A Bioassay to Estimate Root Penetration by Nematodes

An in vitro bioassay with a 96-well microtiter plate was used to study the effect of lectins on burrowing nematode penetration of citrus roots. In each well, one 4-mm root segment, excised from the zone of elongation of rough lemon roots, was buried in 0.88 g dry sand. Addition of a Radopholus citrophilus suspension containing ca. 300 nematodes in 50 μ1 test solution completely moistened the sand in each well. The technique assured uniform treatment concentration throughout the medium. Within 16-24 hours, burrowing nematodes penetrated citrus root pieces, primarily through the cut ends. The lectins (100 μg/ml) Concanavalin A (Con A), soybean agglutinin (SBA), wheat germ agglutinin (WGA), and Lotus tetragonolobus agglutinin (LOT) stimulated an increase in penetration of citrus root segments by Radopholus citrophilus. Concentrations as low as 12.5 μg/ml Con A, LOT, and WGA stimulated burrowing nematode penetration of citrus roots. Heat denaturation of the lectins reversed their effect on penetration; however, incubation of nematodes in lectin (25 μg/ml) with 25 mM competitive sugars did not. The reason for enhanced penetration associated with lectins is unclear.     

Spatial Pattern Analysis of Plant-Parasitic Nematodes

Spatial patterns of Meloidogyne incognita, Tylenchorhynchus claytoni, Helicotylenchus dihystera, and Criconemella ornata were analyzed using Hill''s two-term local quadrat variance method (TTLQV), spectral analysis, and spatial correlation. Data were collected according to a systematic grid sampling plan from seven tobacco fields in North Carolina. Different estimates of nematode cluster size were obtained through TTLQV and spectral analysis. No relationship was observed between either estimate and nematode species, time of sampling (spring vs. fall), or mean density. Cluster size estimates obtained from spectral analysis depended on sampling block size. For each species examined, spatial correlations among nematode population densities were greater within plant rows than across rows, indicating that clusters were ellipsoidal with long axes oriented along plant rows. Analysis of mean square errors indicated that significant gains in sampling efficiency resulted from orienting the long axis of sampling blocks across plant rows. Spatial correlation was greater in the fall than in spring and was greater among 1 × 1-m quadrats than among 3 × 3-m quadrats.     

Artificial Feeding Systems for Plant-Parasitic Nematodes

Feeding of an "obligate" plant-parasitic nematode (nonfungal feeder), Pratylenchus scribneri, in the absence of plant tissue was demonstrated in an artificial system consisting of liquid media and indicator dyes including amaranth and various nontoxic food colors. Among the compounds tested, sucrose, dextrose, Gamborg''s B5 medium, and DL-methionine stimulated a small percentage of feeding (12-36%). A high percentage of feeding (90-100%) occurred in a filtrate from excised corn roots cultured in Gamborg''s B5 medium. This feeding system has the potential to develop an artificial medium for plant-parasitic nematodes and to screen novel nematicides that are stomach poisons.     

Antagonists of Plant-parasitic Nematodes in Florida Citrus

In a survey of antagonists of nematodes in 27 citrus groves, each with a history of Tylenchulus semipenetrans infestation, and 17 noncitrus habitats in Florida, approximately 24 species of microbial antagonists capable of attacking vermiform stages of Radopholus citrophilus were recovered. Eleven of these microbes and a species of Pasteuria also were observed attacking vermiform stages of T. semipenetrans. Verticillium chlamydosporium, Paecilomyces lilacinus, P. marquandii, Streptomyces sp., Arthrobotrys oligospora, and Dactylella ellipsospora were found infecting T. semipenetrans egg masses. Two species of nematophagous amoebae, five species of predatory nematodes, and 29 species of nematophagous arthropods also were detected. Nematode-trapping fungi and nematophagous arthropods were common inhabitants of citrus groves with a history of citrus nematode infestation; however, obligate parasites of nematodes were rare.     

Competition between the Plant-parasitic Nematodes Pratylenchus neglectus and Meloidogyne chitwoodi

In experiments on competition between Pratylenchus neglectus and Meloidogyne chitwoodi in barley, the species that parasitized the roots first inhibited penetration by the latter species. Prior presence of P. neglectus impeded the development of M. chitwoodi. Pratylenchus neglectus reduced egg production, final population levels, and reproductive index of M. chitwoodi. The reduction was linearly related to initial population densities of P. neglectus. Initial population densities of M. chitwoodi had no effect on final population levels of P. neglectus. Carbon assimilation by barley plants was reduced when either nematode species was present alone, but not when both were present together. Both nematode species assimilated lower amounts of carbon when present together than when present alone. A split-root experiment demonstrated that translocatable chemicals were not involved in the competition between the two species.     

Field Application of Entomopathogenic Nematodes for Control of Delia radicum in Collards

Control of Delia radicum (cabbage maggot) in field collards (Brassica oleracea) was compared after one or two applications of entomopathogenic nematodes, Steinernema carpocapsae (All strain) and Heterorhabditis bacterophora (HP88 strain), a single application of granular chlorpyrifos, and a water-only treatment. Nematodes were applied with a sprayer during the egg stage of first-generation D. radicum, and chlorpyrifos was hand placed around collard stems during the same period. A second nematode application was made 10 days later. Chlorpyrifos treatment resulted in fewer puparia per plant, less root damage and higher yield than all other treatments, including the control. Collard yield from nematode-treated beds did not differ from controls. These data indicate that, under these field conditions, the species or strains of entomopathogenic nematodes tested did not reduce the number of active cabbage maggots, nor did they prevent collard root damage.     

Interactions between Nematodes and Earthworms: Enhanced Dispersal of Steinernema carpocapsae

Dispersal of the nematode Steinernema carpocapsae (All strain), applied on the top or the bottom of soil columns, was tested in the presence or absence of two earthworm species, Lumbricus terrestris or Aporrectodea trapezoides. Nematode dispersal was estimated after a 2-week period with a bioassay against the greater wax moth, Galleria mellonella. Vertical dispersal of nematodes was increased in the presence of earthworms. When nematodes were placed on the surface of soil columns, significantly more nematodes dispersed to the lower half of the columns when either earthworm species was present than when earthworms were not present. When nematodes were placed on the bottom of soil columns, significantly more nematodes dispersed to the upper half of the columns when L. terrestris was present than when A. trapezoides was present or in the absence of earthworms. Because nematodes were found on the exterior and in the interior of earthworms, nematode dispersal may be enhanced by direct contact with the earthworms.     

Efficiency of Extraction of Nematodes by Flotation-Sieving Using Molasses and Sugar and by Elutriation

Blackstrap molasses was studied as an economical substitute for sucrose in the preparation of an extracting solution for removal of nematodes from soil by the flotation-sieving technique. The maximal number of nematodes extracted from soil was obtained with molasses solutions with specific gravity values in the range 1.000-1.073. Results of studies on the relation between size of soil sample and the amount of extracting solution are presented. In paired comparisons, a molasses solution with sp. gr. = 1.100 at 27 C extracted greater numbers of plant parasitic, dorylaimoid, mononchoid, and other soil nematodes than did the standard 1.0 M sucrose solution (sp. gr. = 1.100); the superiority of the molasses solution is attributed to its higher viscosity. The molasses method also was superior or equal in efficiency to the elutriation technique.     

Use of Low Temperature Scanning Electron Microscopy to Observe Frozen Hydrated Specimens of Nematodes

Frozen hydrated specimens of Pratylenchus agilis and dauer larvae of Steinernema carpocapsae were observed with low-temperature field emission scanning electron microscopy. This new technique provides information about the surface features of nematodes and also allows specimens to be fractured to reveal their internal structure. Furthermore, both halves of fractured specimens can be retained, examined, and photographed either as two-dimensional micrographs or as three-dimensional images for stereo observation (stereology) or quantitative measurements (stereometry). This technique avoids artifacts normally associated with procedures required to prepare nematodes for examination in the transmission and scanning electron microscopes, such as chemical fixation, dehydration, and sectioning or critical point drying.     

Identification of Entomopathogenic Nematodes in the Steinernematidae and Heterorhabditidae (Nemata: Rhabditida)

This paper contains taxonomic keys for the identification of species of the genera Steinernema and Heterorhabditis. Morphometrics of certain life stages are presented in data tables so that the morphometrics of species identified using the keys can be checked in the tables. Additionally, SEM photographs and diagnoses of the families and genera of Steinernematidae and Heterorhabditidae are presented.     

Spatial Patterns of Entomopathogenic Nematodes in Microcosms: Implications for Laboratory Experiments

Laboratory microcosms were used to: i) measure the effects of soil moisture on survival of Steinernema riobravis and ii) investigate the suitability of using microcosms to study motility and survival of these nematodes. Nematodes recovered from soil contained in petri dishes declined by more than 95% during 7 days, whereas nematodes recovered from the inner surfaces of dishes increased 35-fold. After 7 days in dishes, >20 times as many nematodes were recovered from dish surfaces than from soil. Nematodes exhibited a negative geotropism; greater numbers of nematodes were recovered from the lid surfaces than from the surfaces of dishes. Survivorship of nematodes in soil in plastic centrifuge tubes was somewhat greater than in petri dishes, and fewer nematodes ascended above the soil line in tubes than dishes. Downward migration of nematodes was inversely related to soil column diameter, possibly due to relatively unimpeded movement along container surfaces. An assay was developed by which nematodes were rinsed from the inner surfaces of centrifuge tubes into the soil. The resulting slurry was then processed on Baermann trays to recover motile nematodes. Nematode survival in soil in centrifuge tubes was higher at soil moistures between 2-4% than at lower (0.5-1.0%) and higher (4.0-12.0%) moisture levels. Survival of S. riobravis may be enhanced by quiescence induced by moisture deficits.     

The rDNA Internal Transcribed Spacer Region as a Taxonomic Marker for Nematodes

The ITS region from a wide taxonomic range of nematodes, including secernentean and adenophorean taxa, and free-living, entomopathogenic, and plant-parasitic species, was evaluated as a taxonomic marker. Size of the amplified product aided in the initial determination of group membership, and also suggested groups that may require taxonomic reevaluation. Congeneric species often displayed identically sized ITS regions, but genera such as Pratylenchus and Tylenchorhynchus had species with large differences in size. ITS heterogeneity in individuals and populations was identified in several nematode taxa. PCR-RFLP of ITS1 is advocated as a method of taxonomic analysis in genera such as Helicotylenchus that contain numerous species with few diagnostic morphological characteristics.     

Identification of Cyst Nematodes of Agronomic and Regulatory Concern with PCR-RFLP of ITS1

The first internally transcribed spacer region (ITS1) from cyst nematode species (Heteroderidae) was compared by nucleotide sequencing and PCR-RFLP. European, Asian, and North American isolates of five heterodefid species were examined to assess intraspecific variation. PCR-RFLP patterns of amplified ITS1 DNA from pea cyst nematode, Heterodera goettingiana, from Northern Ireland were identical with patterns from Washington State. Sequencing demonstrated that ITS1 heterogeneity existed within individuals and between isolates, but did not result in different restriction patterns. Three Indian and two U.S. isolates of the corn cyst nematode, Heterodera zeae, were compared. Sequencing detected variation among ITS1 clones from the same individual, between individuals, and between isolates. PCR-RFLP detected several restriction site differences between Indian and U.S. isolates. The basis for the restriction site differences between isolates from India and the U.S. appeared to be the result of additional, variant ITS1 regions amplified from the U.S. isolates, which were not found in the three India isolates. PCR-RFLP from individuals of the U.S. isolates created a composite pattern derived from several ITS1 types. A second primer set was specifically designed to permit discrimination between soybean (H. glycines) and sugar beet (H. schachtii) cyst nematodes. Fok I digestion of amplified product from soybean cyst nematode isolates displayed a uniform pattern, readily discernible from the pattern of sugar beet and clover cyst nematode (H. trifolii).     

Distribution of Field Corn Roots and Parasitic Nematodes in Subsoiled and Nonsubsoiled Soil

A field trial was conducted for 2 years in an Arredondo fine sand containing a tillage pan at 15-20 cm deep to determine the influence of subsoiling on the distribution of corn roots and plant-parasitic nematodes. Soil samples were taken at various depths and row positions at 30, 60, and 90 days after planting in field corn subsoiled under the row with two chisels and in non-subsoiled corn. At 30 and 60 days, in-row nematode population densities to 60 cm deep were not affected by subsoiling compared with population densities in nonsubsoiled plots. After 90 days, subsoiling had not affected total root length or root weight at the 20 depth-row position sampling combinations, but population densities of Meloidogyne incognita and Criconemella spp. had increased in subsoiled corn. Numbers of Pratylenchus zeae were not affected. Subsoiling generally resulted in a change in distribution of corn roots and nematodes in the soil profile but caused little total increase in either roots or numbers of nematodes. Corn yield was increased by subsoiling.     

Sequential Decision Rules for Managing Nematodes with Crop Rotations

A dynamic model of nematode populations under a crop rotation that includes both host and nonhost crops is developed and used to conceptualize the problem of economic control. The steady state of the dynamic system is used to devise an approximately optimal decision policy, which is then applied to cyst nematode (Heterodera schachtii) control in a rotation of sugarbeet with nonhost crops. Long-run economic returns from this approximately optimal decision rule are compared with results from solution of the exact dynamic optimization model. The simple decision rule based on the steady state provides long-run average returns that are similar to the fully optimal solution. For sugarbeet and H. schachtii, the simplified rule can be calculated by maximizing a relatively simple algebraic expression with respect to the number of years in the sequence of nonhost crops. Maximization is easy because only integers are of interest and the number of years in nonhost crops is typically small. Solution of this problem indirectly yields an approximation to the optimal dynamic economic threshold density of nematodes in the soil. The decision rule requires knowledge of annual nematode population change under host and nonhost crops, and the relationship between crop yield and nematode population density.     

Digital holographic microtomography for high‐resolution refractive index mapping of live cells

Quantification of three‐dimensional (3D) refractive index (RI) with sub‐cellular resolution is achieved by digital holographic microtomography (DHμT) using quantitative phase images measured at multiple illumination angles. The DHμT system achieves sensitive and fast phase measurements based on iterative phase extraction algorithm and asynchronous phase shifting interferometry without any phase monitoring or active control mechanism. A reconstruction algorithm, optical diffraction tomography with projection on convex sets and total variation minimization, is implemented to substantially reduce the number of angular scattered fields needed for reconstruction without sacrificing the accuracy and quality of the reconstructed 3D RI distribution. Tomogram of a living CA9‐22 cell is presented to demonstrate the performance of the method. Further, a statistical analysis of the average RI of the nucleoli, the nucleus excluding the nucleoli and the cytoplasm of twenty CA9‐22 cells is performed. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)