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1.  Molecular rDNA phylogeny of Telotylenchidae Siddiqi, 1960 and evaluation of tail termini 
Journal of Nematology  2010;42(4):359-369.
Three stunt nematode species, Tylenchorhynchus leviterminalis, T. dubius and T. claytoni were characterized with segments of small subunit 18S and large subunit 28S rDNA sequence and placed in molecular phylogenetic context with other polyphyletic taxa of Telotylenchidae. Based upon comparably sized phylogenetic breadth of outgroups and ingroups, the 28S rDNA contained three times the number of phylogenetically informative alignment characters relative to the alignment total compared to the larger 18S dataset even though there were fewer than half the number of taxa represented. Tail shapes and hyaline termini were characterized for taxa within these subfamily trees, and variability discussed for some related species. In 18S trees, similar terminal tail thickness was found in a well-supported clade of three Tylenchorhynchus: broad-tailed T. leviterminalis branched outside relatively narrow-tailed T. claytoni and T. nudus. Terminal tail thickness within Merliniinae, Telotylenchinae and related taxa showed a mosaic distribution. Thick-tailed Trophurus, Macrotrophurus and putative Paratrophurus did not group together in the 18S tree. Extremely thickened tail termini arose at least once in Amplimerlinius and Pratylenchoides among ten species of Merliniinae plus three Pratylenchoides, and three times within twelve taxa of Telotylenchinae and Trophurinae. Conflicting generic and family nomenclature based on characters such as pharyngeal overlap are discussed in light of current molecular phylogeny. Contrary to some expectations from current taxonomy, Telotylenchus and Tylenchorhynchus cf. robustus did not cluster with three Tylenchorhynchus spp. Two putative species of Neodolichorhynchus failed to group together, and two populations of Scutylenchus quadrifer demonstrated as much or greater genetic distance between them than among three related species of Merlinius.
PMCID: PMC3380519  PMID: 22736870
character analysis; evolutionary convergence; morphology; nomenclature; phylogeny; stunt nematode; systematics; tail; taxonomy; Tylenchorhynchus
2.  Population Dynamics and Dispersal of Aphelenchoides fragariae in Nursery-grown Lantana 
Journal of Nematology  2010;42(4):332-341.
Population dynamics of Aphelenchoides fragariae were assessed over three growing seasons and during overwintering for naturally-infected, container-grown lantana (Latana camara) plants in a North Carolina nursery. During the growing season, the foliar nematode population in symptomatic leaves peaked in July each year then remained above 100 nematodes/g fresh weight into late summer. Foliar nematodes were also detected in asymptomatic and abscised leaves. Results suggest that leaves infected with foliar nematodes first develop symptoms at populations of about 10 nematodes/g. Foliar nematodes were detected in symptomatic and asymptomatic plant leaves and in abscised leaves during overwintering in a polyhouse, but the number of infected plants was low. A steep disease gradient was found for infection of lantana plants by A. fragariae on a nursery pad with sprinkler irrigation. When the canopies of initially healthy plants were touching the canopies of an infected plants, 100% of the plants became infected within 11 wk, but only 5 to 10% became infected at a canopy distance of 30 cm. Overwintering of A. fragariae in infected plants and a steep disease gradient during the growing season suggests strict sanitation and an increase in plant spacing are needed to mitigate losses from this nematode pest.
PMCID: PMC3380520  PMID: 22736867
Aphelenchoides fragariae; detection; dispersal; ecology; foliar nematode; Lantana camara; management; nursery; ornamental crop; population dynamics; Salvia farinaeae
Journal of Nematology  2010;42(4):313-318.
The objective of this work was to isolate and identify fungi associated with R. reniformis in cotton roots. Soil samples were collected in cotton fields naturally infested with R. reniformis and from cotton stock plants cultured in the greenhouse. Nematodes extracted from the soil were observed under the stereoscope, and discolored eggs and vermiform stages colonized with mycelia were cultured on 1.5% water agar supplemented with antibiotics, and incubated at 27°C. Identification of the nematophagous fungi was based on the morphological characters, and the ITS regions and 5.8S rDNA amplified by PCR using the primers ITS1 and ITS4. The parasitism percentage on vermiform nematodes from greenhouse samples was 21.2%, and the percentages from cotton fields in Limestone, Henry, and Baldwin counties in Alabama were 3%, 23.2%, and 5.6%, respectively. A total of 12 fungi were identified from R. reniformis vermiform stages and eggs. The most frequently isolated fungi were Arthrobotrys dactyloides (46%) and Paecilomyces lilacinus (14%), followed by Phoma exigua (4.8%), Penicillium waksmanii and Dactylaria brochophaga (3.6%), Aspergillus glaucus group (2.4%). Cladosporium herbarum, Cladosporium cladiosporioides, Fusarium oxysporum, Torula herbarum, Aspergillus fumigatus, and an unidentified basidiomycete were less frequent (1.2%). A high percentage (16.8%) of fungi from colonized nematodes was not cultivable on our media. Out of those 12 fungi, only four have been previously reported as nematophagous fungi: three isolates of Arthrobotrys dactyloides, and one isolate of Dactylaria brochopaga, Paecilomyces lilacinus, and Fusarium oxysporum. Molecular identification of Arthrobotrys dactyloides and Dactylaria brochopaga was consistent with the morphological identification, placing these two fungi in the new genus Drechslerella as proposed in the new Orbilaceae classification.
PMCID: PMC3380521  PMID: 22736864
Arthrobotrys dactyloides; Dactylaria brochopaga; Paecilomyces lilacinus; reniform nematode; Rotylenchulus reniformis
4.  Evaluation of Amino Acids as Turfgrass Nematicides1 
Journal of Nematology  2010;42(4):292-297.
Laboratory experiments revealed that DL-methionine, sodium methionate, potassium methionate, and methionine hydroxyl analog at rates of 224 and 448 kg amino acid/ha reduced the number of Belonolaimus longicaudatus mixed life-stages and Meloidogyne incognita J2 in soil, whereas L-threonine and lysine were not effective in reducing the number of either nematode. Futhermore, greenhouse experiments demonstrated that DL-methionine, sodium methionate, potassium methionate, and methionine hydroxyl analog were equally effective against B. longicaudatus at rates of 112, 224, and 448 kg amino acid/ha, and the highest rate (448 kg amino acid/ha) of all amino acids was more effective in reducing the number of B. longicaudatus than the lower rate. However, phytotoxicity was observed on creeping bentgrass (Agrostis palustris) treated with 448 kg amino acid/ha of methionine hydroxyl analog and DL methionine. In addition, in one of two field experiments on bermudagrass (Cynodon dactylon × C. transvaalensis) turf percentage green cover was increased and the number of B. longicaudatus was reduced by 224 kg amino acid/ha of DL-methionine and potassium methionate compared to untreated controls in one of two trials.
PMCID: PMC3380522  PMID: 22736861
amino acid; Belonolaimus longicaudatus; bermudagrass; Cynodon; methionine; nematode management; sting nematode; turfgrass
5.  Natural Migration of Rotylenchulus reniformis In a No-Till Cotton System 
Journal of Nematology  2010;42(4):307-312.
Rotylenchulus reniformis is the most damaging nematode pathogen of cotton in Alabama. It is easily introduced into cotton fields via contaminated equipment and, when present, is difficult and costly to control. A trial to monitor the natural migration of R. reniformis from an initial point of origin was established in 2007 and studied over two growing seasons in both irrigated and non-irrigated no-till cotton production systems. Vermiform females, juveniles and males reached a horizontal distance of 200 cm from the initial inoculation point, and a depth of 91 cm in the first season in both systems. Irrigation had no effect on the migration of vermiform females and juveniles, but males migrated faster in the irrigated trial than in the non-irrigated trial. Population density increased steadily in the irrigated trial during both years, exceeding the economic threshold of 1,000 per 150 cm3, but was highly correlated with rainfall in the non-irrigated trial. The average speed of migration ranged from 0- to 3.3-cm per day over 150 days. R. reniformis was able to establish in both the irrigated and non-irrigated trials in one season and to increase population density significantly.
PMCID: PMC3380523  PMID: 22736863
Behavior; cotton; Gossypium hirsutum; host-parasite relationship; movement; no-till; population dynamics; root growth; Rotylenchulus reniformis
6.  Characterization of New Entomopathogenic Nematodes from Thailand: Foraging Behavior and Virulence to the Greater Wax Moth, Galleria mellonella L. (Lepidoptera: Pyralidae) 
Journal of Nematology  2010;42(4):281-291.
Entomopathogenic nematodes (EPNs) in the genera Steinernema and Heterorhabditis and their associated bacteria (Xenorhabdus spp. and Photorhabdus spp., respectively) are lethal parasites of soil dwelling insects. We collected 168 soil samples from five provinces, all located in southern Thailand. Eight strains of EPNs were isolated and identified to species using restriction profiles and sequence analysis. Five of the isolates were identified as Heterorhabditis indica, and one as Heterorhabditis baujardi. Two undescribed Steinernema spp. were also discovered which matched no published sequences and grouped separately from the other DNA restriction profiles. Behavioral tests showed that all Heterorhabditis spp. were cruise foragers, based on their attraction to volatile cues and lack of body-waving and standing behaviors, while the Steinernema isolates were more intermediate in foraging behavior. The infectivity of Thai EPN strains against Galleria mellonella larvae was investigated using sand column bioassays and the LC50 was calculated based on exposures to nematodes in 24-well plates. The LC50 results ranged from 1.99-6.95 IJs/insect. Nine centimeter columns of either sandy loam or sandy clay loam were used to determine the nematodes’ ability to locate and infect subterranean insects in different soil types. The undescribed Steinernema sp. had the greatest infection rate in both soil types compared to the other Thai isolates and three commercial EPNs (Heterorhabditis bacteriophora, Steinernema glaseri and Steinernema riobrave).
PMCID: PMC3380524  PMID: 22736860
Entomopathogenic Nematodes; Foraging Behavior; Galleria mellonella; Heterorhabditis; Steinernema
7.  Effect of Storage Temperature on Soil Nematode Community Structures as Revealed by PCR-DGGE 
Journal of Nematology  2010;42(4):324-331.
The optimal duration and conditions for storage of soils collected for nematode community analyses are unknown. To study this issue, three types of soils with different geographical origins from the subarctic to cool-temperate Japan were kept at three temperature levels (5, 10, and 20°C) for up to 8 wk following collection. During the storage period, nematode population density was measured, and community structure was assessed by polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). No significant changes in the population density or diversity of nematodes (Shannon-Wiener Diversity Index) were observed during storage compared to initial states, except that density in an andosol collected from Tsukuba, Central Japan decreased significantly after 28 d of storage at 5°C. However, a regression analysis showed a declining trend in nematode density in the latter half of the storage period when soils were stored at 5 or 20°C, depending on the geographic origin of the soil. These results indicate that soils can be stored for 14 d at 5–20°C, with 10°C as optimal. This is the first study to experimentally determine the optimal preservation conditions for nematode assemblages in soils that are to be analyzed using PCR-DGGE.
PMCID: PMC3380525  PMID: 22736866
biodiversity; ecology; method; soil fauna
8.  Inheritance of Resistance to Meloidoygne incognita in Primitive Cotton Accessions from Mexico 
Journal of Nematology  2010;42(4):352-358.
Few sources of resistance to root-knot nematodes (Meloidogyne incognita) in upland cotton (Gossypium hirsutum) have been utilized to develop resistant cultivars, making this resistance vulnerable to virulence in the pathogen population. The objectives of this study were to determine the inheritance of resistance in five primitive accessions of G. hirsutum (TX1174, TX1440, TX2076, TX2079, and TX2107) and to determine allelic relations with the genes for resistance in the genotypes Clevewilt-6 (CW) and Wild Mexico Jack Jones (WMJJ). A half-diallel experimental design was used to create 28 populations from crosses among these seven sources of resistance and the susceptible cultivar DeltaPine 90 (DP90). Resistance to M. incognita was measured as eggs per g roots in the parents, F1 and F2 generations of each cross. The resistance in CW and WMJJ was inherited as recessive traits, as reported previously for CW, whereas the resistance in the TX accessions was inherited as a dominant trait. Chi square analysis of segregation of resistance in the F2 was used to estimate the numbers of genes that conditioned resistance. Resistance in CW and WMJJ appeared to be a multigenic trait whereas the resistance in the TX accessions best fit either a one or two gene model. The TX accessions were screened with nine SSR markers linked to resistance loci in other cotton genotypes. The TX accessions lacked the allele amplified by SSR marker CR316 and linked to resistance in CW and other resistant genotypes derived from this source. Four of five TX genotypes lacked the amplification products from the marker BNL1231 that is also associated with the resistant allele on Chromosome 11 in WMJJ, CW, NemX, M120 RNR and Auburn 634 RNR. However, all five TX genotypes produced the same amplification products from three SSR markers linked to the resistant allele on Chromosome 14 in M120 RNR and M240 RNR. The TX accessions have unique resistance genes that are likely to be useful in efforts to develop resistant cotton cultivars with increased durability.
PMCID: PMC3380526  PMID: 22736869
Allelic relationships; cotton; Gossypium hirsutum; host resistance; inheritance of resistance; Meloidogyne incognita; molecular markers; root-knot nematode
9.  Impact of Rotylenchulus reniformis on Cotton Yield as Affected by Soil Texture and Irrigation 
Journal of Nematology  2010;42(4):319-323.
The effects of soil type, irrigation, and population density of Rotylenchulus reniformis on cotton were evaluated in a two-year microplot experiment. Six soil types, Fuquay sand, Norfolk sandy loam, Portsmouth loamy sand, Muck, Cecil sandy loam, and Cecil sandy clay, were arranged in randomized complete blocks with five replications. Each block had numerous plots previously inoculated with R. reniformis and two or more noninoculated microplots per soil type, one half of which were irrigated in each replicate for a total of 240 plots. Greatest cotton lint yields were achieved in the Muck, Norfolk sandy loam, and Portsmouth loamy sand soils. Cotton yield in the Portsmouth loamy sand did not differ from the Muck soil which averaged the greatest lint yield per plot of all soil types. Cotton yield was negatively related to R. reniformis PI (initial population density) in all soil types except for the Cecil sandy clay which had the highest clay content. Supplemental irrigation increased yields in the higher yielding Muck, Norfolk sandy loam, and Portsmouth loamy sand soils compared to the lower yielding Cecil sandy clay, Cecil sandy loam, and Fuquay sand soils. The Portsmouth sandy loam was among the highest yielding soils, and also supported the greatest R. reniformis population density. Cotton lint yield was affected more by R. reniformis Pi with irrigation in the Portsmouth loamy sand soil with a greater influence of Pi on lint yield in irrigated plots than other soils. A significant first degree PI × irrigation interaction for this soil type confirms this observation.
PMCID: PMC3380527  PMID: 22736865
cotton; Gossypium hirsutum; irrigation; microplot; nematode; reniform nematode; Rotylenchulus reniformis; soil texture; soil moisture; volumetric water content; yield loss
10.  Micoletzkya chinaae n. sp. (Nematoda: Diplogastridae), a potential predacious nematode and Ektaphelenchus macrobulbosus (Nematoda: Ektaphelenchinae) isolated from Simao pine in South-western China 
Journal of Nematology  2010;42(4):298-306.
Detailed morphology of a new diplogastrid and a known ektaphelenchid species isolated from Simao pine in south-western China were illustrated and described/redescribed. Micoletzkya chinaae n. sp. is characterized by a relatively short body length (601-802 μm in female and 505-773 μm in male), undivided cheilorhabdia (forming an entire ring), dimorphic buccal cavity (eury- or stenostomatous), a large claw-like dorsal tooth and a large right subventral tooth in the stoma of eurystomatous form, typical diplogastrid pharynx, didelphic female gonads, nine pairs of genital papillae on male tail region with two ventral pairs (GP1 and GP2) closely associated, a unique gubernaculum morphology, and a long filiform tail in both sexes. The new diplogastrid belongs to the Group 1 category of Micoletzkya species sensu Massey, 1966, which is characterized by stoma equipped with a large dorsal and a large subventral tooth, and both teeth can cross near the center of the pharynx. The new species can be easily distinguished from other species within this group except for M. tomea Massey, 1966 with the long filiform female and male tails. However, it shows great similarities to Mononchoides spp., Koerneria spp., Fictor spp., and Acrostichus members in some aspects. More morphological features as well as molecular data of this clade should be available before relationships between and within these genera can be better interpreted. The two large moveable teeth in eurystomatous worms indicate their potentially predacious habits, and re-isolation of this species is necessary. Morphology of south-western Chinese population of Ektaphelenchus macrobulbosus (Rühm,1956) Massey, 1974 conforms well to the previous descriptions except for a few minor variations. It is characterized by medium-long female and male bodies (676-791 and 613-685 μm, respectively), three incisures in the lateral field, offset cephalic region, knobless stylet 18-20 μm long, oblong median bulb with posteriorly situated valves, two to three rows of developing oocytes, short postuterine sac, absence of female rectum and anus, two pairs of subventral papillae on the male tail region, a cucullus (apophysis) present on the dorsal distal end of the spicule, and the conoid female and male tails.
PMCID: PMC3380528  PMID: 22736862
description; redescription; morphology; morphometrics; new species; pine wood nematode; Pinus kesiya var. langbianensis; SEM; taxonomy; Micoletzkya; Ektaphelenchus
11.  Identification and molecular characterization of a β-1,4-endoglucanase gene (Rr-eng-1) from Rotylenchulus reniformis 
Journal of Nematology  2010;42(4):342-351.
β-1,4-endoglucanses, a.k.a. cellulases, are parasitism genes that facilitate root penetration and migration by plant-parasitic nematodes. Rotylenchulus reniformis is a sedentary semi-endoparasite for which little molecular data has been collected. In this report, we describe the isolation and characterization of a predicted glycosyl hydrolase family 5 cellulase from R. reniformis that we have named Rr-eng-1. The Rr-eng-1 cDNA was 1,341 bp long and was comprised of a 19 bp 5′-untranslated region (UTR), a 1,245 bp open reading frame (ORF), and an 80 bp 3′-UTR. The Rr-eng-1 genomic sequence was 2,325 bp. Alignment of the cDNA and genomic sequences revealed seven introns and eight exons for Rr-eng-1. BLASTN analysis showed the Rr-eng-1 cDNA was most homologous to the Hg-eng-6 mRNA from Heterodera glycines. Southern blot analysis indicated that at least three Rr-eng-1-like sequences were present in the R. reniformis genome. Translation of the Rr-eng-1 ORF yielded a 414 amino acid peptide (Rr-ENG-1) having an N-terminal signal sequence for secretion. No cellulose binding module (CBM) was detected in Rr-ENG-1; however, a putative CBM linker sequence N-terminal to the catalytic domain was present. Rr-ENG-1 was most homologous to Hg-ENG-6 but also shared a number of intron splice positions with Mi-ENG-2. Quantitative RT-PCR indicated that Rr-eng-1 was highly expressed in the J2 and adult vermiform life-stages with a sharp decline in expression detected in sedentary females.
PMCID: PMC3380529  PMID: 22736868
cellulase; endoglucanase; reniform nematode; host parasitic relationship; molecular biology; parasitism gene; phylogenetics; Rotylenchulus reniformis
12.  A High-Throughput Automated Technique for Counting Females of Heterodera glycines using a Fluorescence-Based Imaging System 
Journal of Nematology  2010;42(3):201-206.
The soybean cyst nematode (SCN), Heterodera glycines, is the most damaging pathogen of soybean. Methods to phenotype soybean varieties for resistance to SCN are currently very laborious and time consuming. Streamlining a portion of this phenotyping process could increase productivity and accuracy. Here we report an automated method to count SCN females using a fluorescence-based imaging system that is well suited to high-throughput SCN phenotyping methods used in greenhouse screening. For optimal automated imaging, females were washed from roots at 30 days post-inoculation into small Petri dishes. Using a Kodak Image Station 4000MM Pro, the Petri dishes were scanned using excitation and emission wavelengths of 470 nm and 535 nm, respectively. Fluorescent images were captured and analyzed with Carestream Molecular Imaging Software for automated counting. We demonstrate that the automated fluorescent-based imaging system is just as accurate (r2 ≥ 0.95) and more efficient (>50% faster) than manual counting under a microscope. This method can greatly improve the consistency and turnaround of data while reducing the time and labor commitment associated with SCN female counting.
PMCID: PMC3380484  PMID: 22736857
fluorescence; Heterodera glycines; imaging; phenotyping; resistance; SCN; soybean; soybean cyst nematode
Journal of Nematology  2010;42(3):230-279.
PMCID: PMC3380485
14.  Mortality and behavior in Heterodera glycines juveniles following exposure to isothiocyanate compounds 
Journal of Nematology  2010;42(3):194-200.
For this report, we examined the toxic effects of three plant-derived isothiocyanate compounds on second-stage juveniles (J2) of Heterodera glycines. We found significant differences among compounds in the concentration required to affect nematodes, according to mortality and behavioral measurements. The concentrations required to affect behavior were significantly lower than those required for mortality. Both mortality and behavioral measurements were used to investigate whether nematodes in a quiescent state display decreased sensitivity to isothiocyanates compared with actively moving nematodes. Mortality measurements revealed that quiescent nematodes were significantly less sensitive to isothiocyanates than active nematodes. All behavioral measurements following exposure to benzyl- and phenyl isothiocyanate showed significant differences in sensitivity between quiescent and active nematodes. However, significant differences between quiescent and active nematodes were observed in only one of the five behavioral measurements following exposure to allyl isothiocyanate. These results expand the list of plant-derived compounds toxic to H. glycines and illustrate the impact of behavioral quiescence on nematode sensitivity to exogenous toxins.
PMCID: PMC3380486  PMID: 22736856
soybean cyst nematode; behavior; toxicology; biofumigation; quiescence; chemical penetration; uptake; glucosinolate
15.  Secondary structure models of D2-D3 expansion segments of 28S rRNA for Hoplolaiminae species 
Journal of Nematology  2010;42(3):218-229.
The D2-D3 expansion segments of the 28S ribosomal RNA (rRNA) were sequenced and compared to predict secondary structures for Hoplolaiminae species based on free energy minimization and comparative sequence analysis. The free energy based prediction method provides putative stem regions within primary structure and these base pairings in stems were confirmed manually by compensatory base changes among closely and distantly related species. Sequence differences ranged from identical between Hoplolaimus columbus and H. seinhorsti to 20.8% between Scutellonema brachyurum and H. concaudajuvencus. The comparative sequence analysis and energy minimization method yielded 9 stems in the D2 and 6 stems in the D3 which showed complete or partial compensatory base changes. At least 75% of nucleotides in the D2 and 68% of nucleotides in the D3 were related with formation of base pairings to maintain secondary structure. GC contents in stems ranged from 61 to 73% for the D2 and from 64 to 71% for the D3 region. These ranges are higher than G-C contents in loops which ranged from 37 to 48% in the D2 and 33-45% in the D3. In stems, G-C/C-G base pairings were the most common in the D2 and the D3 and also non-canonical base pairs including A•A and U•U, C•U/U•C, and G•A/A•G occurred in stems. The predicted secondary model and new sequence alignment based on predicted secondary structures for the D2 and D3 expansion segments provide useful information to assign positional nucleotide homology and reconstruction of more reliable phylogenetic trees.
PMCID: PMC3380487  PMID: 22736859
28S; D2-D3; Hoplolaiminae; Hoplolaimus; nematode
16.  Molecular and Morphological Characterization and Biological Control Capabilities of a Pasteuria ssp. Parasitizing Rotylenchulus reniformis, the Reniform Nematode 
Journal of Nematology  2010;42(3):207-217.
Rotylenchulus reniformis is one of 10 described species of reniform nematodes and is considered the most economically significant pest within the genus, parasitizing a variety of important agricultural crops. Rotylenchulus reniformis collected from cotton fields in the Southeastern US were observed to have the nematode parasitic bacterium Pasteuria attached to their cuticles. Challenge with a Pasteuria-specific monoclonal antibody in live immuno-fluorescent assay (IFA) confirmed the discovery of Pasteuria infecting R. reniformis. Scanning and transmission electron microscopy were employed to observe endospore ultrastructure and sporogenesis within the host. Pasteuria were observed to infect and complete their life-cycle in juvenile, male and female R. reniformis. Molecular analysis using Pasteuria species-specific and degenerate primers for 16s rRNA and spoII, and subsequent phylogenetic assessment, placed the Pasteuria associated with R. reniformis in a distinct clade within established assemblages for the Pasteuria infecting phytopathogenic nematodes. A global phylogenetic assessment of Pasteuria 16s rDNA using the Neighbor-Joining method resulted in a clear branch with 100% boot-strap support that effectively partitioned the Pasteuria infecting phytopathogenic nematodes from the Pasteuria associated with bacterivorous nematodes. Phylogenetic analysis of the R. reniformis Pasteuria and Pasteuria spp. parasitizing a number of economically important plant parasitic nematodes revealed that Pasteuria with different host specificities are closely related and likely constitute biotypes of the same species. This suggests host preference, and thus effective differentiation and classification are most likely predicated by an influential virulence determinant(s) that has yet to be elucidated. Pasteuria Pr3 endospores produced by in vitro fermentation demonstrated efficacy as a commercial bionematicide to control R. reniformis on cotton in pot tests, when applied as a seed treatment and in a granular formulation. Population control was comparable to a seed-applied nematicide/insecticide (thiodicarb/imidacloprid) at a seed coating application rate of 1.0 x 108 spores/seed.
PMCID: PMC3380488  PMID: 22736858
biological control; cotton; reniform nematode; endospore; Gossypium hirsutum; molecular biology; morphometrics; Pasteuria spp.; phylogenetics; Rotylenchulus reniformis; ultrastructure
17.  Classification of Rotylenchulus reniformis Numbers in Cotton Using Remotely Sensed Hyperspectral Data on Self-Organizing Maps 
Journal of Nematology  2010;42(3):179-193.
Rotylenchulus reniformis is one of the major nematode pests capable of reducing cotton yields by more than 60%, causing estimated losses that may exceed millions of dollars U.S. Therefore, early detection of nematode numbers is necessary to reduce these losses. This study investigates the feasibility of using remotely sensed hyperspectral data (reflectances) of cotton plants affected with different nematode population numbers with self-organizing maps (SOM) in correlating and classifying nematode population numbers extant in a plant's rhizosphere. The hyperspectral reflectances were classified into three classes based on R. renifomis population numbers present in plant's rhizosphere. Hyperspectral data (350-2500 nm) were also sub-divided into Visible, Red Edge + Near Infrared (NIR) and Mid-IR region to determine the sub-region most effective in spectrally classifying the nematode population numbers. Various combinations of different feature extraction and dimensionality reduction methods were applied in different regions to extract reduced sets of features. These features were then classified using a supervised-SOM classification method. Our results suggest that the overall classification accuracies, in general, for most methods in most regions (except visible region) varied from 60% to 80%, thereby, indicating a positive correlation between the nematode numbers present in plant's rhizosphere and the corresponding plant's hyperspectral signatures. Results showed that classification accuracies in the Mid-IR region were comparable to the accuracies obtained in other sub-regions. Finally, based on our findings, the use of remotely-sensed hyperspectral data with SOM could prove to be extremely time efficient in detecting nematode numbers present in the soil.
PMCID: PMC3380489  PMID: 22736855
Classification; cotton; Gossypium hirsutum; nematode; Rotylenchulus reniformis; Self-Organized Maps
18.  Potential of Leguminous Cover Crops in Management of a Mixed Population of Root-knot Nematodes (Meloidogyne spp.) 
Journal of Nematology  2010;42(3):173-178.
Root-knot nematode is an important pest in agricultural production worldwide. Crop rotation is the only management strategy in some production systems, especially for resource poor farmers in developing countries. A series of experiments was conducted in the laboratory with several leguminous cover crops to investigate their potential for managing a mixture of root-knot nematodes (Meloidogyne arenaria, M. incognita, M. javanica). The root-knot nematode mixture failed to multiply on Mucuna pruriens and Crotalaria spectabilis but on Dolichos lablab the population increased more than 2- fold when inoculated with 500 and 1,000 nematodes per plant. There was no root-galling on M. pruriens and C. spectabilis but the gall rating was noted on D. lablab. Greater mortality of juvenile root-knot nematodes occurred when exposed to eluants of roots and leaves of leguminous crops than those of tomato; 48.7% of juveniles died after 72 h exposure to root eluant of C. spectabilis. The leaf eluant of D. lablab was toxic to nematodes but the root eluant was not. Thus, different parts of a botanical contain different active ingredients or different concentrations of the same active ingredient. The numbers of root-knot nematode eggs that hatched in root exudates of M. pruriens and C. spectabilis were significantly lower (20% and 26%) than in distilled water, tomato and P. vulgaris root exudates (83%, 72% and 89%) respectively. Tomato lacks nematotoxic compounds found in M. pruriens and C. spectabilis. Three months after inoculating plants with 1,000 root-knot nematode juveniles the populations in pots with M. pruriens, C. spectabilis and C. retusa had been reduced by approximately 79%, 85% and 86% respectively; compared with an increase of 262% nematodes in pots with Phaseolus vulgaris. There was significant reduction of 90% nematodes in fallow pots with no growing plant. The results from this study demonstrate that some leguminous species contain compounds that either kill root-knot nematodes or interfere with hatching and affect their capacity to invade and develop within their roots. M. pruriens, C. spectabilis and C. retusa could be used with effect to decrease a mixed field populations of root-knot nematodes.
PMCID: PMC3380490  PMID: 22736854
Crotalaria spectabilis; Crotalaria retusa; Dolichos lablab; Mucuna pruriens; Phaseolus vulgaris; nematicidal compounds; phytoalexins
19.  Resistance to Ditylenchus africanus present in peanut breeding lines 
Journal of Nematology  2010;42(2):159-165.
Peanut is an important cash crop both for commercial and small-scale farmers in South Africa. The effect of Ditylenchus africanus on peanut is mainly qualitative, leading to downgrading of consignments. This nematode is difficult to control because of its high reproductive and damage potential. The objective of this study was to identify peanut genotypes with resistance to D. africanus that would also be sustainable under field conditions. Selected peanut genotypes were evaluated against D. africanus in microplot and field trials. The inbred lines PC254K1 and CG7 were confirmed to be resistant to D. africanus. The resistance expressed by these two genotypes was sustainable under field conditions. The breeding line PC287K5 maintained low nematode numbers in some trials, but its level of resistance was not as strong or as sustainable as that of PC254K1 or CG7. However, PC287K5 could still play an important role in the peanut industry where lower D. africanus populations occur.
PMCID: PMC3380467  PMID: 22736852
Arachis hypogaea; Ditylenchus africanus; management; peanut; peanut pod nematode; resistance
20.  Strip-tilled Cover Cropping for Managing Nematodes, Soil Mesoarthropods, and Weeds in a Bitter Melon Agroecosystem 
Journal of Nematology  2010;42(2):111-119.
A field trial was conducted to examine whether strip-tilled cover cropping followed by living mulch practice could suppress root-knot nematode (Meloidogyne incognita) and enhance beneficial nematodes and other soil mesofauna, while suppressing weeds throughout two vegetable cropping seasons. Sunn hemp (SH), Crotalaria juncea, and French marigold (MG), Tagetes patula, were grown for three months, strip-tilled, and bitter melon (Momordica charantia) seedlings were transplanted into the tilled strips; the experiment was conducted twice (Season I and II). Strip-tilled cover cropping with SH prolonged M. incognita suppression in Season I but not in Season II where suppression was counteracted with enhanced crop growth. Sunn hemp also consistently enhanced bacterivorous and fungivorous nematode population densities prior to cash crop planting, prolonged enhancement of the Enrichment Index towards the end of both cash crop cycles, and increased numbers of soil mesoarthropods. Strip-tilled cover cropping of SH followed by clipping of the living mulch as surface mulch also reduced broadleaf weed populations up to 3 to 4 weeks after cash crop planting. However, SH failed to reduce soil disturbance as indicated by the Structure Index. Marigold suppressed M. incognita efficiently when planted immediately following a M. incognita-susceptible crop, but did not enhance beneficial soil mesofauna including free-living nematodes and soil mesoarthropods. Strip-tilled cover cropping of MG reduced broadleaf weed populations prior to cash crop planting in Season II, but this weed suppression did not last beyond the initial cash crop cycle.
PMCID: PMC3380468  PMID: 22736847
Crotalaria juncea; free-living nematodes; living mulch; Meloidogyne incognita; mesoarthropods; Momordica charantia; nematode community analysis; Tagetes patula
21.  Host Status of Endophyte-Infected and Noninfected Tall Fescue Grass to Meloidogyne spp. 
Journal of Nematology  2010;42(2):151-158.
Tall fescue grass cultivars with or without endophytes were evaluated for their susceptibility to Meloidogyne incognita in the greenhouse. Tall fescue cultivars evaluated included, i) wild-type Jesup (E+, ergot-producing endophyte present), ii) endophyte-free Jesup (E-, no endophyte present), iii) Jesup (Max-Q, non-ergot producing endophyte) and iv) Georgia 5 (E+). Peach was included as the control. Peach supported greater (P ≤ 0.05) reproduction of M. incognita than all tall fescue cultivars. Differences in reproduction were not detected among the tall fescue cultivars and all cultivars were rated as either poor or nonhosts for M. incognita. Suppression of M. incognita reproduction was not influenced by endophyte status. In two other greenhouse experiments, host susceptibility of tall fescue grasses to two M. incognita isolates (BY-peach isolate and GA-peach isolate) did not appear to be related to fungal endophyte strain [i.e., Jesup (Max-Q; nontoxic endophyte strain) vs. Bulldog 51 (toxic endophyte strain)]. Host status of tall fescue varied with species of root-knot nematode. Jesup (Max-Q) was rated as a nonhost for M. incognita (BY-peach isolate and GA-peach isolate) and M. hapla, a poor host for M. javanica and a good host for M. arenaria. Bulldog 51 tall fescue was also a good host for M. arenaria and M. javanica, but not M. incognita. Jesup (Max-Q) tall fescue may have potential as a preplant control strategy for M. incognita and M. hapla in southeastern and northeastern United States, respectively.
PMCID: PMC3380469  PMID: 22736851
Endophyte; Festuca arundinacea; host-parasitic relationship; management; Meloidogyne arenaria; Meloidogyne hapla; Meloidogyne incognita; Meloidogyne javanica; resistance; rootknot nematode; Schedonorus arundinaceus; tall fescue grass
22.  A Novel Strain of Steinernema riobrave (Rhabditida: Steinernematidae) Possesses Superior Virulence to Subterranean Termites (Isoptera: Rhinotermitidae) 
Journal of Nematology  2010;42(2):91-95.
Subterranean termites are major global pests of wood structures and wood products. Among the most economically important subterranean termite species in the US are Heterotermes aureus, Reticulitermes flavipes, and Coptotermes formosanus. In prior studies, the entomopathogenic nematode, Steinernema riobrave strain 355, exhibited a high level of virulence to H. aureus compared with other nematode species. However, S. riobrave 355 was reported to be poorly or only moderately virulent to R. flavipes and C. formosanus, respectively. We hypothesized that other strains of S. riobrave may possess a high level of virulence to all three termite species. Under laboratory conditions we compared three novel strains of S. riobrave (3-8b, 7-12, and TP) with the 355 strain for virulence to H. aureus, R. flavipes, and C. formosanus workers. H. aureus was very susceptible to all the S. riobrave strains, and termites in all nematode treatments were dead after 4 d. The TP strain of S. riobrave caused greater mortality in R. flavipes and C. formosanus compared to the other nematode strains. Specifically, the TP strain caused 75% and 91% mortality in R. flavipes and C. formosanus, respectively, which was more than 300% and 70% higher than the mortality caused by other strains. Additional studies are warranted to determine the ability of S. riobrave (TP) to control the targeted termite species under field conditions.
PMCID: PMC3380470  PMID: 22736844
Coptotermes formosanus; entomopathogenic nematode; Heterotermes aureus; Reticulitermes flavipes; Steinernema riobrave; termite; biological control
23.  Vittatidera zeaphila (Nematoda: Heteroderidae), a new genus and species of cyst nematode parasitic on corn (Zea mays) 
Journal of Nematology  2010;42(2):139-150.
A new genus and species of cyst nematode, Vittatidera zeaphila, is described from Tennessee. The new genus is superficially similar to Cactodera but is distinguished from other cyst-forming taxa in having a persistent lateral field in females and cysts, persistent vulval lips covering a circumfenestrate vulva, and subventral gland nuclei of the female contained in a separate small lobe. Infective juveniles (J2) are distinguished from all previously described Cactodera spp. by the short stylet in the second-stage juvenile (14-17 μm); J2 of Cactodera spp. have stylets at least 18 μm long. The new species also is unusual in that the females produce large egg masses. Known hosts are corn and goosegrass. DNA analysis suggests that Vittatidera forms a separate group apart from other cyst-forming genera within Heteroderinae.
PMCID: PMC3380471  PMID: 22736850
cyst nematode; Eleusine indica; goosegrass; maize; molecular analysis; new genus; taxonomy; Vittatidera zeaphila; Zea mays
24.  Lethal Temperature for Pinewood Nematode, Bursaphelenchus xylophilus, in Infested Wood Using Microwave Energy 
Journal of Nematology  2010;42(2):101-110.
To reduce the risks associated with global transport of wood infested with pinewood nematode Bursaphelenchus xylophilus, microwave irradiation was tested at 14 temperatures in replicated wood samples to determine the temperature that would kill 99.9968% of nematodes in a sample of ≥ 100,000 organisms, meeting a level of efficacy of Probit 9. Treatment of these heavily infested wood samples (mean of > 1,000 nematodes/g of sapwood) produced 100% mortality at 56 °C and above, held for 1 min. Because this “brute force” approach to Probit 9 treats individual nematodes as the observational unit regardless of the number of wood samples it takes to treat this number of organisms, we also used a modeling approach. The best fit was to a Probit function, which estimated lethal temperature at 62.2 (95% confidence interval 59.0-70.0) °C. This discrepancy between the observed and predicted temperature to achieve Probit 9 efficacy may have been the result of an inherently limited sample size when predicting the true mean from the total population. The rate of temperature increase in the small wood samples (rise time) did not affect final nematode mortality at 56 °C. In addition, microwave treatment of industrial size, infested wood blocks killed 100% of > 200,000 nematodes at ≥ 56 °C held for 1 min in replicated wood samples. The 3rd-stage juvenile (J3) of the nematode, that is resistant to cold temperatures and desiccation, was abundant in our wood samples and did not show any resistance to microwave treatment. Regression analysis of internal wood temperatures as a function of surface temperature produced a regression equation that could be used with a relatively high degree of accuracy to predict internal wood temperatures, under the conditions of this study. These results provide strong evidence of the ability of microwave treatment to successfully eradicate B. xylophilus in infested wood at or above 56 °C held for 1 min.
PMCID: PMC3380472  PMID: 22736846
Pinewood nematode; quarantine; microwave; dielectric heating; international trade; embargo; eradication; Probit 9; International Standard of Phytosanitary Measures No. 15
25.  Effect of Mowing Cotton Stalks and Preventing Plant Re-Growth on Post-Harvest Reproduction of Meloidogyne incognita 
Journal of Nematology  2010;42(2):96-100.
The southern root-knot nematode (Meloidogyne incognita) is a major parasite of cotton in the U.S., and management tactics for this nematode attempt to minimize population levels. We compared three post-harvest practices for their ability to reduce nematode population levels in the field, thereby reducing initial nematode population for the next year's crop. The three practices tested were: 1) chemical defoliation before harvest plus cutting cotton stalks after harvest, 2) chemical defoliation plus applying a herbicide to kill plants prior to cutting the stalks, and 3) chemical defoliation without cutting stalks. Experiments were conducted in both the greenhouse and in the field. The greenhouse experiments demonstrated that M. incognita reproduction (measured as egg counts and root gall rating indices) was significantly greater when stalks were not cut. Cutting stalks plus applying herbicide to kill cotton roots did not significantly reduce nematode reproduction compared to cutting stalks alone. In field experiments, cutting stalks reduced egg populations and root galling compared to defoliation without stalk cutting. In a greenhouse bioassay which used soil from the field plots, plants grown in soil from the defoliation only treatment had greater root gall ratings and egg counts than in the stalk cutting plus herbicide treatment. Therefore, we conclude that cutting cotton stalks immediately after harvest effectively reduces M. incognita reproduction, and may lead to a lower initial population density of this nematode in the following year.
PMCID: PMC3380473  PMID: 22736845
Cotton; cultural control; defoliation; Gossypium hirsutum; herbicide; Meloidogyne incognita; nematode management; post-harvest; reproduction; roots; southern root-knot nematode

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