Optimization of Inoculation for In Vivo Production of Entomopathogenic Nematodes

Entomopathogenic nematodes are potent biopesticides that can be mass-produced by in vitro or in vivo methods. For in vivo production, consistently high infection rates are critical to efficiency of the process. Our objective was to optimize in vivo inoculation of Steinernema carpocapsae and Heterorhabditis bacteriophora in Galleria mellonella and Tenebrio molitor by determining effects of inoculation method, nematode concentration, and host density. We found immersing hosts in a nematode suspension to be approximately four times more efficient in time than pipeting inoculum onto the hosts. The number of hosts exhibiting signs of nematode infection increased with nematode concentration and decreased with host density per unit area. This is the first report indicating an effect of host density on inoculation efficiency. We did not detect an effect of nematode inoculum concentration on nematode yield per host or per gram of host. Yield was affected by host density in one of the four nematode-host combinations (S. carpocapsae and T. molitor). We conclude that optimization of inoculation parameters is a necessary component of developing an in vivo production system for entomopathogenic nematodes.     

Influence of Salinity on Survival and Infectivity of Entomopathogenic: Nematodes

Exposure to NaC1, KCI, and CaCl₂ affected the entomopathogenic nematodes Heterorhabditis bacteriophora and Steinernema glaseri differently. Survival, virulence, and penetration efficiency of S. glaseri were not affected by these salts. At high concentrations, however, all three salts inhibited its ability to move through a soil column and locate and infect a susceptible host. Calcium chloride and KCl had no effect on H. bacteriophora survival, penetration efficiency, or movement through a soil column, but moderate concentrations of these salts enhanced H. bacteriophora virulence. NaCl, however, adversely affected each of these parameters at high salinities (>16 dS/m). Salt effects on S. glaseri are attributed solely to interference with nematode host-finding ability, whereas the NaCl effects on H. bacteriophora are attributed to its toxicity and possibly to interference with host-finding behavior.     

Effects of host nutrition on virulence and fitness of entomopathogenic nematodes: Lipid- and protein-based supplements in Tenebrio molitor diets

Entomopathogenic nematodes, Heterorhabditis indica and Steinernema riobrave, were tested for virulence and reproductive yield in Tenebrio molitor that were fed wheat bran diets with varying lipid- and protein-based supplements. Lipid supplements were based on 20% canola oil, peanut, pork or salmon, or a low lipid control (5% canola). Protein treatments consisted of basic supplement ingredients plus 0, 10, or 20% egg white; a bran-only control was also included. Some diet supplements had positive effects on nematode quality, whereas others had negative or neutral effects. All supplements with 20% lipids except canola oil caused increased T. molitor susceptibility to H. indica, whereas susceptibility to S. riobrave was not affected. Protein supplements did not affect host susceptibility, and neither lipid nor protein diet supplements affected reproductive capacity of either nematode species. Subsequently, we determined the pest control efficacy of progeny of nematodes that had been reared through T. molitor from different diets against Diaprepes abbreviatus and Otiorhynchus sulcatus. All nematode treatments reduced insect survival relative to the control (water only). Nematodes originating from T. molitor diets with the 0% or 20% protein exhibited lower efficacy versus D. abbreviatus than the intermediate level of protein (10%) or bran-only treatments. Nematodes originating from T. molitor lipid or control diets did not differ in virulence. Our research indicates that nutritional content of an insect host diet can affect host susceptibility to entomopathogenic nematodes and nematode fitness; therefore, host media could conceivably be optimized to increase in vivo nematode production efficiency.     

Dynamics of Belonolaimus longicaudatus Parasitism on a Susceptible St. Augustinegrass Host

St. Augustinegrass (Stenotaphrum secundatum) cv FX-313 was used as a model laboratory host for monitoring population growth of the sting nematode, Belonolaimus longicaudatus, and for quantifying the effects of sting nematode parasitism on host performance in two samples of autoclaved native Margate fine sand with contrasting amounts of organic matter (OM = 7.9% and 3.8%). Following inoculation with 50 Belonolaimus longicaudatus per pot, nematodes peaked at a mean of 2,139 nematodes per pot 84 days after inoculation, remained stable through 168 days at 2,064 nematodes per pot, and declined at 210 days. The relative numbers of juveniles and adults demonstrated senescence after 84 days. Root dry weight of nematode-inoculated plants increased briefly to an apparent equilibrium 84 days after inoculation, whereas root weights of uninoculated controls continued to increase, exceeding those of inoculated plants from 84 to 210 days (P < 0.01). At 210 days, uninoculated plants had 227% the root dry weight of inoculated plants. Transpiration of FX-313 was reduced by nematodes (P < 0.0001) at 84 and 126 days after inoculation; reduction was first observed at 42 days and last observed 168 days after inoculation (P < 0.05). OM content affected all plant performance variables at multiple dates, and generally there were no inoculation x OM content interactions. OM content had no effect on nematode numbers per pot, although there was a slight (P < 0.05) increase in the number of nematodes per gram root dry weight in the low-OM soil compared with the high-OM soil.     

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.     

Use of Nematodes as Biomonitors of Nonfumigant Nematicide Movement through Field Soil

Three field experiments were established in a loamy sand soil in the Coastal Plain of North Carolina to determine downward movement of aldicarb and fenamiphos with a nematode bioassay. Penetration of bioassay plant roots by Meloidogyne incognita was measured at 1, 3, 7, 14, 21, and 28 days after treatment in the greenhouse as a means of determining nematicide effectiveness. Chemical movement was similar in planted and fallow soil. Nematicidal activity was greater in soil collected from the 0 to 10 cm depth than from the 10 to 20 cm depth. Fenamiphos suppressed host penetration by the nematode more than aldicarb under the high rainfall (19 cm) and low soil temperatures that occurred soon after application in the spring. During the summer, which had 13 cm precipitation and warmer soil temperatures, both chemicals performed equally well at the 0 to 10 cm depth. At the lower soil level (10 to 20 cm), aldicarb limited nematode penetration of host roots more quickly than fenamiphos. Both of these chemicals moved readily in the sandy soil in concentrations sufficient to control M. incognita. Although some variability was encountered in similar experiments, nematodes such as M. incognita have considerable potential as biomonitors of nematicide movement in soil.     

Inheritance of Resistance to Pratylenchus penetrans in Alfalfa

Fifty-two alfalfa (Medicago sativa L.) clones, randomly selected from the cultivar Baker and the experimental line MNGRN-4, were evaluated for resistance (based on nematode reproduction) to Pratylenchus penetrans in growth chamber tests (25 C). Twenty-five clones, representing the range of nematodes and eggs per plant, were selected and retested. Four moderately resistant and two susceptible alfalfa clones were identified. Inheritance of resistance to P. penetrans was studied in these six clones using a diallel mating design. The S₁, Fl, and reciprocal progenies differed for numbers of nematodes and eggs per g dry root and for shoot and root weights (P < 0.05). Resistance, measured as numbers of nematodes in roots, was correlated between parental clones and their S₁ families (r = 0.94), parental clones and their half-sib families (r = 0.81), and S₁ and half-sib families (r = 0.88). General combining ability (GCA) effects were significant for nematode resistance traits. Both GCA and specific combining ability (SCA) effects were significant for plant size traits, but SCA was more important than GCA in predicting progeny plant size. Reciprocal effects were significant for both nematode resistance and plant size traits, which may slow selection progress in long-term selection programs. However, the GCA effects are large enough that breeding procedures that capitalize on additive effects should be effective in developing alfalfa cultivars with resistance to P. penetrans.     

Host Status of Five Weed Species and Their Effects on Pratylenchus zeae Infestation of Maize

The host suitability of five of the most common weed species occurring in maize (Zea mays L.) fields in South Africa to Pratylenchus zeae was tested. Based on the number of nematodes per root unit, mealie crotalaria (Crotalaria sphaerocarpa) was a good host; goose grass (Eleusine indica), common pigweed (Amaranthus hybridus), and thorn apple (Datura stramonium) were moderate hosts; and khaki weed (Tagetes minuta) was a poor host. Only the root residues of khaki weed suppressed the P. zeae infestation of subsequently grown maize. When goose grass, khaki weed, and mealie crotalaria were grown in association with maize in soil infested with P. zeae, goose grass and khaki weed severely suppressed maize root development; this resulted in a low number of nematodes per maize root system and a high number of nematodes per maize root unit. Mealie crotalaria did not restrict maize root growth and did not affect nematode densities per maize root system or maize root unit. Special attention should be given to the control of mealie crotalaria, which is a good host for P. zeae, and goose grass, which, in addition to its ability to compete with maize, is also a suitable host for P. zeae.     

Penetration of Crotalaria juncea,Dolichos lablab,and Sesamum indicum Roots by Meloidogyne javanica

Penetration of Crotalaria juncea (PI 207657 and cv. Tropic Sun) Dolichos lablab cv. Highworth, and Sesamum indicum by juveniles (J2) of Meloidogyne javanica was assessed to investigate the mechanism by which these plants may reduce nematode numbers in the field. Growth chamber experiments were conducted at 25 C, with vials containing 90 g sand infested with 450 J2; tomato (UC 204 C) was included as a susceptible host. Fifteen days after inoculation, roots were stained and the nematodes within stained roots were counted. Both C. juncea lines were highly resistant to penetration, as they contained significantly fewer nematodes per cm of root and per root system than the other plants. Although containing more nematodes per cm of root than C. juncea, S. indicum and D. lablab had significantly fewer nematodes per root system and per cm of root than tomato. Roots were significantly longer in the plants with the lowest nematode penetration. Although C. juncea, D. lablab, and S. indicum may have potential utility as cover or rotation crops in soil infested with M. javanica, further quantitative information on the reproduction of M. javanica and other nematodes in these plants is needed.     

Population Dynamics of Heterodera schachtii on Tomato and Sugar Beet

Experiments showed that development of male and female Heterodera schachtii on tomato and sugarbeet are disproportionately influenced by the nematode inoculum level and root size, which together determine the density of invading larvae. Slight overcrowding favored development of males over females, whereas severe overcrowding equally affected development of males and females. Differential population changes of host-selected races on tested cultivars was attributable to selective development of male and female nematodes.     

Evaluation of 31 Potential Biofumigant Brassicaceous Plants as Hosts for Three Meloiodogyne Species

Brassicaceous cover crops can be used for biofumigation after soil incorporation of the mowed crop. This strategy can be used to manage root-knot nematodes (Meloidogyne spp.), but the fact that many of these crops are host to root-knot nematodes can result in an undesired nematode population increase during the cultivation of the cover crop. To avoid this, cover crop cultivars that are poor or nonhosts should be selected. In this study, the host status of 31 plants in the family Brassicaceae for the three root-knot nematode species M. incognita, M. javanica, and M. hapla were evaluated, and compared with a susceptible tomato host in repeated greenhouse pot trials. The results showed that M. incognita and M. javanica responded in a similar fashion to the different cover cultivars. Indian mustard (Brassica juncea) and turnip (B. rapa) were generally good hosts, whereas most oil radish cultivars (Raphanus. sativus ssp. oleiferus) were poor hosts. However, some oil radish cultivars were among the best hosts for M. hapla. The arugula (Eruca sativa) cultivar Nemat was a poor host for all three nematode species tested. This study provides important information for chosing a cover crop with the purpose of managing root-knot nematodes.     

Parasitism of Heterodera schachtii and Meloidogyne javanica by Hirsutella rhossiliensis in Microplots over Two Growing Seasons

Numbers of cyst and root-knot nematodes and percentage parasitism by the nematophagous fungus Hirsutella rhossiliensis were quantified in microplots over 2 years. The microplots contained either sugarbeets in loam infested with Heterodera schachtii or tomatoes in sand infested with Meloidogyne javanica. The fungus was added to half of the microplots for each crop. Although H. rhossiliensis established in both microplot soils, the percentage of nematodes parasitized did not increase with nematode density and nematode numbers were not affected by the fungus. The results indicate that long-term interactions between populations of the fungus and cyst or root-knot nematodes will not result in biological control.     

Recycling Potential and Fitness of Steinernematid Nematodes Cultured in Curculio caryae and Galleria mellonella

The recycling potential of entomopathogenic nematodes in the pecan weevil, Curculio caryae, following inundative applications is an important factor in considering whether nematodes could be incorporated into a C. caryae management strategy. Our objective was to determine the recycling potential and fitness of Steinernema carpocapsae and S. riobrave cultured in C. caryae. To estimate fitness and quality, we reared nematodes in larvae of C. caryae and in the commonly used standard host, the greater wax moth, Galleria mellonella. Nematode lipid content, infectivity (power to invade), virulence (power to kill), and reproductive capacity (yield per insect) in C. caryae larvae were compared with G. mellonella data. Lipid content was higher in S. carpocapsae cultured in C. caryae than in G. mellonella, but S. riobrave lipid content was not affected by host source. Host source did not affect subsequent infectivity or virulence to C. caryae (P > 0.05) but did affect reproductive capacity (P < 0.0001). Both nematode species produced more progeny in C. caryae when they were first cultured in G. mellonella than when they were first passed through C. caryae. In terms of potential to recycle under field conditions, we predict that nematodes resulting from one round of recycling in C. caryae larvae would be equally capable of infecting and killing more weevils, but the potential to continue recycling in C. caryae would diminish over time due to reduced reproduction in that host.     

Review of Pasteuria penetrans: Biology,Ecology, and Biological Control Potential

Pasteuria penetrans is a mycelial, endospore-forming, bacterial parasite that has shown great potential as a biological control agent of root-knot nematodes. Considerable progress has been made during the last 10 years in understanding its biology and importance as an agent capable of effectively suppressing root-knot nematodes in field soil. The objective of this review is to summarize the current knowledge of the biology, ecology, and biological control potential of P. penetrans and other Pasteuria members. Pasteuria spp. are distributed worldwide and have been reported from 323 nematode species belonging to 116 genera of free-living, predatory, plant-parasitic, and entomopathogenic nematodes. Artificial cultivation of P. penetrans has met with limited success; large-scale production of endospores depends on in vivo cultivation. Temperature affects endospore attachment, germination, pathogenesis, and completion of the life cycle in the nematode pseudocoelom. The biological control potential of Pasteuria spp. have been demonstrated on 20 crops; host nematodes include Belonolaimus longicaudatus, Heterodera spp., Meloidogyne spp., and Xiphinema diversicaudatum. Pasteuria penetrans plays an important role in some suppressive soils. The efficacy of the bacterium as a biological control agent has been examined. Approximately 100,000 endospores/g of soil provided immediate control of the peanut root-knot nematode, whereas 1,000 and 5,000 endospores/g of soil each amplified in the host nematode and became suppressive after 3 years.     

Effects of Nematophagous Fungi on Numbers and Death Rates of Bacterivorous Nematodes in Arable Soil

In a series of microcosm experiments with an arable, sandy loam soil amended with sugarbeet leaf, the short-term (8 weeks) dynamics of numbers of nematodes were measured in untreated soil and in γ-irradiated soil inoculated with either a field population of soil microorganisms and nematodes or a mixed population of laboratory-propagated bacterivorous nematode species. Sugarbeet leaf stimulated an increase in bacterivorous Rhabditidae, Cephalobidae, and a lab-cultivated Panagrolaimus sp. Differences were observed between the growth rates of the nematode population in untreated and γ-irradiated soils, which were caused by two nematophagous fungi, Arthrobotrys oligospora and Dactylaria sp. These fungi lowered the increase in nematode numbers due to the organic enrichment in the untreated soil. We estimated the annually produced bacterivous nematodes to consume 50 kg carbon and 10 kg nitrogen per ha, per year, in the upper, plowed 25 cm of arable soil.     

Response of Resistant and Susceptible Bell Pepper (Capsicum annuum) to a Southern California Meloidogyne incognita Population from a Commercial Bell Pepper Field

To determine the presence and level of root-knot nematode (Meloidogyne spp.) infestation in Southern California bell pepper (Capsicum annuum) fields, soil and root samples were collected in April and May 2012 and analyzed for the presence of root-knot nematodes. The earlier samples were virtually free of root-knot nematodes, but the later samples all contained, sometimes very high numbers, of root-knot nematodes. Nematodes were all identified as M. incognita. A nematode population from one of these fields was multiplied in a greenhouse and used as inoculum for two repeated pot experiments with three susceptible and two resistant bell pepper varieties. Fruit yields of the resistant peppers were not affected by the nematodes, whereas yields of two of the three susceptible pepper cultivars decreased as a result of nematode inoculation. Nematode-induced root galling and nematode multiplication was low but different between the two resistant cultivars. Root galling and nematode reproduction was much higher on the three susceptible cultivars. One of these susceptible cultivars exhibited tolerance, as yields were not affected by the nematodes, but nematode multiplication was high. It is concluded that M. incognita is common in Southern California bell pepper production, and that resistant cultivars may provide a useful tool in a nonchemical management strategy.     

Transmission of Bursaphelenchus xylophilus through Oviposition Wounds of Monochamm carolinensis (Coleoptera: Cerambycidae)

Transmission of pinewood nematode through Monochamus carolinensis oviposition wounds was documented. Nematode transmission was measured as the average number of nematodes isolated per oviposition wound excavated and also as the percentage of oviposition wounds from which nematodes were isolated. The influence of three factors that might affect nematode transmission was investigated: age of the beetle vector, number of nematodes carried per beetle, and egg deposition in the oviposition wound. Only the number of nematodes carried by the beetle was found to have a significant effect on transmission. Nematodes were transmitted more frequently and in slightly greater numbers by beetles carrying more nematodes. The influence of pinewood on nematode exit from beetles were investigated by comparing nematode exit from beetles placed over pine chips with those placed over distilled water. Nematodes exited in greater numbers and at a higher frequency from beetles over pine chips than from beetles over distilled water. Apparently, the nematodes are able to detect a factor from the pine chips that promotes their exit from the beetles.     

Fergusobia/Fergusonina-induced Shoot Bud Gall Development on Melaleuca quinquenervia

Fergusobia nematodes and Fergusonina flies are mutualists that cause a variety of gall types on myrtaceous plant buds and young leaves. The biology of an isolate of the gall complex was studied in its native range in Australia for possible use in southern Florida as a biological control agent against the invasive broad-leaved paperbark tree, Melaleuca quinquenervia. Timed studies with caged Fergusonina flies on young branches of M. quinquenervia revealed that females are synovigenic with lifetime fecundities of 183 ± 42 (standard error; SE) eggs and longevities of 17 ± 2 days. None of the male flies but all dissected female flies contained parasitic female nematodes (range = 3-15), nematode eggs (12-112), and nematode juveniles (78-1,750). Female flies deposited eggs (34 ± 6; 8-77 per bud) and nematode juveniles (114 ± 15; 44-207 per bud) into bud apices within 15 days. Histological sections of shoot buds suggested that nematodes induce the formation of hypertrophied, uninucleate plant cells prior to fly larval eclosion. Enlarged size, granular cytoplasm, and enlarged nucleus and nucleolus characterized these cells, which appeared similar to those of other species galled by nematodes in the Anguinidae. Observations of ovipositional behavior revealed that female Fergusonina sp. create diagnostic oviposition scars. The presence of these scars may facilitate recognition of host use during specificity screening.     

Effect of Temperature on Infection and Survival of Rotylenchulus reniformis

From infestation of lettuce with preinfective females to egg deposition, populations of Rotylenchulus reniformis from Baton Rouge, Louisiana; Lubbock and Weslaco, Texas; and Mayaguez, Puerto Rico, required 41, 13, 7, and 7 days at 15, 20, 25, and 34 C, respectively. No nematode infection occurred at 10 C with any R. reniformis population, and the population from Puerto Rico did not reproduce at 15 C. Nematode survival was not influenced by temperature, since populations from Texas and Louisiana survived for 6 months without a host at - 5 , - 1 , 4, and 25 C. Survival of R. reniformis was substantially influenced by soil moisture. Soil moistures greater than 7% (< 1 bar) aided nematode survival at storage temperature of 25 C, whereas moisture adversely affected nematode survival below freezing. Soil moisture below 4% (> 15 bars) favored nematode survival below freezing but adversely affected nematodes in soils stored at 25 C. Soil moisture effects on nematode survival were less accentuated at 4 and 0 C.