Camera-Based Short Physical Performance Battery and Timed Up and Go Assessment for Older Adults With Cancer.

This paper presents an automatic camera-based device to monitor and evaluate the gait speed, standing balance, and 5 times sit-stand (5TSS) tests of the Short Physical Performance Battery (SPPB) and the Timed Up and Go (TUG) test. The proposed design measures and calculates the parameters of the SPPB tests automatically. The SPPB data can be used for physical performance assessment of older patients under cancer treatment. This stand-alone device has a Raspberry Pi (RPi) computer, three cameras, and two DC motors. The left and right cameras are used for gait speed tests. The center camera is used for standing balance, 5TSS, and TUG tests and for angle positioning of the camera platform toward the subject using DC motors by turning the camera left/right and tilting it up/down. The key algorithm for operating the proposed system is developed using Channel and Spatial Reliability Tracking in the cv2 module in Python. Graphical User Interfaces (GUIs) in the RPi are developed to run tests and adjust cameras, controlled remotely via smartphone and its Wi-Fi hotspot. We have tested the implemented camera setup prototype and extracted all SPPB and TUG parameters by conducting several experiments on a human subject population of 8 volunteers (male and female, light and dark complexions) in 69 test runs. The measured data and calculated outputs of the system consist of tests of gait speed (0.041 to 1.92 m/s with average accuracy of >95%), and standing balance, 5TSS, TUG, all with average time accuracy of >97%.

The Smart Soil Organism Detector: An instrument and machine learning pipeline for soil species identification.

Understanding the diversity of soil organisms is complicated by both scale and substrate. Every footprint we leave in the soil covers hundreds to millions of organisms yet we cannot see them without extremely laborious extraction and microsopy endeavors. Studying them is also challenging. Keeping them alive so that we can understand their lifecycles and ecological roles ranges from difficult to impossible. Functional and taxonomic identification of soil organisms, while possible, is also challenging. Here we present the Smart Soil Organism Detector, an instrument and machine learning pipeline that combines high-resolution imaging, multi-spectral sensing, large-bore flow cytometry, and machine learning to extract, isolate, count, identify, and separate soil organisms in a high-throughput, high-resolution, non-destructive, and reproducible manner. This system is not only capable of separating alive nematodes, dead nematodes, and nematode cuticles from soil with 100% out-of-sample accuracy, but also capable of identifying nematode strains (sub-species) with 95.5% out-of-sample accuracy and 99.4% specificity. Soil micro-arthropods were identified to class with 96.1% out-of-sample accuracy. Broadly applicable across soil taxa, the Smart SOD system is a tool for understanding global soil biodiversity.

Camera-Based Human Gait Speed Monitoring and Tracking for Performance Assessment of Elderly Patients with Cancer.

This paper presents a camera-based device for monitoring walking gait speed. The walking gait speed data will be used for performance assessment of elderly patients with cancer and calibrating wearable walking gait speed monitoring devices. This standalone device has a Raspberry Pi computer, three cameras (two cameras for finding the trajectory and gait speed of the subject and one camera for tracking the subject), and two stepper motors. The stepper motors turn the camera platform left and right and tilt it up and down by using video footage from the center camera. The left and right cameras are used to record videos of the person walking. The algorithm for operating the proposed system is developed in Python. The measured data and calculated outputs of the system consist of times for frames, distances from the center camera, horizontal angles, distances moved, instantaneous gait speed (frame-by-frame), total distance walked, and average speed. This system covers a large Lab area of 134.3 m2 and has achieved errors of less than 5% for gait speed calculation.Clinical Relevance- This project will help specialists to adjust the chemo dosage for elderly patients with cancer. The results will be used to analyze the human walking movements for estimating frailty and rehabilitation applications, too.

Novel associations in antibiosis stemming from an insect pupal cell.

The pupal soil cell of the pecan weevil, Curculio caryae (Coleoptera: Curculionidae), was reported previously to exhibit antibiosis to an entomopathogenic fungus, Beauveria bassiana. The objectives of this study were to examine 1) if the antimicrobial effect occurs in other insects that form pupal cells, 2) whether the effect extends to plant pathogenic fungi, and 3) identify the source of antibiosis in pupal soil cells of C. caryae. Antibiosis of pupal cells against B. bassiana was confirmed in-vitro in three additional curculionids, Diaprepes abbreviatus, Conotrachelus nenuphar, and Pissodes nemorensis, all of which had fewer fungal colonies relative to controls. Pupal soil cells were found to suppress phytopathogenic fungi in-vitro, including suppression of Alternaria solani by D. abbreviatus pupal cell, and that of Monilinia fructicola by C. caryae. The detection of antibiosis of soil cells formed by surface-sterilized insects using sterile soil implies the antimicrobial effect stemmed from inside the insect. Further, a novel biotic mechanism was identified: a bacterium related to Serratia nematodiphila was isolated from C. caryae pupal soil cells and was found to be associated with antibiosis. The bacterial cultures with or without autoclave had similar effects but were not as potent as pupal soil cells for suppressing B. bassiana. Also, autoclaved soil cells and autoclaved bacterial culture suppressed M. fructicola but were not as inhibitory as non-autoclaved soil cells. This indicates that antibiosis may be due to bacterial metabolites, although other factors may also be involved. Our findings suggest potential to develop the antibiotic compounds as novel bio-fungicides to control plant diseases.

Optimizing for taxonomic coverage: a comparison of methods to recover mesofauna from soil.

Manipulating soil properties to modify the dynamics between nematodes and their natural enemies has been proposed to conserve services such as the biological control of insect pests by entomopathogenic nematodes. Many soil microarthropods including acari mites and collembola are natural enemies of nematodes; however, little is known about the naturally occurring assemblages of these two soil dwelling groups and how they might be influenced by soil conditions. A method to efficiently recover both nematodes and microarthropods from environmental samples would be helpful to characterize communities of these two groups in different habitats. Because samples of nematodes extracted from soil by sucrose centrifugation (SC) also contain soil mites, collembola, protozoans, and fungal and bacterial propagules, the efficiency of SC to recover microarthropods was compared to more conventional methods of microarthropod recovery such as heptane flotation (HF), Berlese funnels (BF), and a modified flotation Berlese method (FBF). Microarthropods were identified using an inverted microscope to class in one experiment and to order in a second. Significantly more microarthropods of all taxa were recovered by SC than with either Berlese method (BF or FBF). In total, 40% more microarthropods comprising seven orders were recovered by HF compared to SC, but the difference was not significant. Ecological indices (diversity, richness, and evenness) derived from HF and SC were congruent and significantly higher than those derived from BF. Excessive organic matter in the HF extractions, compared to those of SC, BF, and FBF, made mite detection and identification difficult and time consuming. Moreover, unlike SC, neither HF nor any Berlese method recovered nematodes. Accordingly, we found SC to be the most efficient method for microarthropod extraction, making it an ideal method for studies of communities of nematodes and many of their natural enemies in the soil.

Responses of Anastrepha suspensa, Diachasmimorpha longicaudata, and Sensitivity of Guava Production to Heterorhabditis bacteriophora in Fruit Fly Integrated Pest Management.

Caribbean fruit fly, also known as Caribfly or Anastrepha suspensa , is a major tephritid pest of guavas. A virulent entomopathogenic nematode (EPN) species was investigated to suppress the fruit-to-soil stages of Caribflies, which are also attacked by the koinobiont parasitoid Diachasmimorpha longicaudata in south Florida. The main objective was to develop a feasible and cost-effective EPN-application method for integrated pest management (IPM) of Caribfly to improve guava production. Naturally infested guavas were treated with increasing Heterorhabditis bacteriophora infective juvenile (IJ) concentration or rate (0, 25, 50, …, 1,600 IJs cm -2 ) in field trials to measure the optimum IJ rate and then examine sensitivity of producing guavas to inclusion of Heterorhabditis bacteriophora in Caribfly IPM plans. Relative survival of Caribfly in treatments significantly decreased with increasing IJ rate from 0 to 100 IJs cm -2 . Similarly, probability of observing large numbers of parasitoid wasps ( Diachasmimorpha longicaudata ) in EPN treatments significantly declined with increasing IJ rate (0-100 IJs cm -2 ), even though the non-target effects of Heterorhabditis bacteriophora on relative survival of Diachasmimorpha longicaudata could not be determined because of few emerging parasitoid wasps. Optimum suppression (⩾ 60%) of Caribfly was consistently achieved at 100 IJs cm -2 or 17,500 IJs fruit -1 . Profitability analysis showed that Heterorhabditis bacteriophora can be included in Caribfly IPM tactics to produce guavas. Costs of EPNs in Caribfly IPM are minimized if Heterorhabditis bacteriophora is strategically applied by spot treatment of fruit. Repayment of costs of EPN-augmentation by spot treatments appears achievable by recovering 5.71% of the annual yield losses (⩾1,963 kg ha -1 ≈ US$ 8,650 ha -1 ), which are largely due to Caribfly infestation. Hectare-wide EPN-augmentation (or broadcasting) method requires more fruit recovery than the total annual yield losses to repay its high costs. Profitability of guava production in south Florida will not be very sensitive to marginal costs of the spot treatment method, when compared to the field-wide broadcasting of Heterorhabditis bacteriophora .

The saprophytic fungus Fusarium solani increases the insecticidal efficacy of the entomopathogenic nematode Steinernema diaprepesi.

In two field surveys, high proportions of Galleria mellonella L. (Lepidoptera: Pyralidae) sentinel larval cadavers were infected by Fusarium solani without evidence of concomitant entomopathogenic nematode (EPN) or entomopathogenic fungus (EPF) reproduction. Because F. solani is not considered entomopathogenic, the survey suggested the possibility that F. solani competes with EPNs. We tested the hypotheses that F. solani attracts the EPN, Steinernema diaprepesi, to facilitate infection of Diaprepes root weevils (Diaprepes abbreviatus L.) and thereafter competes with the nematode in the insect cadaver. In two-choice olfactometer assays where one side was treated with F. solani mycelia and conidia, juvenile S. diaprepesi were attracted to the fungus, in either raw soil, or in autoclaved soil in the presence or absence of insects. However, this attraction was attenuated as the habitat became more complex, by using raw soil in combination with insect larvae. Fusarium oxysporum did not recruit the nematode. When soil microcosms were tested with F. solani conidia and S. diaprepesi, the concomitant infection increased the mortality of the insect (P = 0.02) to 83%, compared to 58% and 0% mortality when nematodes or fungi were individually applied, respectively. Concomitant inoculation also increased the number of cadavers that supported nematode reproduction and increased the population density of fungus in soil. The number of IJs entering the host insect was not affected by F. solani. These results support the possibility that F. solani can facilitate the insecticidal efficiency of S. diaprepesi in order to exploit the resources in the cadaver.

Spatial relationships between entomopathogenic nematodes and nematophagous fungi in Florida citrus orchards.

Relationships between entomopathogenic nematodes (EPNs), nematophagous fungi (NF) and soil physical and chemical properties were studied in a survey of 53 citrus orchards in central ridge and flatwoods ecoregions of Florida. Seven species of NF associated with nematodes were quantified directly using a real time qPCR assay. All nematophagous fungi studied except Arthrobotrys musiformis and Hirsutella rhossiliensis were frequently detected (24-56%) in both regions. Paecilomyces lilacinus and Gamsylella gephyropagumwere encountered more frequently in the flatwoods (P=0.03) and on the ridge (P=0.02), respectively. Redundancy analysis revealed seven abiotic and biotic factors as significantly related to the NF occurrence. Multiple regression of fungi on these variables explained 78%, 66%, 48%, 36%, 23% and 4% of the variation in Catenaria sp., A. musiformis, A. dactyloides, P. lilacinus, A. oligospora and G. gepharopagum, respectively. When the data from citrus were pooled with those reported previously from natural areas and subjected to principle component analysis, the first two principle components explained 43% of the variation in NF communities. The surveys (citrus vs natural areas) were discriminated by PC2 (P<0.001) and the ecoregion by PC1 (P<0.002), and all but one NF species were related (P<0.01) to one or both components. NF communities tended to have more species and greater diversity in the flatwoods, where EPN richness and diversity were the least. However, the strength of associations between individual EPN and NF species as measured by SADIE reflected the associations between each species and ground water depth, suggesting that ecoregion preferences affected the species associations. Within each ecoregion, significant relationships between the individual NF and EPN species measured by stepwise regression tended to be positive. The results did not support the hypothesis that NF modulate the spatial patterns of EPN species between or within these two ecoregions.

Biological control potential of entomopathogenic nematodes for management of Caribbean fruit fly, Anastrepha suspensa Loew (Tephritidae).

BACKGROUND: Caribbean fruit fly (Caribfly) is a serious economic insect pest because of development of larvae that hatch from eggs oviposited into fruits by female adults. This study assessed the virulence of twelve entomopathogenic nematode (EPN) isolates to Caribfly in laboratory bioassays as a starting point toward evaluation of management strategies for the fruit-to-soil-dwelling stages of A. suspensa in fields infested by Caribfly. RESULTS: Inoculation of A. suspensa with 1 mL of ca 200 IJs larva-1 killed Caribfly at either larval or pupal stage. Pupae were more resistant to EPN infections than larvae. Adult emergence from inoculated pupae in soil microcosms was significantly lower than that observed in filter paper assays. Longest or largest steinernematids suppressed emergence of more adult Caribfly from pupae in soils, whereas shorter heterorhabditids were more infectious to Caribfly larvae. The highest mortalities of A. suspensa were caused by exotic nematodes Steinernema feltiae and Heterorhabditis bacteriophora, followed by the native Heterorhabditis indica and the exotic Steinernema carpocapsae. CONCLUSION: Entomopathogenic nematodes reduced the development of Caribfly larvae and pupae to adult in our bioassays, suggesting that EPNs have potential for biological control of A. suspensa. Future work will assess management strategies, using the virulent EPNs, in orchards infested by A. suspensa. © 2016 Society of Chemical Industry.

Eliciting maize defense pathways aboveground attracts belowground biocontrol agents.

Plant defense pathways mediate multitrophic interactions above and belowground. Understanding the effects of these pathways on pests and natural enemies above and belowground holds great potential for designing effective control strategies. Here we investigate the effects of aboveground stimulation of plant defense pathways on the interactions between corn, the aboveground herbivore adult Diabrotica speciosa, the belowground herbivore larval D. speciosa, and the subterranean ento-mopathogenic nematode natural enemy Heterorhabditis amazonensis. We show that adult D. speciosa recruit to aboveground herbivory and methyl salicylate treatment, that larval D. speciosa are relatively indiscriminate, and that H. amazonensis en-tomopathogenic nematodes recruit to corn fed upon by adult D. speciosa. These results suggest that entomopathogenicnematodes belowground can be highly attuned to changes in the aboveground parts of plants and that biological control can be enhanced with induced plant defense in this and similar systems.

Entomopathogenic nematode food webs in an ancient, mining pollution gradient in Spain.

Mining activities pollute the environment with by-products that cause unpredictable impacts in surrounding areas. Cartagena-La Unión mine (Southeastern-Spain) was active for >2500years. Despite its closure in 1991, high concentrations of metals and waste residues remain in this area. A previous study using nematodes suggested that high lead content diminished soil biodiversity. However, the effects of mine pollution on specific ecosystem services remain unknown. Entomopathogenic nematodes (EPN) play a major role in the biocontrol of insect pests. Because EPNs are widespread throughout the world, we speculated that EPNs would be present in the mined areas, but at increased incidence with distance from the pollution focus. We predicted that the natural enemies of nematodes would follow a similar spatial pattern. We used qPCR techniques to measure abundance of five EPN species, five nematophagous fungi species, two bacterial ectoparasites of EPNs and one group of free-living nematodes that compete for the insect-cadaver. The study comprised 193 soil samples taken from mining sites, natural areas and agricultural fields. The highest concentrations of iron and zinc were detected in the mined area as was previously described for lead, cadmium and nickel. Molecular tools detected very low numbers of EPNs in samples found to be negative by insect-baiting, demonstrating the importance of the approach. EPNs were detected at low numbers in 13% of the localities, without relationship to heavy-metal concentrations. Only Acrobeloides-group nematodes were inversely related to the pollution gradient. Factors associated with agricultural areas explained 98.35% of the biotic variability, including EPN association with agricultural areas. Our study suggests that EPNs have adapted to polluted habitats that might support arthropod hosts. By contrast, the relationship between abundance of Acrobeloides-group and heavy-metal levels, revealed these taxa as especially well suited bio-indicators of soil mining pollution.

Stimulation of the Salicylic Acid Pathway Aboveground Recruits Entomopathogenic Nematodes Belowground.

Plant defense pathways play a critical role in mediating tritrophic interactions between plants, herbivores, and natural enemies. While the impact of plant defense pathway stimulation on natural enemies has been extensively explored aboveground, belowground ramifications of plant defense pathway stimulation are equally important in regulating subterranean pests and still require more attention. Here we investigate the effect of aboveground stimulation of the salicylic acid pathway through foliar application of the elicitor methyl salicylate on belowground recruitment of the entomopathogenic nematode, Steinernema diaprepesi. Also, we implicate a specific root-derived volatile that attracts S. diaprepesi belowground following aboveground plant stimulation by an elicitor. In four-choice olfactometer assays, citrus plants treated with foliar applications of methyl salicylate recruited S. diaprepesi in the absence of weevil feeding as compared with negative controls. Additionally, analysis of root volatile profiles of citrus plants receiving foliar application of methyl salicylate revealed production of d-limonene, which was absent in negative controls. The entomopathogenic nematode S. diaprepesi was recruited to d-limonene in two-choice olfactometer trials. These results reinforce the critical role of plant defense pathways in mediating tritrophic interactions, suggest a broad role for plant defense pathway signaling belowground, and hint at sophisticated plant responses to pest complexes.

Concilience in Entomopathogenic Nematode Responses to Water Potential and Their Geospatial Patterns in Florida.

The geospatial patterns of four species of native entomopathogenic nematodes in Florida were previously shown to be related to soil properties that affect soil water potential. Here we compared the responses to water potential of third stage, infective juvenile (IJ), Steinernema sp. (Sx), and Steinernema diaprepesi (Sd) in controlled conditions. The two species were selected because they are closely related (Steinernema glaseri-group), but tend to occupy different habitats. In columns of sandy soil with moisture gradients ranging from field capacity (6% w:w) to saturated (18%), Sx migrated toward wetter soil whereas Sd migrated toward drier soil. Survival of two isolates each of Sx and Sd for 7 days in the absence of food was greatest at 18 and 6% soil moisture, respectively. After three cycles of migration through soil to infect insect larvae 10 cm distant, Sd dominated EPN communities when soil columns were maintained at 6% moisture, whereas Sx was dominant in soil maintained at 18% moisture. When rehydrated after 24 h on filter paper at 90% RH, 50% of Sd survived compared to no Sx. Two isolates of Sd also survived better than two isolates of Sx during up to 24 h in a hypertonic solution (30% glycerol). The behavioral responses of both species to water potential and osmotic gradients were consistent with surveys in which Sx was recovered only from flatwoods ecoregions with shallow water tables and poorly drained soils, whereas Sd most frequently inhabited the central ridge ecoregion comprising well-drained soils and deeper water tables. Comparative proteomic analysis revealed differential expression of proteins involved in thermo-sensation (guanylyl cyclase and F13E6-4) and mechano-sensation and movement (paramyosin, Actin 3, LET-99, CCT-2), depending on whether Sd was in soil at 6 or 18% moisture. Proteins involved in metabolism, lectin detoxification, gene regulation, and cell division also differed between the two conditions. Our data suggest the plausibility of modifying soil moisture conditions in flatwoods orchards in ways that favor more desirable (effective) EPN species. Similarly, these particular behavioral traits are likely to be useful in guiding the selection or engineering of EPN species for use in different ecoregions.

Social Networks of Educated Nematodes.

Entomopathogenic nematodes are obligate lethal parasitoids of insect larvae that navigate a chemically complex belowground environment while interacting with their insect hosts, plants, and each other. In this environment, prior exposure to volatile compounds appears to prime nematodes in a compound specific manner, increasing preference for volatiles they previously were exposed to and decreasing attraction to other volatiles. In addition, persistence of volatile exposure influences this response. Longer exposure not only increases preference, but also results in longer retention of that preference. These entomopathogenic nematodes display interspecific social behavioral plasticity; experienced nematodes influence the behavior of different species. This interspecific social behavioral plasticity suggests a mechanism for rapid adaptation of belowground communities to dynamic environments.

Characterization of Biocontrol Traits in Heterorhabditis floridensis: A Species with Broad Temperature Tolerance.

Biological characteristics of two strains of the entomopathogenic nematode, Heterorhabditis floridensis (332 isolated in Florida and K22 isolated in Georgia) were described. The identity of the nematode's symbiotic bacteria was elucidated and found to be Photorhabdus luminescens subsp. luminescens. Beneficial traits pertinent to biocontrol (environmental tolerance and virulence) were characterized. The range of temperature tolerance in the H. floridensis strains was broad and showed a high level of heat tolerance. The H. floridensis strains caused higher mortality or infection in G. mellonella at 30°C and 35°C compared with S. riobrave (355), a strain widely known to be heat tolerant, and the H. floridensis strains were also capable of infecting at 17°C whereas S. riobrave (355) was not. However, at higher temperatures (37°C and 39°C), though H. floridensis readily infected G. mellonella, S. riobrave strains caused higher levels of mortality. Desiccation tolerance in H. floridensis was similar to Heterorhabditis indica (Hom1) and S. riobrave (355) and superior to S. feltiae (SN). H. bacteriophora (Oswego) and S. carpocapsae (All) exhibited higher desiccation tolerance than the H. floridensis strains. The virulence of H. floridensis to four insect pests (Aethina tumida, Conotrachelus nenuphar, Diaprepes abbreviatus, and Tenebrio molitor) was determined relative to seven other nematodes: H. bacteriophora (Oswego), H. indica (Hom1), S. carpocapsae (All), S. feltiae (SN), S. glaseri (4-8 and Vs strains), and S. riobrave (355). Virulence to A. tumida was similar among the H. floridensis strains and other nematodes except S. glaseri (Vs), S. feltiae, and S. riobrave failed to cause higher mortality than the control. Only H. bacteriophora, H. indica, S. feltiae, S. riobrave, and S. glaseri (4-8) caused higher mortality than the control in C. nenuphar. All nematodes were pathogenic to D. abbreviatus though S. glaseri (4-8) and S. riobrave (355) were the most virulent. S. carpocapsae was the most virulent to T. molitor. In summary, the H. floridensis strains possess a wide niche breadth in temperature tolerance and have virulence and desiccation levels that are similar to a number of other entomopathogenic nematodes. The strains may be useful for biocontrol purposes in environments where temperature extremes occur within short durations.

Sending mixed messages: a trophic cascade produced by a belowground herbivore-induced cue.

Plants defend themselves against herbivores both directly (chemical toxins and physical barriers) and indirectly (attracting natural enemies of their herbivores). Previous work has shown that plant roots of citrus defend against root herbivores by releasing an herbivore-induced plant volatile (HIPV), pregeijerene (1,5-dimethylcyclodeca-1,5,7-triene), that attracts naturally occurring entomopathogenic nematodes (EPNs) to Diaprepes abbreviatus larvae when applied in the field. However, the soil community is complex and contains a diversity of interspecific relationships that modulate food web assemblages. Herein, we tested the hypothesis that other nematode types beyond EPNs, as well as, nematophagous fungi are affected by the same HIPV that attracts EPNs to herbivore-damaged roots. We employed molecular probes designed to detect and quantify nematodes from the Acrobeloides-group (free-living bacterivorous nematodes, FLBNs), some of which compete with EPNs by 'hyperparasitizing' insect cadavers, and five species of nematophagous fungi (NF), which attack and kill EPNs. In two different agricultural systems (citrus and blueberry), we detected diverse species of nematodes and fungi; however, only the behavior of FLBNs was affected in a manner similar to that reported previously for EPNs. Although detected, NF abundance was not statistically affected by the presence of the belowground HIPV. We provide the first evidence showing subterranean HIPVs behave much the same as those aboveground, attracting not only parasitoids, but also hyperparasites and other food web members.

Wide interguild relationships among entomopathogenic and free-living nematodes in soil as measured by real time qPCR.

Entomopathogenic nematodes (EPNs) are promising biological control agents of soil-dwelling insect pests of many crops. These nematodes are ubiquitous in both natural and agricultural areas. Their efficacy against arthropods is affected directly and indirectly by food webs and edaphic conditions. It has long been suggested that a greater understanding of EPN ecology is needed to achieve consistent biological control by these nematodes and the development of molecular tools is helping to overcome obstacles to the study of cryptic organisms and complex interactions. Here we extend the repertoire of molecular tools to characterize soil food webs by describing primers/probe set to quantify certain free-living, bactivorous nematodes (FLBNs) that interact with EPNs in soil. Three FLBN isolates were recovered from soil baited with insect larvae. Morphological and molecular characterization confirmed their identities as Acrobeloides maximum (RT-1-R15C and RT-2-R25A) and Rhabditis rainai (PT-R14B). Laboratory experiments demonstrated the ability of these FLBNs to interfere with the development of Steinernema diaprepesi, Steinernema riobrave and Heterorhabditis indica parasitizing the weevil Diaprepes abbreviatus (P<0.001), perhaps due to resource competition. A molecular probe was developed for the strongest competitor, A. maximum. We selected the highly conserved SSU rDNA sequence to design the primers/probe, because these sequences are more abundantly available for free-living nematodes than ITS sequences that can likely provide better taxonomic resolution. Our molecular probe can identify organisms that share ⩾98% similarity at this locus. The use of this molecular probe to characterize soil communities from samples of nematode DNA collected within a citrus orchard revealed positive correlations (P<0.01) between Acrobeloides-group nematodes and total numbers of EPNs (S. diaprepesi, H. indica and Heterorhabditis zealandica) as well as a complex of nematophagous fungi comprising Catenaria sp. and Monachrosporium gephyropagum that are natural enemies of EPNs. These relationships can be broadly interpreted as supporting Linford's hypothesis, i.e., decomposition of organic matter (here, insect cadavers) greatly increases bactivorous nematodes and their natural enemies.

Subterranean, herbivore-induced plant volatile increases biological control activity of multiple beneficial nematode species in distinct habitats.

While the role of herbivore-induced volatiles in plant-herbivore-natural enemy interactions is well documented aboveground, new evidence suggests that belowground volatile emissions can protect plants by attracting entomopathogenic nematodes (EPNs). However, due to methodological limitations, no study has previously detected belowground herbivore-induced volatiles in the field or quantified their impact on attraction of diverse EPN species. Here we show how a belowground herbivore-induced volatile can enhance mortality of agriculturally significant root pests. First, in real time, we identified pregeijerene (1,5-dimethylcyclodeca-1,5,7-triene) from citrus roots 9-12 hours after initiation of larval Diaprepes abbreviatus feeding. This compound was also detected in the root zone of mature citrus trees in the field. Application of collected volatiles from weevil-damaged citrus roots attracted native EPNs and increased mortality of beetle larvae (D. abbreviatus) compared to controls in a citrus orchard. In addition, field applications of isolated pregeijerene caused similar results. Quantitative real-time PCR revealed that pregeijerene increased pest mortality by attracting four species of naturally occurring EPNs in the field. Finally, we tested the generality of this root-zone signal by application of pregeijerene in blueberry fields; mortality of larvae (Galleria mellonella and Anomala orientalis) again increased by attracting naturally occurring populations of an EPN. Thus, this specific belowground signal attracts natural enemies of widespread root pests in distinct agricultural systems and may have broad potential in biological control of root pests.

Use of real-time PCR to discriminate parasitic and saprophagous behaviour by nematophagous fungi.

Entomopathogenic nematodes (EPNs) are important pathogens of soilborne insects and are sometimes developed commercially to manage insect pests. Numerous nematophagous fungal species (NF) prey on nematodes and are thought to be important in regulating natural or introduced EPN populations. However, nematophagy by these fungi in nature cannot be inferred using existing methods to estimate their abundance in soil because many of these fungi are saprophytes, resorting to parasitism primarily when certain nutrients are limiting. Therefore, we developed an assay to quantify NF DNA in samples of nematodes. Species-specific primers and TaqMan probes were designed from the ITS rDNA regions of Arthrobotrys dactyloides, Arthrobotrys oligospora, Arthrobotrys musiformis, Gamsylella gephyropagum and Catenaria sp. When tested against 23 non-target fungi, the TaqMan real-time PCR assay provided sensitive and target-specific quantification over a linear range. The amount of A. dactyloides or Catenaria sp. DNA in 20 infected nematodes, measured by real-time PCR, differed between fungal species (P=0.001), but not between experiments (P>0.05). However, estimates of relative NF parasitism using a bioassay with 20 nematodes infected by either species, differed greatly (P<0.001) depending on whether the fungi were alone or combined in the samples used in the assay. Tests done to simulate detection of NF DNA in environmental samples showed that, for all species, background genomic DNA and/or soil contaminants reduced the quantity of DNA detected. Nested PCR was ineffective for increasing the detection of NF in environmental samples. Indeed, real-time PCR detected higher amounts of NF DNA than did nested PCR. The spatial patterns of NF parasitism in a citrus orchard were derived using real-time PCR and samples of nematodes extracted from soil. The parasitism by Catenaria sp. was positively related to the abundance of both heterorhabditid and steinernematid EPNs. The possible significance of the associations is ambiguous because NF attack a broad range of nematode taxa whereas EPNs are a small minority of the total nematode population in a soil sample. These studies demonstrate the potential of real-time PCR to study the role of NF parasitism in soil food webs.

Entomopathogenic nematodes, root weevil larvae, and dynamic interactions among soil texture, plant growth, herbivory, and predation.

Greenhouse experiments were conducted to assess the influence of soil texture on the persistence, efficacy and plant protection ability of entomopathogenic nematodes (EPNs) applied to control larvae of the Diaprepes root weevil (DRW), Diaprepes abbreviatus, infesting potted citrus seedlings. Seedlings were grown in pots containing either coarse sand, fine sand, or sandy loam. Three DRW larvae were added to each of 80 pots of each soil type. 24 h later, 20 pots of each soil type that had received weevil larvae were inoculated with EPN infective juveniles (IJs) of one of the following species: Steinernema diaprepesi, Steinernema riobrave and Heterorhabditis indica. Pots of each soil without EPNs were established as controls with DRW and controls without DRWs. Subsequently, pots with larvae received three additional larvae monthly, and the experiment continued for 9 months. Plant root and top weights at the end of the experiment were affected by both soil (P≤0.0001) and nematodes (P≤0.0001), and nematode species protected plants differently in different soils (interaction P≤0.0001). Soil porosity was inversely related to plant damage by DRW, whether or not EPNs were present; and porosity was directly related to the level of plant protection by EPNs. Mortality of caged sentinel weevil larvae placed in pots near the end of the experiment was highest in pots treated with S. diaprepesi. In a second, similar experiment that included an additional undescribed steinernematid of the Steinernema glaseri-group, soil type affected root damage by DRW and root protection by EPNs in the same manner as in the first experiment. Final numbers of S. diaprepesi and Steinernema sp. as measured by real-time PCR were much greater than those of S. riobrave or H. indica in all soils. Across all treatments, the number of weevil larvae in soil at the end the experiment was inversely related to soil porosity. In all soils, fewer weevil larvae survived in soil treated with S. diaprepesi or Steinernema sp. than in controls with DRW or treatments with S. riobrave or H. indica. The results of these experiments support the hypothesis that EPNs provide greater protection of seedlings against DRW larvae in coarse textured soil than in finer textured soil. However, less vigorous growth of the control without DRW seedlings in the two finer textured soils suggests that unidentified factors that stressed seedlings in those soils also impaired the ability of seedlings to tolerate weevil herbivory.