Sand Column Assay Research Articles

Interactions between the entomopathogenic nematode Heterorhabditis sonorensis (Nematoda: Heterorhabditidae) and the saprobic fungus Fusarium oxysporum (Ascomycota: Hypocreales)

In this study, we assessed the effect of the saprobic fungus, Fusarium oxysporum (Ascomycota: Hypocreales) on the fitness of the entomopathogenic nematode Heterorhabditis sonorensis (Caborca strain). Sand column assays were considered to evaluate the effect of fungal mycelia on infective juvenile (IJ) movement and host access. Additionally, we investigated the effect of fungal spores on the nematodes’ ability to search for a host, its virulence, penetration efficiency and reproduction. Three application timings were considered to assess interactions between the fungus and the nematodes. In vitro assays were also considered to determine the effect of fungal extracts on the nematode’s symbiotic bacteria. Our observations indicate that presence and age of fungal mycelia significantly affect IJ movement in the sand columns and their ability to establish in the host. These results were also reflected in a reduced insect mortality. In particular, treatments with the 15days old mycelia showed a significant reduction in insect mortality and penetration efficiency. Presence of fungal spores also impacted nematode virulence and reproduction. In particular, two of the application timings tested (simultaneous [EPN and fungal spores applied at the same time] and alternate I [EPN applied first, fungus applied 24h later]) resulted in antagonistic interactions. Moreover, IJ progeny was reduced to half in the simultaneous application. In vitro assays revealed that fungal extracts at the highest concentration tested (10mg/ml) inhibited the growth of the symbiotic bacteria. Overall, these results suggest that saprobic fungi may play an important role in regulating. EPN populations in the soil, and that they may be one of the factors that impact nematode survival in the soil and their access to insect hosts.

OmpA influences Escherichia coli biofilm formation by repressing cellulose production through the CpxRA two‐component system

Previously we discovered that OmpA of Escherichia coli increases biofilm formation on polystyrene surfaces (González Barrios et al., Biotechnol Bioeng, 93:188-200, 2006a). Here we show OmpA influences biofilm formation differently on hydrophobic and hydrophilic surfaces since it represses cellulose production which is hydrophilic. OmpA increased biofilm formation on polystyrene, polypropylene, and polyvinyl surfaces while it decreased biofilm formation on glass surfaces. Sand column assays corroborated that OmpA decreases attachment to hydrophilic surfaces. The ompA mutant formed sticky colonies, and the extracellular polysaccharide that caused stickiness was identified as cellulose. A whole-transcriptome study revealed that OmpA induces the CpxRA two-component signal transduction pathway that responds to membrane stress. CpxA phosphorylates CpxR and results in reduced csgD expression. Reduced CsgD production represses adrA expression and results in reduced cellulose production since CsgD and AdrA are responsible for 3,5-cyclic diguanylic acid and cellulose synthesis. Real-time polymerase chain reaction confirmed csgD and adrA are repressed by OmpA. Biofilm and cellulose assays with double deletion mutants adrA ompA, csgB ompA, and cpxR ompA confirmed OmpA decreased cellulose production and increased biofilm formation on polystyrene surfaces through CpxR and AdrA. Further evidence of the link between OmpA and the CpxRA system was that overproduction of OmpA disrupted the membrane and led to cell lysis. Therefore, OmpA inhibits cellulose production through the CpxRA stress response system, and this reduction in cellulose increases biofilm formation on hydrophobic surfaces.

Toxin-Antitoxin Systems in Escherichia coli Influence Biofilm Formation through YjgK (TabA) and Fimbriae

The roles of toxin-antitoxin (TA) systems in bacteria have been debated. Here, the role of five TA systems in regard to biofilm development was investigated (listed as toxin/antitoxin: MazF/MazE, RelE/RelB, ChpB, YoeB/YefM, and YafQ/DinJ). Although these multiple TA systems were reported previously to not impact bacterial fitness, we found that deletion of the five TA systems decreased biofilm formation initially (8 h) on three different surfaces and then increased biofilm formation (24 h) by decreasing biofilm dispersal. Whole-transcriptome profiling revealed that the deletion of the five TA systems induced expression of a single gene, yjgK, which encodes an uncharacterized protein; quantitative real-time PCR (qRT-PCR) confirmed consistent induction of this gene (at 8, 15, and 24 h). Corroborating the complex phenotype seen upon deleting the TA systems, overexpression of YjgK decreased biofilm formation at 8 h and increased biofilm formation at 24 h; deletion of yjgK also affected biofilm formation in the expected manner by increasing biofilm formation after 8 h and decreasing biofilm formation after 24 h. In addition, YjgK significantly reduced biofilm dispersal. Whole-transcriptome profiling revealed YjgK represses fimbria genes at 8 h (corroborated by qRT-PCR and a yeast agglutination assay), which agrees with the decrease in biofilm formation upon deleting the five TA systems at 8 h, as well as that seen upon overexpressing YjgK. Sand column assays confirmed that deleting the five TA systems reduced cell attachment. Furthermore, deletion of each of the five toxins increased biofilm formation at 8 h, and overexpression of the five toxins repressed biofilm formation at 8 h, a result that is opposite that of deleting all five TA systems; this suggests that complex regulation occurs involving the antitoxins. Also, the ability of the global regulator Hha to reduce biofilm formation was dependent on the presence of these TA systems. Hence, we suggest that one role of TA systems is to influence biofilm formation.

Investigations on the efficacy of entomopathogenic nematodes against Spodoptera littoralis (Biosd.) and Galleria mellonella (L.)

The infectivity of seven entomopathogenic nematode strains of the families Steinernematidae and Heterorhabditidae were tested against the last instar larvae of the cotton leafworm, Spodoptera littoralis (Biosd.) and the greater wax moth, Galleria mellonella (L.) in a Petri dish and sand column assay. Exposure of the insect species was done at a dose of 100 IJ/ ml. Heterorhabditis sp. ELG., H. indica, Heterorhabditis sp. ELB. were the highest in activity, they gave a 100% mortality to S. littoralis larvae in a Petri dish assay 24 h postexposure. The rest of the strains tested gave mortalities ranged between 50 and 90% at 24 h then 100% after 48 h postexposure . In the sand-column assay strains, Heterorhabditis sp. ELB., S. riobravae and S. carpocapsae were the highest in activity against S. littoralis with 100% mortality within 24 h postexposure. The results of infectivity against G. mellonella showed that H. indica was the highest in the Petri dish assay and H. egyptii was the highest in the sand column assay with 100% mortality after 24 h of exposure. The penetration rate as an indicator of entomopathogenic nematode infection was also evaluated. The highest value was recorded for Heterorhabditis sp. ELB. (62.3%) and S. carpocapsae (61.4%) against S. littoralis in both the Petri dish and sand-column assay, respectively. In the case of G. mellonella, the highest value of penetration was recorded for Heterorhabditis sp. ELG. (68.3%) and S. riobravae (65.00%) in both the Petri dish and sand-column assay, respectively.

Virulence of entomopathogenic nematodes to larvae of the guava weevil, Conotrachelus psidii (Coleoptera: Curculionidae), in laboratory and greenhouse experiments

Abstract The guava weevil, Conotrachelus psidii, is a major pest of guava in Brazil and causes severe reduction in fruit quality. This weevil is difficult to control with insecticides because adults emerge over a long period, and larvae develop to the fourth-instar inside the fruit and move to the soil for pupation. We assessed the virulence of entomopathogenic nematodes to fourth-instar larvae in soil by comparing their susceptibility to nine species or strains: Heterorhabditis bacteriophora HP88, H. baujardi LPP7, and LPP1, H. indica Hom1, Steinernema carpocapsae All and Mexican, S. feltiae SN, S. glaseri NC, and S. riobrave 355. In petri dish assays with sterile sand at a concentration of 100 infective juveniles (IJs) of a given nematode species/strain, larval mortality ranged from 33.5 to 84.5%, with the heterorhabditids being the most virulent. In sand column assays with H. baujardi LPP7, H. indica Hom1, or S. riobrave 355 at concentrations of 100, 200, and 500 IJs, mortality was greater than the control only for H. baujardi (62.7%) and H. indica (68.3%) at the highest concentration. For H. baujardi LPP7 in a petri dish assay, the time required to kill 50 and 90% of the larvae (LT50 and LT90) for 100 IJs was 6.3 and 9.9 days, whereas the lethal concentration required to kill 50 and 90% of the larvae (LC50 and LC90) over 7 days was 52 and 122.2 IJs. In a greenhouse study with guava trees in 20-L pots, 10 weevil larvae per pot, and concentrations of 500, 1000 or 2000 IJs, H. baujardi LPP7 caused 30 and 58% mortality at the two highest concentrations. These results show that H. baujardi is virulent to fourth-instar larvae and has potential as a biological control agent in IPM programs.

Alterations in adhesion, transport, and membrane characteristics in an adhesion-deficient pseudomonad.

A stable adhesion-deficient mutant of Burkholderia cepacia G4, a soil pseudomonad, was selected in a sand column assay. This mutant (ENV435) was compared to the wild-type strain by examining the adhesion of the organisms to silica sand and their transport through two aquifer sediments that differed in their sand, silt, and clay contents. We compared the longitudinal transport of the wild type and the adhesion mutant to the transport of a conservative chloride tracer in 25-cm-long glass columns. The transport of the wild-type strain was severely retarded compared to the transport of the conservative tracer in a variety of aquifer sediments, while the adhesion mutant and the conservative tracer traveled at similar rates. An intact sediment core study produced similar results; ENV435 was transported at a faster rate and in much greater numbers than G4. The results of hydrophobic interaction chromatography revealed that G4 was significantly more hydrophobic than ENV435, and polyacrylamide gel electrophoresis revealed significant differences in the lipopolysaccharide O-antigens of the adhesion mutant and the wild type. Differences in this cell surface polymer may explain the decreased adhesion of strain ENV435.

Differential susceptibility of Dysmicoccus vaccinii (Homoptera: Pseudococcidae) to entomopathogenic nematodes (Rhabditida: Heterorhabditidae and Steinernematidae).

The susceptibility of the mealy bug, Dysmicoccus vaccinii Miller & Polavarapu, to infection by various species and strains of entomopathogenic nematodes was investigated in laboratory sand-dish and sand-column assays. Steinernemo carpocapsae (Weiser) (All strain), S. feltiae (Filipjev) (AB [Australia] strain), and S. glaseri (Steiner) (NC strain) were ineffective against individual mealybugs in sand-dish assays conducted in small petri dishes (1 cm high by 3.5 cm diameter) at 25 degrees C with doses of infective juvenile nematodes ranging up to 500 or 1,000 infective juveniles and exposure periods up to 5 d. However, Heterorhabditis bacteriophora Poinar (HP88 strain and 2 New Jersey isolates). H. hawaiiensis Gardner, Stock & Kaya (MG-13 strain), and H. indicus Poinar, Karunakar & David (EMS-13 strain) induced significant mortality (65.0-90.0%) at doses as low as 100 infective juveniles and an exposure of 5 d. H. bacteriophora (HBNJ strain) was effective at doses of 500 and 1,000 infective juveniles but, together with H. zealandica Poinar (V16 strain) and 4 other H. bacteriophora isolates from New Jersey, was ineffective at doses of 100 infective juveniles. Removal of the waxy coating from the mealybugs did not influence susceptibility to H. bacteriophora (HP88 strain). In the sand-column assay (5.5 cm high by 5 cm diameter, 5-d exposure, 25 degrees C), which more closely resembles host-finding in the field, H. bacteriophora (HP88 strain) induced no significant mortality against individual mealybugs at doses of 100 infective juveniles but produced 93.8% mortality at 500 infective juveniles, whereas H. indicus (EMS-13 strain) induced 56.3 and 100% mortality at 100 and 500 infective juveniles, respectively. H. bacteriophora (HP88 strain and some New Jersey isolates), H. hawaiiensis (MG-13 strain), and H. indicus (EMS-13 strain) successfully reproduced in and emerged from mealybug cadavers. This study demonstrates strong variability in the susceptibility of D. vaccinii to different species and strains of entomopathogenic nematodes, and implicates certain heterorhabditids as promising candidates for the biological control of this insect.

The Effects of Aging On the Foraging Behaviour of Steinernema Carpocapsae (Rhabdita: Steinernematidae)

Steinernema carpocapsae infective juvenile behaviour changes with age. Soon after emergence from the host cadaver, S. carpocapsae infective juveniles spend most of their time nictating. This behaviour enhances their host finding (ambushing) success. Between two and four weeks after emergence, their mobility increases and their nictation rate decreases. These alterations in behaviour suggested that a shift in foraging strategy from ambushing to a more mobile mode may occur. We tested this hypothesis by measuring changes in behaviours that may confer success to mobile foragers, including response to host cues and attachment to sedentary vs mobile hosts. We found that the success rate of ambushing declined with age, but there was no corresponding increase in the success rate of S. carpocapsae infective juveniles as mobile searchers, as measured in a sand column assay. We suggest that entomopathogenic nematodes do not alter their foraging strategy in response to unsuccessful search or aging.

Comparison of Bioassays to Measure Virulence of Different Entomopathogenic Nematodes

Five bioassays were compared for their usefulness to determine the virulence of four nematode strains. The objective of this study was to develop standard assays for particular nematode species. In all assays, the nematodes Steinernema feltiae (strain UK), S. riobravis, S. scapterisci Argentina and Heterorhabditis bacteriophora HP88 were exposed to Galleria mellonella larvae. All bioassays except the sand column assay were conducted in multi-well plastic dishes. In the penetration rate assay, the number of individual nematodes invading the insect was determined after a 48-h exposure to 200 infective juveniles (IJs). In the one-on-one assay, each larva was exposed to an individual nematode for 72 h before insect mortality was recorded. In the exposure time assay, insect mortality was recorded after exposure to 200 IJs for variable time periods. The dose-response assay involved exposing larvae to different nematode concentrations over the range 1-200 IJs/insect and recording mortality every 24 h for a 96-h ...

Infection of Entomopathogenic Nematodes Steinernema glaseri and Heterorhabditis bacteriophora against Popillia japonica (Coleoptera: Scarabaeidae) Larvae

Defense mechanisms of Japanese beetle larvae, Popillia japonica, and their effectiveness against entomopathogenic nematodes were investigated in the laboratory. Behavioral grooming by P. japonica larvae was effective in reducing invasion of both Steinernema glaseri and Heterorhabditis bacteriophora. Forty percent of H. bacteriophora infective juveniles were inactivated or killed in the host gut fluid within 8 hr, whereas 11% of S. glaseri were killed. Host melanotic encapsulation, induced by injection of 20 H. bacteriophora infective juveniles, caused 75% nematode mortality. S. glaseri was less susceptible to the host immune response, which caused only 12% mortality of injected nematodes. In a sand column assay, S. glaseri was found in the host hemocoel 8 hr earlier than H. bacteriophora. However, Photorhabdus luminescens, the bacterium of H. bacteriophora, was detected 16 hr earlier in the host's hemolymph than S. glaseri's bacterium, Xenorhabdus poinarii. Despite the differences in effectiveness of host defenses, S. glaseri and H. bacteriophora caused similar host mortality in the sand column assay. We demonstrated that the two nematode species use different strategies for invasion of P. japonica larvae. S. glaseri, which tolerated P. japonica gut fluid and avoided the host immune response, has a high initial rate of penetration and survival; H. bacteriophora, which exhibited poor tolerance to the host gut fluid and induced a strong host immune response, releases its bacteria during or soon after penetration to kill the host.

Injection‐attachment of Methylosinus trichosporium OB3b in a two‐dimensional miniature sand‐filled aquifer simulator

For some potentially useful and emerging in situ bioremediation technologies it is important to control bacterial attachment to subsurface materials during the injection of microbial cell suspensions. In this study the attachment patterns of Methylosinus trichosporium OB3b were measured after horizontal injections into a two‐dimensional miniature aquifer simulator containing a wet homogeneous sand. In preliminary sand column assays, bacterial attachment to the sand was increased nearly 2 orders of magnitude compared to attachment in the presence of distilled water by raising the concentration of a pH 7.0 sodium‐potassium phosphate buffer to 10 mM. The maximal concentration of attached cells was ∼4×108 cells/g dry sand with both sand minicolumns and the wet sand aquifer simulator. For the latter this occurred on streamlines directly between the horizontal injector and withdrawal ports, where injection‐withdrawal velocities were the highest. The effects of a simulated groundwater cross flow during suspension injection on bacterial attachment to the aquifer simulator sands were also studied, and a peristaltic pumping method to counteract these groundwater flow effects resulted in a more localized pattern, i.e., without extensive downstream skewing of the bacterial attachment zone. Phenol red was utilized as a nonbinding, red‐colored tracer compound. It proved to be very convenient for quantitatively measuring the earlier breakthroughs of cells versus an inert tracer during the aquifer simulator and subsequent capillary tube cell injection experiments and also for visualizing the anticipated boundaries of cell attachment in the aquifer simulator. The effect of injection velocity on the observed bacterial attachment patterns in these experiments appears to be accounted for by colloid filtration theory.

Development of an Adhesion Assay and Characterization of an Adhesion-Deficient Mutant of Pseudomonas fluorescens

A sand column adhesion assay was developed which distinguishes the adhesion abilities of a number of pseudomonads isolated from fine sandy loam. Pseudomonas fluorescens Pf0-1 which adhered at >90% of the total cells added was subjected to transposon Tn5 insertion mutagenesis. From 2,500 Pf0-1::Tn5 mutants examined in the sand column assay, two adhesion-deficient Pf0-1 mutants showing <50% attachment were isolated. Marker exchange analysis of one of these mutants, Pf0-5, confirmed that the decreased adhesion was linked to the Tn5 insertion in the chromosome. The growth rate of Pf0-5 in enriched media and sterile soil was similar to that of the wild type; in minimal medium, however, Pf0-5 grew faster. In a soil column assay, less Pf0-5 than wild-type bacteria were recovered, suggesting a decreased ability to persist in soil. A 34-kilodalton major outer membrane protein present in the wild type was missing in Pf0-5. Transmission electron microscopy of the cell surface revealed that the wild-type possessed polar flagella which were absent in the mutant.