 0.4% and 0.6% agar produced higher nematode yields compared to media without agar. Media with 0.2% and 0.8% agar resulted in intermediate levels of nematode production. At the higher agitation speed, media supplemented with 0.8% agar resulted in the lowest yield of nematodes when compared to the other media tested. Results indicated that increasing nutrient concentration levels was detrimental to nematode production. Also, media containing agar (0.4% and 0.6%) increased nematode yields when cultures were grown at low agitation speed. When IJs were used as the inoculum, 0.2% agar also enhanced recovery and nematode yield at the higher agitation speed.
PMCID: PMC4930316  PMID: 27418706
biocontrol; entomopathogenic nematode; in vitro production; liquid fermentation
19.  Reproduction and Damage Potential of Five Geographical Ditylenchus africanus Populations on Peanut 
Journal of Nematology  2016;48(2):72-78.
Ditylenchus africanus affects peanut quality, which leads to downgrading of consignments and economic losses for producers. This nematode is difficult to control and host-plant resistance may be the most effective way to control it. Recently, the peanut breeding line PC254K1 has been identified as resistant to a D. africanus population from Vaalharts and will be included into the peanut breeding program. The objectives of our study were to compare the reproduction potential of D. africanus geographic populations from five different areas in the peanut production area of South Africa and to assess whether PC254K1 is resistant to all five D. africanus populations. Reproduction of the D. africanus populations was evaluated on peanut callus in growth cabinets at 21°C, 28°C, and 35°C. The peanut cv. Sellie was included in the study as the D. africanus-susceptible reference genotype in the greenhouse and microplots. Reproduction potential of all five of the D. africanus populations was similar. Resistance of PC254K1 was confirmed to all five D. africanus populations. The resistance trait of a D. africanus-resistant cultivar developed from PC254K1 should, therefore, be sustainable over the five localities tested during this study.
PMCID: PMC4930318  PMID: 27418699
Arachis hypogaea; Ditylenchus africanus; peanut; pod nematode; resistance
20.  The Effect of Endophytic Fungi on Nematode Populations in Summer-dormant and Summer-active Tall Fescue 
Journal of Nematology  2016;48(2):87-94.
Summer-active (continental) and summer-dormant (Mediterranean) tall fescue morphotypes are each adapted to different environmental conditions. Endophyte presence provides plant parasitic nematode resistance, but not with all endophyte strains and cultivar combinations. This study sought to compare effects of four nematode genera on continental and Mediterranean cultivars infected with common toxic or novel endophyte strains. A 6-mon greenhouse study was conducted with continental cultivars, Kentucky 31 (common toxic) and Texoma MaxQ II (novel endophyte) and the Mediterranean cultivar Flecha MaxQ (novel endophyte). Endophyte-free plants of each cultivar were controls. Each cultivar × endophyte combination was randomly assigned to a control, low or high inoculation rate of a mixed nematode culture containing stunt nematodes (Tylenchorhynchus spp.), ring nematodes (Criconemella spp.), spiral nematodes (Helicotylenchus spp.), and lesion nematodes (Pratylenchus spp.). Endophyte infection had no effect on nematode population densities. The cultivar × endophyte interaction was significant. Population densities of stunt nematode, spiral nematode, and ring nematodes were higher for Flecha MaxQ than other cultivar × endophyte combinations. Novel endophyte infection enhances suitability of Flecha MaxQ as a nematode host.
PMCID: PMC4930320  PMID: 27418701
endophyte; host-parasite relationship; lesion nematode (Pratylenchus spp.); ring nematode (Criconemella spp.); spiral nematode (Helicotylenchus spp.); stunt nematode (Tylenchorhynchus spp.); summer active; summer dormant; tall fescue
21.  Occurrence of Panagrellus (Rhabditida: Panagrolaimidae) Nematodes in a Morphologically Aberrant Adult Specimen of Rhynchophorus ferrugineus (Coleoptera: Dryophthoridae) 
Journal of Nematology  2016;48(1):1-6.
An aberrant specimen of Rhynchophorus ferrugineus (Coleoptera: Dryophthoridae) also known as red palm weevil (RPW), the most economically important insect pest of palms in the world, was found among a batch of conspecifics reared for research purposes. A morphological analysis of this weevil revealed the presence of nematodes associated with a structured cuticle defect of the thorax. These nematodes were not able to be cultured, but were characterized by molecular analysis using 28S and 18S ribosomal DNA and shown to belong to the family Panagrolaimidae (Rhabditida), within a clade of Panagrellus. While most nematodes in the insect were juveniles, a single male adult was partially characterized by light microscopy. Morphometrics showed similarities to a species described from Germany. Excluding the entomopathogenic nematodes (EPN), only five other genera of entomophilic or saprophytic rhabditid nematodes are associated with this weevil. This is the first report of panagrolaimid nematodes associated with this invasive pest. Possible mechanisms of nematode-insect association are discussed.
PMCID: PMC4859612  PMID: 27168645
insect thorax defect; invasive species; nematode phoresy; physiological ecology; saprophagous nematode; sour paste nematode
22.  Resistance to Southern Root-knot Nematode (Meloidogyne incognita) in Wild Watermelon (Citrullus lanatus var. citroides) 
Journal of Nematology  2016;48(1):14-19.
Southern root-knot nematode (RKN, Meloidogyne incognita) is a serious pest of cultivated watermelon (Citrullus lanatus var. lanatus) in southern regions of the United States and no resistance is known to exist in commercial watermelon cultivars. Wild watermelon relatives (Citrullus lanatus var. citroides) have been shown in greenhouse studies to possess varying degrees of resistance to RKN species. Experiments were conducted over 2 yr to assess resistance of southern RKN in C. lanatus var. citroides accessions from the U.S. Watermelon Plant Introduction Collection in an artificially infested field site at the U.S. Vegetable Laboratory in Charleston, SC. In the first study (2006), 19 accessions of C. lanatus var. citroides were compared with reference entries of Citrullus colocynthis and C. lanatus var. lanatus. Of the wild watermelon accessions, two entries exhibited significantly less galling than all other entries. Five of the best performing C. lanatus var. citroides accessions were evaluated with and without nematicide at the same field site in 2007. Citrullus lanatus var. citroides accessions performed better than C. lanatus var. lanatus and C. colocynthis. Overall, most entries of C. lanatus var. citroides performed similarly with and without nematicide treatment in regard to root galling, visible egg masses, vine vigor, and root mass. In both years of field evaluations, most C. lanatus var. citroides accessions showed lesser degrees of nematode reproduction and higher vigor and root mass than C. colocynthis and C. lanatus var. lanatus. The results of these two field evaluations suggest that wild watermelon populations may be useful sources of resistance to southern RKN.
PMCID: PMC4859613  PMID: 27168648
Citrullus lanatus var. citroides; Citrullus lanatus var. lanatus; Meloidogyne incognita; plant introduction; resistance; southern root-knot nematode; wild watermelon
23.  Molecular and Morphological Characterization of Xiphinema chambersi Population from Live Oak in Jekyll Island, Georgia, with Comments on Morphometric Variations 
Journal of Nematology  2016;48(1):20-27.
A population of Xiphinema chambersi from the root zone around live oak (Quercus virginiana Mill.) trees on Jekyll Island, GA, is described using both morphological and molecular tools and compared with descriptions of type specimens. Initially, because of a few morphological differences, this nematode was thought to represent an undescribed species. However, on further examination, the morphometrics of the nematodes from live oak tend to agree with most of the morphometrics in the original description and redescription of X. chambersi except for few minor differences in V% relative to body length, slightly shorter stylet length, different c value, and the number of caudal pores. We consider these differences to be part of the normal variation within this species and accordingly image this new population of X. chambersi and redescribe the species. The new population is characterized by having females with a body length of 2.1 to 2.5 mm; lip region slightly rounded and set off from head; total stylet length 170 to 193 µm; vulva at 20.4% to 21.8% of body length; a monodelphic, posterior reproductive system; elongate, conoid tail with a blunt terminus and four pairs of caudal pores, of which two pairs are subdorsal and two subventral. Sequence data from the D2–D3 region of the 28S rRNA molecule subjected to GenBank sequence comparison using BLAST showed that the sequence had 96% and 99% similarity with X. chambersi from Alabama and Florida, respectively. Phylogenetic relationships of X. chambersi with other xiphinematids based on analysis of this DNA fragment are presented. This finding represents a new location of X. chambersi in Georgia on live oak for this species.
PMCID: PMC4859614  PMID: 27168649
28S rRNA; live oak; morphology; morphometrics; phylogeny; redescription; taxonomy; tree; Xiphinema chambersi
24.  Morphological and Molecular Characterization of Two Aphelenchoides Endophytic in Poplar Leaves 
Journal of Nematology  2016;48(1):28-33.
During a long-term, large network study of the ecology of plant endophytes in native habitats, various nematodes have been found. Two poplar species, Populus angustifolia (narrowleaf cottonwood) and Populus trichocarpa (black cottonwood), are important ecological and genomic models now used in ongoing plant–pathogen–endophyte interaction studies. In this study, two different aphelenchid nematodes within surface-sterilized healthy leaves of these two Populus spp. in northwestern North America were discovered. Nematodes were identified and characterized microscopically and molecularly with 28S ribosomal RNA (rRNA) and 18S rRNA molecular markers. From P. angustifolia, Aphelenchoides saprophilus was inferred to be closest to another population of A. saprophilus among sequenced taxa in the 18S tree. From P. trichocarpa, Laimaphelenchus heidelbergi had a 28S sequence only 1 bp different from that of a Portuguese population, and 1 bp different from the original Australian type population. The 28S and 18S rRNA trees of Aphelenchoides and Laimaphelenchus species indicated L. heidelbergi failed to cluster with three other Laimaphelenchus species, including the type species of the genus. Therefore, we support a conservative definition of the genus Laimaphelenchus, and consider these populations to belong to Aphelenchoides, amended as Aphelenchoides heidelbergi n. comb. This is the first report of these nematode species from within aboveground leaves. The presence of these fungal-feeding nematodes can affect the balance of endophytic fungi, which are important determinants of plant health.
PMCID: PMC4859615  PMID: 27168650
nematode ecology; phylogeny; ribosomal DNA; systematics; taxonomy
25.  Molecular and Morphological Characterization of Aphelenchoides fuchsi sp. n. (Nematoda: Aphelenchoididae) Isolated from Pinus eldarica in Western Iran 
Journal of Nematology  2016;48(1):34-42.
Aphelenchoides fuchsi sp. n. is described and illustrated from bark and wood samples of a weakened Mondell pine in Kermanshah Province, western Iran. The new species has body length of 332 to 400 µm (females) and 365 to 395 µm (males). Lip region set off from body contour. The cuticle is weakly annulated, and there are four lines in the lateral field. The stylet is 8 to 10 μm long and has small basal swellings. The excretory pore is located ca one body diam. posterior to metacorpus valve or 51 to 62 μm from the head. The postuterine sac well developed (60–90 µm). Spicules are relatively short (15–16 μm in dorsal limb) with apex and rostrum rounded, well developed, and the end of the dorsal limb clearly curved ventrad like a hook. The male tail has usual three pairs of caudal papillae (2+2+2) and a well-developed mucro. The female tail is conical, terminating in a complicated step-like projection, usually with many tiny nodular protuberances. The new species belongs to the Group 2 sensu Shahina, category of Aphelenchoides species. Phylogenetic analysis based on small subunit (SSU) and partial large subunit (LSU) sequences of rRNA supported the morphological results.
PMCID: PMC4859616  PMID: 27168651
Aphelenchoides; LSU; molecular; morphology; morphometrics; new species; phylogeny; SSU; taxonomy

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