Biological Control of the Pecan Weevil,Curculio caryae (Coleoptera: Curculionidae), with Entomopathogenic Nematodes

Steinernema carpocapsae (Weiser) strain A11, S. feltiae (Filipjev) strain SN, and Heterorhabditis bacteriophora Poinar strains HP88 and Georgia were tested for their efficacy as biological control agents of the pecan weevil, Curculio caryae (Horn), in pecan orchard soil-profile containers under greenhouse conditions. Percentage C. caryae parasitism by S. carpocapsae and H. bacteriophora strain HP88 and Georgia was consistently poor when applied either prior to or following C. caryae entry into the soil, suggesting that these nematode species and (or) their enterobacteria are poor biological control agents of weevil larvae. Soil taken 21 days following application of S. carpocapsae or H. bacteriophora strain HP88 induced a low rate of infection of Galleria mellonella larvae, whereas soil that had been similarily treated with H. bacteriophora strain Georgia induced a moderate rate of infection. Percentage C. caryae parasitism by S. feltiae was consistently low when applied following C. caryae entry into the soil and was inconsistent when applied as a barrier prior to entry of weevil larvae into the soil. Soil taken 21 days following application of S. feltiae induced a high rate of infection of G. mellonella larvae.     

Temperature Effects on Heterorhabditis megidis and Steinernema carpocapsae Infectivity to Galleria mellonella

The effect of temperature on the infection of larvae of the greater wax moth, Galleria mellonella, by Heterorhabditis megidis H90 and Steinernema carpocapsae strain All, was determined. For both species, infection, reproduction, and development were fastest at 20 to 24 °C. Infection by both H. megidis and S. carpocapsae occurred between 8 and 16 °C; however, neither species reproduced at 8 °C. Among the nematodes used in experiments at 8 °C, no H. megidis and very few S. carpocapsae developed beyond the infective juvenile stage. Compared with H. megidis, S. carpocapsae invaded and killed G. mellonella larvae faster at 8 to 16 °C. By comparing invasion rates, differences in infectivity between the two nematode species were detected that could not be detected in conventional petri dish bioassays where mortality was measured after a specified period. Invasion of G. mellonella larvae by H. megidis was faster at 24 than at 16 °C.     

Comparison of Two Steinernematid Species for Control of the Root Weevil Diaprepes abbreviatus

Steinernema carpocapsae Weiser All strain was compared to Steinernema riobravis Cabanillas, Poinar, and Raulston for control of the root weevil, Diaprepes abbreviatus (L.), in the laboratory and in potted citrus. In the laboratory bioassay, D. abbreviatus larvae were exposed to 30, 60, and 120 nematodes/cm³ in sand. Insect mortality 1 week after application was greater (P ≤ 0.05) for S. riobravis than for S. carpocapsae in the laboratory bioassay. In the greenhouse bioassay, D. abbreviatus larvae were exposed to 3 and 9 nematodes per cm³ of soil in potted citrus. Again, at each rate, mortality was greater (P ≤ 0.05) in pots treated with S. riobravis than in pots treated with S. carpocapsae. The results of this study suggest that S. riobravis is a better biological control agent against D. abbreviatus larvae in potted plants than S. carpocapsae.     

Effect of Soil Moisture and a Surfactant on Entomopathogenic Nematode Suppression of the Pecan Weevil, Curculio caryae

Our overall goal was to investigate several aspects of pecan weevil, Curculio caryae, suppression with entomopathogenic nematodes. Specifically, our objectives were to: 1) determine optimum moisture levels for larval suppression, 2) determine suppression of adult C. caryae under field conditions, and 3) measure the effects of a surfactant on nematode efficacy. In the laboratory, virulence of Heterorhabditis megidis (UK211) and Steinernema carpocapsae (All) were tested in a loamy sand at gravimetric water contents of negative 0.01, 0.06, 0.3, 1.0, and 15 bars. Curculio caryae larval survival decreased as moisture levels increased. The nematode effect was most pronounced at –0.06 bars. At –0.01 bars, larval survival was ≤5% regardless of nematode presence, thus indicating that intense irrigation alone might reduce C. caryae populations. Overall, our results indicated no effect of a surfactant (Kinetic) on C. caryae suppression with entomopathogenic nematodes. In a greenhouse test, C. caryae larval survival was lower in all nematode treatments compared with the control, yet survival was lower in S. carpocapsae (Italian) and S. riobrave (7–12) treatments than in S. carpocapsae (Agriotos), S. carpocapsae (Mexican), and S. riobrave (355) treatments (survival was reduced to approximately 20% in the S. riobrave [7–12] treatment). A mixture of S. riobrave strains resulted in intermediate larval survival. In field experiments conducted over two consecutive years, S. riobrave (7–12) applications resulted in no observable control, and, although S. carpocapsae (Italian) provided some suppression, treatment effects were generally only detectable one day after treatment. Nematode strains possessing both high levels of virulence and a greater ability to withstand environmental conditions in the field need to be developed and tested.     

Curative Control of the Peachtree Borer Using Entomopathogenic Nematodes

The peachtree borer, Synanthedon exitiosa (Say 1823), is a major pest of stone fruit trees in North America. Current management relies upon preventative control using broad-spectrum chemical insecticides, primarily chlorpyrifos, applied in the late summer or early fall. However, due to missed applications, poor application timing, or other factors, high levels of S. exitiosa infestation may still occur and persist through the following spring. Curative treatments applied in the spring to established infestations would limit damage to the tree and prevent the next generation of S. exitiosa from emerging within the orchard. However, such curative measures for control of S. exitiosa do not exist. Our objective was to measure the efficacy of the entomopathogenic nematode, Steinernema carpocapsae, as a curative control for existing infestations of S. exitiosa. In peach orchards, spring applications of S. carpocapsae (obtained from a commercial source) were made to infested trees and compared with chlorpyrifos and a water-only control in 2014 and 2015. Additionally, types of spray equipment were compared: nematodes were applied via boom sprayer, handgun, or trunk sprayer. To control for effects of application method or nematode source, in vivo laboratory-grown S. carpocapsae, applied using a watering can, was also included. Treatment effects were assessed 39 d (2014) or 19 d (2015) later by measuring percentage of trees still infested, and also number of surviving S. exitiosa larvae per tree. Results indicated that S. carpocapsae provided significant curative control (e.g., >80% corrected control for the handgun application). In contrast, chlorpyrifos failed to reduce S. exitiosa infestations or number of surviving larvae. In most comparisons, no effect of nematode application method was detected; in one assessment, only the handgun and watering can methods reduced infestation. In conclusion, our study indicates that S. carpocapsae may be used as an effective curative measure for S. exitiosa infestations.     

Entomopathogenic Nematodes for Control of Insect Pests Above and Below Ground with Comments on Commercial Production

Entomopathogenic nematodes (EPNs) have been utilized in classical, conservation, and augmentative biological control programs. The vast majority of applied research has focused on their potential as inundatively applied augmentative biological control agents. Extensive research over the past three decades has demonstrated both their successes and failures for control of insect pests of crops, ornamental plants, trees and lawn and turf. In this paper we present highlights of their development for control of insect pests above and below ground. The target insects include those from foliar, soil surface, cryptic and subterranean habitats. Advances in mass-production and formulation technology of EPNs, the discovery of numerous efficacious isolates/strains, and the desirability of reducing pesticide usage have resulted in a surge of commercial use and development of EPNs. Commercially produced EPNs are currently in use for control of scarab larvae in lawns and turf, fungus gnats in mushroom production, invasive mole crickets in lawn and turf, black vine weevil in nursery plants, and Diaprepes root weevil in citrus in addition to other pest insects. However, demonstrated successful control of several other insects, often has not lead to capture of a significant share of the pesticide market for these pests.     

Comparison of Three Methods for Estimating the Number of Entomopathogenic Nematodes Present in Soil Samples

Numbers of Steinernema sp. (CB2B) and S. carpocapsae (Agriotos) exponentially declined after application into a clay loam soil. Over a 35-day sampling period, Steinernema sp. (CB2B) was more persistent than S. carpocapsae (Agriotos). The presence or absence of the second-stage cuticle on the third-stage juveniles (J3) at the time of application did not alter the rate of population decline of Steinernema sp. (CB2B). Nearly all J3 of Steinernema sp. (CB2B) and S. carpocapsae (Agriotos) lost their cuticle within 24 hours of being in soil. Centrifugal flotation recovered the greatest number of nematodes, with a lower variance than either the live bait or Baermann funnel techniques. A strong positive linear relationship was evident between numbers of nematodes present in the soil and the numbers that established in a bait insect. Approximately 40% of Steinernema sp. (CB2B) and 30% of the S. carpocapsae (Agriotos) present in the soil established in Galleria mellonella larvae. The extraction techniques had different efficiencies and gave different relative estimates of persistence for the two species. Persistence and infectivity was best measured using a combination of live bait and flotation techniques.     

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

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

Effect of Entomopathogenic Nematodes on Mesocriconema xenoplax Populations in Peach and Pecan

The effect of Steinernema riobrave and Heterorhabditis bacteriophora on population density of Mesocriconema xenoplax in peach was studied in the greenhouse. Twenty-one days after adding 112 M. xenoplax adults and juveniles/1,500 cm³ soil to the soil surface of each pot, 50 infective juveniles/cm² soil surface of either S. riobrave or H. bacteriophora were applied. Another entomopathogenic nematode application of the same density was administered 3 months later. The experiment was repeated once. Mesocriconema xenoplax populations were not suppressed (P ≤ 0.05) in the presence of either S. riobrave or H. bacteriophora 180 days following ring nematode inoculation. On pecan, 200 S. riobrave infective-stage juveniles/cm² were applied to the soil surface of 2-year-old established M. xenoplax populations in field microplots. Additional applications of S. riobrave were administered 2 and 4 months later. This study was terminated 150 days following the initial application of S. riobrave. Populations of M. xenoplax were not suppressed in the presence of S. riobrave.     

Susceptibility of the Plum Curculio,Conotrachelus nenuphar,to Entomopathogenic Nematodes

The plum curculio, Conotrachelus nenuphar, is a major pest of pome and stone fruit. Our objective was to determine virulence and reproductive potential of six commercially available nematode species in C. nenuphar larvae and adults. Nematodes tested were Heterorhabditis bacteriophora (Hb strain), H. marelatus (Point Reyes strains), H. megidis (UK211 strain), Steinernema riobrave (355 strain), S. carpocapsae (All strain), and S. feltiae (SN strain). Survival of C. nenuphar larvae treated with S. feltiae and S. riobrave, and survival of adults treated with S. carpocapsae and S. riobrave, was reduced relative to non-treated insects. Other nematode treatments were not different from the control. Conotrachelus nenuphar larvae were more susceptible to S. feltiae infection than were adults, but for other nematode species there was no significant insect-stage effect. Reproduction in C. nenuphar was greatest for H. marelatus, which produced approximately 10,000 nematodes in larvae and 5,500 in adults. Other nematodes produced approximately 1,000 to 3,700 infective juveniles per C. nenuphar with no significant differences among nematode species or insect stages. We conclude that S. carpocapsae or S. riobrave appears to have the most potential for controlling adults, whereas S. feltiae or S. riobrave appears to have the most potential for larval control.     

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.     

Influence of Soil pH and Oxygen on Persistence of Steinernema spp.

Survival of infective juveniles of Steinernema carpocapsae and Steinernema glaseri gradually declined during 16 weeks of observation as the tested soil pH decreased from pH 8 to pH 4. Survival of both species of Steinernema dropped sharply after 1 week at pH 10. Survival or S. carpocapsae and S. glaseri was similar at pH 4, 6, and 8 during the first 4 weeks, but S. carpocapsae survival was significantly greater than S. glaseri at pH 10 through 16 weeks. Steinernema carpocapsae and S. glaseri that had been stored at pH 4, 6, and 8 for 16 weeks, and at pH 10 for 1 or more weeks were not infective to Galleria mellonella larvae. Steinernema carpocapsae survival was significantly greater than that of S. glaseri at oxygen:nitrogen ratios of 1:99, 5:95, and 10:90 during the first 2 weeks, and survival of both nematode species declined sharply to less than 20% after 4 weeks. Survival of both nematode species significantly decreased after 8 weeks as the tested oxygen concentrations decreased from 20 to 1%, and no nematode survival was recorded after 16 weeks. Steinernema carpocapsae pathogenicity was significantly greater than that of S. glaseri during the first 2 weeks. No nematode pathogenicity was recorded at oxygen concentrations of 1, 5, and 10% after 2 weeks and at 20% after 16 weeks.     

Development Rates in Entomopathogenic Nematodes: Infected Hosts vs. Aqueous Suspension

Rearing conditions have been shown to affect several aspects of entomopathogenic nematode biology, including dispersal behavior and infectivity. The present study explores the differences in development rate of Heterorhabditis bacteriophora and Steinernema carpocapsae when infective juveniles (IJ) were collected in water using the standard White trap method vs. natural emergence from cadavers into sand. We exposed Galleria mellonella to IJ entompopathogenic nematodes treated in one of three ways: collected in a White trap, allowed to emerge directly into sand, or collected in a White trap and treated with a cadaver homogenate. When S. carpocapsae IJ were allowed to emerge from cadavers directly into sand and then allowed to infect new hosts, they developed into adults at a faster rate than IJ that were collected with White traps. The difference in development was not due to differential infection rates. No difference in development stages was detected amount the same H. bacteriophora treatments.     

Compatibility of Soil Amendments with Entomopathogenic Nematodes

The impact of inorganic and organic fertilizers on the infectivity, reproduction, and population dynamics of entomopathogenic nematodes was investigated. Prolonged (10- to 20-day) laboratory exposure to high inorganic fertilizer concentrations inhibited nematode infectivity and reproduction, whereas short (1-day) exposures increased infectivity. Heterorhabditis bacteriophora was more sensitive to adverse effects than were two species of Steinernema. In field studies, organic manure resulted in increased densities of a native population of Steinernema feltiae, whereas NPK fertilizer suppressed nematode densities regardless of manure applications. Inorganic fertilizers are likely to be compatible with nematodes in tank mixes and should not reduce the effectiveness of nematodes used for short-term control as biological insecticides, but may interfere with attempts to use nematodes as inoculative agents for long-term control. Organic manure used as fertilizer may encourage nematode establishment and recycling.     

Cryopreservation of Steinernema carpocapsae and Heterorhabditis bacteriophora

A method for the cryopreservation of third-stage infective juveniles (IJ) of Steinernema carpocapsae and Heterorhabiditis bacteriophora was developed. Cryoprotection was achieved by incubating the nematodes in 22% glycerol (S. carpocapsae) or 14% glycerol (H. bacteriophora) for 24 hours, followed by 70% methanol at 0 C for 10 minutes. The viability of S. carpocapsae frozen in liquid nitrogen as 20 μl volumes spread over cover slip glass was > 80%. Survival of H. bacteriophora frozen on glass varied from 10 to 60% but was improved to > 80% by replacing the glass with filter paper. Cryopreservation and storage of 1-ml aliqots of S. carpocapsae IJ resulted in > 50% survival after 8 months; pathogenicity was retained and normal in vitro development took place. Trehalose and glycerol levels increased and glycogen levels decreased during incubation of S. carpocapsae IJ in glycerol. Normal levels of trehalose, glycerol and glycogen were restored during post freezing rehydration.     

Soil Applications of Steinernematid and Heterorhabditid Nematodes for Control of Colorado Potato Beetles,Leptinotarsa decemlineata (Say)

Three strains of Steinernema feltiae Filipjev (All, Mexican, and Breton strains) and one of Heterorhabditis heliothidis (Khan, Brooks, and Hirschmann) were evaluated for their potential to control Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say), larvae and pupae in the soil. In laboratory studies, H. heliothidis and S. feltiae (Mexican strain) produced the highest mortality (6 days posttreatment) of CPB when applied to the surface of a soil column containing mature CPB larvae 5 cm below. Mortality ranged from 80 to 90% at rates of 79-158 nematodes/cm². Similar results were seen in a field microplot study with all four nematodes; S. feltiae (Mexican strain) and H. heliothidis were most effective. Adult CPB emergence was reduced 86.5-100% after application of 31-93 H. heliothidis/cm² and 88.4-100% with 93-155 S. feltiae (Mexican strain)/cm². The All strain of S. feltiae was moderately effective (ca. 80% reduction at 93-155 nematodes/cm²), while the Breton strain was ineffective (< 40% reduction at 155 nematodes/cm²). In small plots of potatoes enclosed in field cages, application of H. heliothidis and S. feltiae (Mexican strain) at rates of 93-155 nematodes/cm² before larval CPB burial in the soil resulted in 66-77% reduction in adult CPB emergence. Soil applications of these nematodes show potential for biological control of CPB.     

Nonsusceptibility of the Honey Bee,Apis mellifera (Hymenoptera: Apidae), to Steinernematid and Heterorhabditid Nematodes

We exposed honey bee workers and brood to four entomopathogenic nematode species under conditions normally encountered in the hive by spraying nematodes onto combs. Mortality of adult bees exposed to any of the nematode species was less than 10%, and there was no evidence of nematode infection when dead adults were dissected. To assess the impact of nematodes on brood, we used smaller-size honey combs placed in the second story (super) of a hive and large brood combs placed in the main section of the hive. Our results were inconsistent between these two experimental designs. The smaller honey combs sprayed with Steinernema carpocapsae contained the largest number of uncapped ceils, those sprayed with Heterorhabditis baeteriophora or S. riobravis contained an intermediate number of uncapped cells, and control combs and those sprayed with S. glaseri contained the fewest nmnber of uncapped cells. Large combs sprayed with S. riobravis contained more uncapped ceils than controls or those sprayed with S. carpocapsae, although the differences were not significant. Our results do not support the hypothesis that high-temperature-tolerant species of nematodes are necessarily more infective to honey bees.     

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.     

Entomopathogenic Nematodes and Bacteria Applications for Control of the Pecan Root-Knot Nematode, Meloidogyne partityla, in the Greenhouse

Meloidogyne partityla is a parasite of pecan and walnut. Our objective was to determine interactions between the entomopathogenic nematode-bacterium complex and M. partityla. Specifically, we investigated suppressive effects of Steinernema feltiae (strain SN) and S. riobrave (strain 7–12) applied as infective juveniles and in infected host insects, as well as application of S. feltiae''s bacterial symbiont Xenorhabdus bovienii on M. partityla. In two separate greenhouse trials, the treatments were applied to pecan seedlings that were simultaneously infested with M. partityla eggs; controls received only water and M. partityla eggs. Additionally, all treatment applications were re-applied (without M. partityla eggs) two months later. Four months after initial treatment, plants were assessed for number of galls per root system, number of egg masses per root system, number of eggs per root system, number of eggs per egg mass, number of eggs per gram dry root weight, dry shoot weight, and final population density of M. partityla second-stage juveniles (J2). In the first trial, the number of egg masses per plant was lower in the S. riobrave-infected host treatment than in the control (by approximately 18%). In the second trial, dry root weight was higher in the S. feltiae-infected host treatment than in the control (approximately 80% increase). No other treatment effects were detected. The marginal and inconsistent effects observed in our experiments indicate that the treatments we applied are not sufficient for controlling M. partityla.     

Control of the Oriental Fruit Moth, Grapholita molesta, Using Entomopathogenic Nematodes in Laboratory and Fruit Bin Assays

The oriental fruit moth (OFM), Grapholita molesta (Busck), which is among the most important insect pests of peaches and nectarines, has developed resistance to a wide range of insecticides. We investigated the ability of the entomopathogenic nematodes (EPN) Steinernema carpocapsae (Weiser), S. feltiae (Filipjev), S. riobrave (Cabanillas et al.), and Heterorhabditis marelatus (Liu and Berry) to control OFM under laboratory and fruit bin conditions. At a dosage of 10 infective juveniles (IJ)/cm² in the laboratory, S. carpocapsae caused 63%, S. feltiae 87.8%, S. riobrave 75.6%, and H. marelatus 67.1% OFM mortality. All four nematode species caused significant OFM larval mortality in comparison to the nontreated controls. Steinernema feltiae was used for the bin assays due to the higher OFM mortality it caused than the other tested EPN species and to its ability to find OFM under cryptic environments. Diapausing cocooned OFM larvae in miniature fruit bins were susceptible to IJ of S. feltiae in infested corner supports and cardboard strips. Treatment of bins with suspensions of 10 or 25 S. feltiae IJ/ml water with wetting agent (Silwet L77) resulted in 33.3 to 59% and 77.7 to 81.6% OFM mortality in corner supports and cardboard strips, respectively. This paper presents new information on the use of EPN, specifically S. feltiae, as nonchemical means of OFM control.