Steinernema khuongi n. sp. (Panagrolaimomorpha, Steinernematidae), a new entomopathogenic nematode species from Florida, USA.

In this study, molecular (ribosomal sequence data), morphological and cross-hybridization properties were used to identify a new Steinernema sp. from Florida, USA. Molecular and morphological data provided evidence for placing the novel species into Clade V, or the 'glaseri-group' of Steinernema spp. Within this clade, analysis of sequence data of the rDNA genes, 28S and internal transcribed spacer (ITS), depicted the novel species as a distinctive entity and closely related to S. glaseri and S. cubanum. Additionally, cross-hybridization assays showed that the new species is unable to interbreed with either of the latter two species, reinforcing its uniqueness from a biological species concept standpoint. Key morphological diagnostic characters for S. khuongi n. sp. include the mean morphometric features of the third-stage infective juveniles: total body length (average: 1066 µm), tail length (average: 65 µm), location of the excretory pore (average: 80.5 µm) and the values of c (average: 16.4), D% (average: 60.5), E% (average: 126) and H% (average: 46.6). Additionally, males can be differentiated from S. glaseri and S. cubanum by the values of several ratios: D% (average: 68), E% (average: 323) and SW% (average: 120). The natural distribution of this species in Florida encompasses both natural areas and citrus groves, primarily in shallow groundwater ecoregions designated as 'flatwoods'. The morphological, molecular, phylogenetic and ecological data associated with this nematode support its identity as a new species in the S. glaseri-group.

Grower acceptance of entomopathogenic nematodes: case studies on three continents.

Projects to manage arthropod pests using entomopathogenic nematodes (EPNs) in Brazil, Korea and USA are reviewed to identify conditions and practices that affected the use of EPNs for pest management. A proliferation of covered agriculture in Korea, the growth in demand for high value, pesticide-free produce in Korea and Brazil, and the cost-effectiveness of EPNs created favorable conditions for the widespread adoption of EPN products in Brazilian guava orchards and Korean vegetable greenhouses. In Florida, EPNs imported from South America function successfully as classical biocontrol agents against invasive mole crickets attacking pasture and turf. However, the low value of pasture and the availability of cost-effective chemical insecticides in turf have depressed the demand for EPN products to control mole crickets. In Florida citrus orchards, a recent, dramatic increase in the use of chemical insecticides to control an arthropod vector of a devastating bacterial disease of citrus (huanglongbing) reduced the demand for EPN products to control Diaprepes root weevils. Nevertheless, a rich and diverse EPN fauna in the Florida peninsula provides significant control of subterranean stages of root weevils in some habitats, and is the focus of research to develop cultural practices that exploit the potential for increased pest management through EPN conservation.

From Augmentation to Conservation of Entomopathogenic Nematodes: Trophic Cascades, Habitat Manipulation and Enhanced Biological Control of Diaprepes abbreviatus Root Weevils in Florida Citrus Groves.

The use of entomopathogenic nematodes (EPN) for management of the root weevil, Diaprepes abbreviatus, in Florida citrus groves is considered a biological control success story and typically involves augmentation in which EPN are applied inundatively as biopesticides to quickly kill the pest. However, recent evidence indicates that efficacy of EPN applications in Florida citrus depends on soil type. They are very effective in the well drained coarse sands of the Central Ridge but often less so in poorly drained fine-textured soils of the Flatwoods. Moreover, groves on the Central Ridge can harbor rich communities of endemic EPN that might often suppress weevil populations below economic thresholds, whereas Flatwoods groves tend to have few endemic EPN and frequent weevil problems. Current research is examining the ecological dynamics of EPN in Florida citrus groves, the potential impact of EPN augmentation on soil food webs, especially endemic EPN, and whether habitat manipulation and inoculation strategies might be effective for conserving and enhancing EPN communities to achieve long-term control in problem areas. Conservation biological control could extend the usefulness of EPN in Florida citrus and be especially appropriate for groves with persistent weevil problems.

Augmenting Entomopathogenic Nematodes in Soil from a Florida CitrusOrchard: Non-Target Effects of a Trophic Cascade.

Laboratory experiments were conducted to study non-target effects of augmenting entomopathogenic nematode (EPN)communities in soil. When raw soil from a citrus orchard was augmented with either 2,000 Steinernema riobrave or S. diaprepesi, fewer EPN (P

Food web responses to augmenting the entomopathogenic nematodes in bare and animal manure-mulched soil.

Factorial treatments of entomopathogenic nematodes (EPN) and composted, manure mulches were evaluated for two years in a central Florida citrus orchard to study the post-application biology of EPN used to manage the root weevil, Diaprepes abbreviatus. Mulch treatments were applied once each year to study the effects of altering the community of EPN competitors (free-living bactivorous nematodes) and antagonists (nematophagous fungi (NF), predaceous nematodes and some microarthro-pods). EPN were augmented once with Steinernema riobrave in 2004 and twice in 2005. Adding EPN to soil affected the prevalence of organisms at several trophic levels, but the effects were often ephemeral and sometimes inconsistent. EPN augmentation always increased the mortality of sentinel weevil larvae, the prevalence of free-living nematodes in sentinel cadavers and the prevalence of trapping NF. Subsequent to the insecticidal effects of EPN augmentation in 2004, but not 2005, EPN became temporarily less prevalent, and fewer sentinel weevil larvae died in EPN-augmented compared to non-augmented plots. Manure mulch had variable effects on endoparasitic NF, but consistently decreased the prevalence of trapping NF and increased the prevalence of EPN and the sentinel mortality. Both temporal and spatial abundance of NF were inversely related to the prevalence of Steinernema diaprepesi, whereas Heterorhabditis zealandica prevalence was positively correlated with NF over time. The number of weevil larvae killed by EPN was likely greatest in 2005, due in part to non-target effects of augmentation on the endemic EPN community in 2004 that occurred during a period of peak weevil recruitment into the soil.

Immunocytological detection of residual marrow disease at clinical remission predicts metastatic relapse in small cell lung cancer.

A panel of monoclonal antibodies against neural and epithelial associated antigens was used to examine bone marrow from patients in clinical remission from small cell lung cancer (SCLC). A standard peroxidase-antiperoxidase technique and Ficoll-Hypaque enrichment were used to detect SCLC-like cells at the 1-2% level of contamination in 8 of 12 patients who were disease free by conventional criteria, including routine marrow cytology and histology and endobronchoscopic biopsy or cytology. Six of these patients ultimately relapsed, with metastatic sites found between 2 and 6 months after restaging. Furthermore, 6 patients had undergone chemointensification including autologous marrow rescue with radical irradiation to the primary lung tumor. Four of these 6 subsequently relapsed, also with metastatic sites. Of the 4 patients without bone marrow metastases at restaging using this technique, 2 relapsed, with cells found at the primary site, and 2 remained in complete remission. Serum free cell culture was attempted in 9 of 12 cases and SCLC-like cell colonies grew, in suspension, in 4. The SCLC-like nature of these cells has been confirmed by electron microscopy in 1 case and by repeat immunocytochemistry for small cell associated antigens in 3 cases. Bone marrow positivity using these techniques appears to predict a high risk of metastatic relapse regardless of further therapy.

Bionomics of a Phoretic Association Between Paenibacillus sp. and the Entomopathogenic Nematode Steinernema diaprepesi.

Spores of an unidentified bacterium were discovered adhering to cuticles of third-stage infective juvenile (IJ) Steinernema diaprepesi endemic in a central Florida citrus orchard. The spores were cup-shaped, 5 to 6 mm in length, and contained a central endospore. Based on 16S rDNA gene sequencing, the bacterium is closely related to the insect pathogens Paenibacillus popilliae and P. lentimorbus. However, unlike the latter bacteria, the Paenibacillus sp. is non-fastidious and grew readily on several standard media. The bacterium did not attach to cuticles of several entomopathogenic or plant-parasitic nematodes tested, suggesting host specificity to S. diaprepesi. Attachment of Paenibacillus sp. to the third-stage cuticle of S. diaprepesi differed from Paenibacillus spp. associated with heterorhabditid entomopathogenic nematodes, which attach to the IJ sheath (second-stage cuticle). The inability to detect endospores within the body of S. diaprepesi indicates that the bacterial association with the nematode is phoretic. The Paenibacillus sp. showed limited virulence to Diaprepes abbreviatus, requiring inoculation of larvae with 108 spores to achieve death of the insect and reproduction of the bacterium. The effect of the bacterium on the nematode population biology was studied in 25-cm-long vertical sand columns. A single D. abbreviatus larva was confined below 15-cm depth, and the soil surface was inoculated with either spore-free or spore-encumbered IJ nematodes. After 7 days, the proportion of IJ below 5-cm depth was seven-fold greater for spore-free IJ than for spore-encumbered nematodes. Mortality of D. abbreviatus larvae was 72% greater (P <= 0.01) for spore-free compared to spore-encumbered S. diaprepesi. More than 5 times as many progeny IJs (P <= 0.01) were produced by spore-free compared to spore-encumbered nematodes. These data suggest that the bacterium is a component of the D. abbreviatus food web with some potential to regulate a natural enemy of the insect.

Changes in arginine, PAL activity, and nematode behavior in salinity-stressed citrus.

De novo arginine biosynthesis has been described as a response of citrus to a range of stresses. It is often noted that stress in plants enhances susceptibility to herbivory and pathogenic attack. Using a citrus and nematode (Tylenchulus semipenetrans) system, the effects of salinity stress on nematode behavior, amino acids (particularly arginine), and phenylalanine ammonia lyase (PAL) activity was investigated. The hypothesis was tested that under salinity stress, citrus grows more slowly and produces arginine in response to high levels of in vivo ammonia, resulting in lower PAL activity and increased susceptibility to nematode attack. After 30 days of high salinity (0.1 M NaCl), plants exhibited a 38% reduction in growth, 35% reduction in PAL activity, and had 54% higher infection rates. PAL activity was inversely correlated (P < or = 0.05) with salinity level and with increase in arginine concentration.

Abundance of Diaprepes abbreviatus (L.) (Coleoptera: Curculionidae) neonates falling to the soil under tree canopies in Florida citrus groves.

The purpose of these experiments was to estimate the number and distribution of Diaprepes abbreviatus (L.) neonate larvae dropping from the canopy of infested citrus trees. The number of neonates was monitored in the field using passive funnel traps in two simultaneous experiments and a separate experiment for an additional year. In one experiment, traps were placed from trunk to dripline in the cardinal directions under each of five trees (132 traps total). In a second experiment, eight traps were placed under each tree in the cardinal directions, one trap 30 cm from the trunk and one trap 30 cm from the dripline/direction for 25 trees (200 traps total). Larvae were collected weekly for 50 wk in conical tubes containing ethylene glycol as a preservative. Traps closer to the tree trunk captured more larvae than traps nearer the dripline. The area under the tree canopy was positively correlated with the total estimated number of larvae captured per tree. The estimated number of total larvae/tree over the course of our experiments ranged from 955 to 7,290. The highest number of neonate larvae observed in 1 wk was 67 +/- 6/m2. There was an inverse relationship between the number of traps beneath a tree and the number of trees that needed to be sampled to estimate mean population density with a given precision. However, there was a direct relationship between number of traps/tree and the total number of traps needed for a given precision. This passive technique could be used to quantify the destructive larval stage and to assess D. abbreviatus management strategies.

Effect of Fenamiphos Placement on Tylenchulus semipenetrans and Yield in a Florida Citrus Orchard.

Grapefruit trees on sour orange rootstock on the east coast of Florida were treated with 22.3 kg a.i./ha fenamiphos (broadcast equivalent) in 1.52-m bands extending from the dripline to beneath the canopy, in 1.52-m bands extending from the dripline toward the row middle, or left as untreated controls. During the course of the experiment, mean density of feeder roots and Tylenchulus semipenetrans in control plots was 3.8 and 5.8 times greater, respectively, in samples from beneath tree canopies than from 90 cm beyond the dripline on the top of beds. Population densities of T. semipenetrans in each zone (under canopy vs. row middle) were smallest when treatment occurred in that zone. Nematode levels beneath the canopy were 11% of control levels 19 weeks following treatment when fenamiphos was applied beneath the canopy and 52% of controls when treated at the dripline. The number of female T. semipenetrans per gram root weight was also reduced under the canopy by the under-canopy treatment. Fruit yield 5 months following nematicide application was not affected by treatment. Percentage change in yield between that harvest and a harvest 16 months after treatment was + 17% in the under-canopy treatment, - 1% in the dripline treatment, and - 17% in the untreated controls.

Responses of Tylenchulus semipenetrans to Citrus Fruit Removal: Implications for Carbohydrate Competition.

Sixteen mature Valencia orange trees on rough lemon rootstock were selected on the basis of approximately equal, naturally occurring populations of Tylenchulus semipenetrans in soil. In March, fruit 1 cm in diameter or less were removed from eight of the trees, which were kept free of fruit for 15 months. In July, 4 months after fruit removal, fibrous root (<2 mm d) mass density of defruited trees was 51% greater and insoluble starch in fibrous roots was 24% less than on control trees with fruit. Female T. semipenetrans per gram of root were 64% more numerous on roots of control trees than on defruited trees at this time. Numbers of female nematodes per tree and of juveniles and males in soil did not differ between treatments 4 months after fruit removal. Root mass density remained higher on defruited than control trees for the remaining 13 months that the trees were studied, while nematode density in soil beneath defruited trees rapidly increased to levels proportionate to the additional root mass density. Nine months after fruit removal (December), starch concentration was 84% higher in roots of defruited trees compared to controls and remained 28% higher than in controls 15 months (May) following fruit removal. Between months 9 and 15 following fruit removal, nematode density in soil beneath defruited trees increased at a rate five times that of nematode density beneath control trees. In May, female fecundity (eggs/female) on defruited trees was 41% greater than on control trees. The data were consistent with the hypothesis that carbohydrate competition between developing citrus fruit and T. semipenetrans influences seasonal fluctuations in nematode population densities.

Hydraulic Lift Increases Herbivory by Diaprepes abbreviatus Larvae and Persistence of Steinernema riobrave in Dry Soil.

Citrus seedlings were grown in double pots that separated the root systems into discrete lower and upper zones to test the hypothesis that hydraulic lift affects persistence and efficacy of entomopathogenic nematodes. Three treatments were established: (i) both pots were irrigated at water potential

Validation of a model for prediction of host damage by two nematode species.

PLANT ROOTS WERE MECHANICALLY INJURED OR SUBJECTED TO NEMATODE PARASITISM TO TEST THE MODEL OF HOST DAMAGE BY TWO NEMATODE SPECIES: y = m' + (l - m')c'z(P)z(P) for y 1.0, where m' = m + (m - m) (1 - y)/[(1 - y) + (l - y)] and c' = (z(-T) + z(-T))/2. Damage functions for greenhouse-grown radish plants (cv. Cherry Belle) mechanically injured with small or large steel needles were used to predict growth of plants injured by both needles. Growth predictions accounted for 94%, 87%, and 82% of mean treatment variation in plant height, stem weight, and root weight, respectively. Cowpea (cv. California Blackeye No. 5) damage functions, based on preplant population levels of Meloidogyne incognita and M. javanica, were used to predict seed yield of plants concomitantly infected with various levels of each species. Single species damage functions and population growth curves indicated significant host resistance to M. incognita and significantly lower virulence of that species compared to M. javanica. Model predictions accounted for 88% of mean seed yield variation in two-species treatments. In a separate experiment, mean top weights of 30-day-old cowpea plants, nniformly inoculated with 20,000 M. javanica eggs, increased with increasing levels of concomitantly inoculated M. incognita eggs. It is speculated that competitive interactions between M. incognita and M. javanica mitigated host damage by the more virulent species.

Damage Potential and Reproduction of Belonolaimus longicaudatus and Hoplolaimus galeatus on Alyceclover.

Alyceclover (Alysicarpus spp.) is an annual, high-quality leguminous forage, suitable for production under tropical and subtropical climates where the husbandry of conventional leguminous forages, Trifolium spp., is uneconomical. The damage potential and reproduction of Belonolairaus longicaudatus and Hoplolaimus galeatus on alyceclover were studied under greenhouse conditions, using sand and sandy clay loam soil materials, respectively. Both nematode species reproduced on alyceclover, but only B. longicaudatus was pathogenic. Symptoms of B. longicaudatus damage were suppression of shoot yield, limited root system, stunting, incipient wilting, and occasional seedling mortality. In one experiment, the threshold-damage density was three nematodes/100 cm(3) sand, whereas in the other experiment it was zero nematodes.

Population Changes of Tylenchulus semipenetrans Under Localized Versus Uniform Drought in the Citrus Root Zone.

Population development of Tylenchulus semipenetrans in dry soil was investigated in a greenhouse study. Citrus seedlings were grown in sandy soil in vertical tubes with upper and lower sections. Nematode population densities in the upper tubes were measured at 16, 23, and 37 days, post-treatment. Three treatments consisted of i) irrigating both tubes when soil water potential reached -1 5 kPa (non-drought), ii) irrigating only the bottom tube (local drought), and iii) no irrigation (uniform drought). Soil water potential in the upper tubes did not differ under local and uniform drought during the first 16 days post-treatment, when it approached - 125 kPa. Thereafter, the water potential of soil under uniform drought continued to decrease, while that under local drought stabilized at approximately -150 kPa. Treatments had no consistent effects on female T. semipenetrans counts from soil or roots. However, after 37 days, numbers of eggs, juvenile, and male nematodes per gram of root under local drought were more than 2.4-fold greater than those under non-drought or uniform drought. Numbers of juvenile and male nematodes in soil were 6.5 times higher under local drought than under non-drought after 37 days. Nematodes did not survive in soil under uniform drought. Most of the eggs recovered on each date, from roots under local and non-drought, hatched within 35 days. Sixteen days of uniform drought reduced cumulative egg hatch to 51%, and almost no eggs hatched after 23 and 37 days of uniform drought. Thus, the response of T. semipenetrans to dry soil is fundamentally different, depending on whether all or part of the rhizosphere experiences drought. These data and field observations suggest that hydraulic lift via the root xylem may prolong the activity of some nematodes and possibly other rhizosphere-inhabiting organisms in dry soil.

Sampling Citrus Fibrous Roots and Tylenchulus semipenetrans.

Sampling precision was investigated for Tylenchulus semipenetrans juveniles and males in soil and females from roots and for citrus fibrous root mass density. For the case of two composite samples of 15 cores each, counts of juvenile and male nematodes were estimated to be within 40% of mu, at P < 0.06 (alpha) in orchards where x > 1,500 nematodes/100 cm(3) soil. A similar level of alpha was estimated for measurements of fibrous root mass density, but at a precision level of 25% of mu. Densities of female nematodes were estimated with less precision than juveniles and males. Precision estimates from a general sample plan derived from Taylor's Power Law were in good agreement with estimates from individual orchards. Two aspects involved in deriving sampling plans for management advisory purposes were investigated. A minimum of five to six preliminary samples were required to appreciably reduce bias toward underestimation of sigma. The use of a Student's t value rather than a standard normal deviate in formulae to estimate sample size increased the estimates by an average of three units. Cases in which the use of z rather than Student's t is appropriate for these formulae are discussed.

Leaching Soluble Salts Increases Population Densities of Tylenchulus semipenetrans.

The effect of salinity on population densities of Tylenchulus semipenetrans was measured on 3-month-old salt-tolerant Rangpur lime growing on either loamy sand, sand, or organic mix and on 4-month-old salt-sensitive Sweet lime in organic mix. Salinity treatments were initiated by watering daily with 25 mol/m(3) NaCl + 3.3 mol/m(3) CaCl for 3 days and every other day with 50 mol/m(3) NaC1 + 6.6 mol/m(3) CaC1 for one week, with no salt (NS) treatments as controls. Salinity was discontinued in one treatment (DS) by leaching with tap water prior to inoculation with nematodes, whereas the continuous salinity (CS) treatment remained unchanged. Overall, in Rangpur lime organic soil supported the highest population densities of T. semipenetrans, followed by loamy sand and sand. The DS treatment resulted in the highest (P

Effects of Perennial Peanut (Arachis glabrata) Ground Cover on Nematode Communities in Citrus.

The effects of perennial peanut (Arachis glabrata) ground cover on the nematode community in a citrus orchard were examined. Samples were taken from two different ground cover treatments (perennial peanut or bare ground) at each of three distances from the tree trunk. Richness, measured as total numbers of nematode genera per sample, and total numbers of nematodes were greatest in the perennial peanut treatment (P < 0.05). Abundance of many genera of bacterivores, fungivores, and omnivores were increased by the perennial peanut ground cover. Total numbers of plant parasites were greater in perennial peanut treatments on three of the five sampling dates (P < 0.05), mainly due to trends in numbers of Mesocriconema. Distance from a tree trunk and the interaction of ground cover treatments and proximity to a tree trunk were most influential for Belonolaimus and Hoplolaimus. Although differences among treatments were observed for nematode genera and trophic groups, ecological indices were not consistently sensitive to treatments. Among several ecological indices evaluated, richness was most often affected by ground cover treatment.

Competition Between Entomopathogenic and Free-Living Bactivorous Nematodes in Larvae of the Weevil Diaprepes abbreviatus.

Field and laboratory experiments were conducted to determine the degree to which free-living, bactivorous nematodes (FLBN) are able to competitively displace entomopathogenic nematodes (EPN) from insect cadavers. Two hundred larvae of the insect Diaprepes abbreviatus were buried at regular intervals during 2 years in experimental plots that were untreated or treated twice annually with Steinernema riobrave. Larvae were recovered after 7 days, and nematodes emerging from cadavers during the next 30 days were identified. The monthly prevalence of FLBN was directly related to that of S. riobrave (r = 0.38; P = 0.001) but was not related to the prevalence of the endemic EPN, S. diaprepesi, Heterorhabditis zealandica, H. indica, or H. bacteriophora (r = 0.02; P = 0.80). In a second experiment, treatment of small field plots with S. riobrave increased the prevalence of insect cadavers in which only FLBN were detected compared to untreated controls (30% vs. 14%; P = 0.052), and increased numbers of FLBN per buried insect by more than 10-fold. In the laboratory, sand microcosms containing one D. abbreviatus larva were treated with (i) the FLBN, Pellioditis sp.; (ii) S. riobrave; (iii) S. riobrave + Pellioditis; or (iv) neither nematode. Insect mortality was higher in the presence of both nematodes (57%) than when S. riobrave was alone (42%) (P = 0.01). An average of 59.2 Pellioditis sp. g(-1) insect body weight emerged in the presence of S. riobrave, whereas 6.2 nematodes g(-1) insect were recovered in the absence of the EPN (P = 0.01). Pellioditis sp. reduced the number of S. riobrave per cadaver by 84%; (P = 0.03), and per available insect by 82% (P = 0.001), compared to S. riobrave alone. Population size of S. diaprepesi was not affected by Pellioditis sp. in experiments of the same design. Faster development (P = 0.05) and nutrient appropriation within the insect cadaver by S. diaprepesi compared to S. riobrave may increase the fitness of the former species to compete with Pellioditis sp. The results of these studies demonstrate the potential of FLBN to regulate population densities of EPN and to dampen estimates of EPN-induced mortality of insect pests in the field.

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.