At present, synthetic insecticides are still the primary means of controlling white grubs. With the implementation of the Food Quality Protection Act of 1996, golf turf managers have already lost many options for curative white grub control.

Neonicotinoid insecticides (imidacloprid, clothianidin) and insect growth regulators (halofenozide) are less hazardous than organophosphates and carbamates, but they are only effective when used preventively, resulting in the treatment of large areas that otherwise would have needed only partial or no treatment. In the long term, these insecticides' high efficacy against many turfgrass pests combined with their large-area applications is likely to reduce predators, parasitoids, and pathogens of white grubs and other insect pests by depriv-ing them of prey/hosts. Ultimately, this approach may increase dependency on chemical control.

ENTOMOPATHOGENIC NEMATODES AS AN ALTERNATIVE

Entomopathogenic nematodes (Hetero-rhabditidae and Steinernematidae) offer a non-toxic, environmentally safe, and IPM-compatible alternative to synthetic insecticides in turfgrass. These nematodes occur in natural and agricultural soils around the world and are used for biological control of insects, primarily soil-dwelling insects.

The only free-living stage of entomopathogenic nematodes is the infective juvenile that must persist in the soil until it can locate and infect a suitable host. After penetrating into the host's body cavity, the infective juvenile regurgitates species-specific symbiotically associated bacteria. The nematode and bacteria cooperate to kill the host within a few days. The developing nematodes feed on the bacteria and host tissues digested by the bacteria and develop through up to three generations, until hundreds to hundreds of thousands of new infective juveniles emerge from the depleted host cadaver to search for new hosts.

Research in the U.S. has shown that these nematodes can be as effective as synthetic insecticides against Japanese beetle larvae. However, recent research has shown that the masked chafer, oriental beetle, European chafer, Asiatic garden beetle, and other white grub species are less susceptible than the Japanese beetle to the commonly used nematode species such as Heterorhabditis bacteriophora and Steinernema glaseri.

STEINERNEMA SCARABAEI: A NEW HIGHLY VIRULENT NEMATODE

	Rutgers University scientists investigated the effects of soil moisture and soil type on the infectivity and persistence of nematodes for short- and long-term suppression of white grubs. Shown above is a white grub infected with Steinernema scarabaei.

In laboratory, greenhouse, and field studies, S. scarabaei has shown exceptional virulence to a wide range of white grub species, including the Japanese beetle, oriental beetle, Euro-pean chafer, Asiatic garden beetle, and several May/June beetle species. It dramatically outperformed any other nematode species tested in greenhouse and field studies, even at rates as low as one fourth of the other species applied. In ongoing field studies supported by the USGA, we have seen significant suppression of oriental beetle larval populations by S. scarabaei at least one year, often two years, after application. This long-term effect is due to the high virulence of S. scarabaei, the long persistence of its infec-tive juveniles, and its effective reproduction and recycling in the infected white grubs.

FACTORS AFFECTING NEMATODE EFFICACY AND SURVIVAL

The performance of entomopathogenic nematodes can be affected by many environmental factors. Two of the most important factors are soil moisture and soil type/texture. In soil, infective juveniles move through the water film that coats the pore spaces. If this film becomes too thin (in dry soil) or the pore spaces are completely filled with water (in water-saturated soil), nematode movement can be restricted. In field studies, soil moisture was positively related to H. bacteriophora efficacy against Japanese beetle larvae. Infective juveniles can survive dessiccation to relatively low moisture levels if water removal is gradual, giving them time to adapt to an inactive stage.

Generally, nematode survival and dispersal tend to be lower in fine-textured soils, but the effect of soil moisture and texture on nematode infectivity and survival varies with nematode species and may depend on nematode size, behavior, and physiology. In turfgrass trials against Japanese beetle larvae, H. bacteriophora was more effective in fine-textured soils than sandy soil, probably because finer soils retain moisture better and restrict nematode movement to the upper soil layers where most of the white grubs are found.

STUDIES ON THE EFFECT OF SOIL TYPE AND SOIL MOISTURE

To improve the predictability of S. scarabaei applications in the field, both for short-term and long-term white grub management, we conducted a series of laboratory and greenhouse experiments studying the effect of different soil types and moisture levels on the infectivity and survival of this species. For comparison, the well-known and widely available species H. bacteriophora was included in the study.

Five typical mineral soils were collected from turfgrass areas, acidic sand from a blueberry field, and a typical potting mix from a nursery. Soil characteristics and soil moisture release curves were established. Third-instar oriental beetles and Japanese beetles were collected in turf areas and stored individually at 50°F for one to ten weeks in a mixture of organic compost and loamy sand. The nematodes H. bacteriophora (GPS11 strain) and S. scarabaei (AMK001 strain) were cultured and stored following standard procedures.

EFFECT OF SOIL TYPE ON NEMATODE INFECTIVITY

Two laboratory experiments tested the effect of six substrates on nematode infectivity. Experiment 1 tested the infectivity of S. scarabaei (0 or 200 infective juveniles per vial) against oriental beetle larvae. Experiment 2 tested the infectivity of H. bacteriophora (0 or 500 infective juveniles per vial) against Japanese beetle larvae. At seven days after treatment (DAT), the larvae were recovered and infected larvae were dissected and digested in a pepsin solution, and the number of nematodes established in them were counted.

Figure 1. Effect of different substrates on infectivity (A-B) and efficacy (C-D) of the entomo-pathogenic nematode Steinernema scarabaei against the third-instar oriental beetle (A, C) and Heterorhabditis bacteriophora against the third-instar Japanese beetle (B, D). Columns with the same letter are not significantly different (P<0.05).

The number of S. scarabaei established in the larvae was significantly lower in acidic sand than in potting mix, and significantly lower in potting mix than in loam, sandy loam, and loamy sand, and significantly lower in silt loam than in loamy sand (Figure 1A). Larval mortality followed a very similar pat-tern, with significantly lower mortality in acidic sand (50%) and potting mix (67%) than in the other soils (90-100%). H. bacteriophora establishment in larvae (Figure 1B) was the highest in potting mix and the lowest in acidic sand and did not differ significantly among loamy sand, sandy loam, silt loam, and clay loam. Larval mortality also was the highest in potting mix (93%) and the lowest in acidic sand (13%) and did not differ significantly among loamy sand, sandy loam, silt loam, and clay loam (57-63%).

Two greenhouse experiments were with perennial ryegrass growing on the various substrates. Experiment 1 tested S. scarabaei (0 or 156 infective juveniles per pot) against oriental beetles. Experiment 2 tested H. bacterio-phora (0 or 625 infective juveniles per pot) against Japanese beetles. The pots were destructively sampled at 14 DAT to determine the number of surviving larvae.

Figure 2. Persistence of Steinernema scarabaei (A) and Heterorhabditis bacteriophora (B) in different substrates. Asterisk (*) indicates significant differences in recovery among substrate types per baiting date (P<0.05). A significant decline in recovery was found in no substrate for S. scarabaei and in all substrates for H. bacteriophora.

Mortality in the greenhouse experi-ment followed a trend similar to the mortality in the laboratory experiment, except that the negative effect of acidic sand was less pronounced, possibly modulated by the presence of grass roots (Figure 1). S. scarabaei caused higher mortality in loamy sand than in silt loam and potting mix, with sandy loam, loam, and acidic sand not significantly different from either group (Figure 1C). H. bacteriophora caused higher mortality in potting mix than in sandy loam, and the remaining soils were not significantly different from either group (Figure 1D).

EFFECT OF SOIL TYPE ON NEMATODE PERSISTENCE

Two laboratory experiments tested the effect of six soil types on persistence of S. scarabaei and H. bacteriophora. At different times after adding 200 infective juveniles per cup treatment (Figure 2), five cups from each treatment were opened and the soil baited with five wax moth larvae for four three-day baiting rounds. Infected larvae were dissected and digested to count the number of nematodes established in them.

Figure 3. Effect of soil water potential on infectivity of the entomopathogenic nematodes Steinernema scarabaei (A) and Heterorhabditis bacteriophora (B) in different substrates against the third-instar Popillia japonica. Columns with the same letter are not significantly different (P<0.05). Asterisk (*) indicates that no infection and mortality occurred at these data points.

S. scarabaei showed excellent persistence with no significant decline in recovery over time in any of the substrates (Figure 2A). Over the entire experiment, recovery was significantly higher in loamy sand than in all other soils, and significantly higher in sandy loam than in potting mix. H. bacteriophora persistence was much shorter, and recovery significantly declined in all substrates (Figure 2B). Overall, recovery was significantly higher in clay loam than in sandy loam and acidic sand, and was lower in potting mix than in all other soils.

EFFECT OF SOIL MOISTURE ON NEMATODE INFECTIVITY

Sandy loam was prepared at different soil water potentials (from wettest to driest: -1, -10, -100, -1000, -3000 kPa), allowed to equilibrate for four days, mixed again, and filled into plastic vials. Into each vial, one third-instar Japanese beetle was released and, one day later, 0 nematodes, 200 S. scarabaei, or 1,000 H. bacteriophora were added to the soil. At seven days after treatment (DAT), the larvae were recovered and infected larvae were dissected and the nematodes established in them counted.

Figure 4. Persistence of Steinernema scarabaei or Heterorhabditis bacteriophora in sandy loam at four soil water potentials. Asterisk (*) indicates significant differences in recovery among water potential per baiting date (P<0.05). A significant linear decline in recovery was found for -100 kPa and -1000 kPa in S. scarabaei and for -10 kPa to -3000 kPa in H. bacteriophora (P<0.05).

The number of S. scarabaei established in larvae was higher at -10 kPa and -100 kPa than at -1 kPa and -1000 kPa, but was the lowest at -3000 kPa (Figure 3A). Larval mortality by S. scarabaei was significantly higher at -100 kPa (100%), -10 kPa (97%), and -1000 kPa (87%), than at -3000 kPa (50%), with mortality at -1 kPa (77%) not significantly different from either group. H. bacteriophora establishment followed a similar pattern as for S. scarabaei at -1 kPa to -100 kPa, but was more restricted in drier soil with very low establishment at -1000 kPa and no establishment at -3000 kPa (Figure 3B). Similarly, larval mortality by H. bacteriophora was significantly higher at -1 kPa (57%), -10 kPa (77%), and -100 kPa (67%) than at -1000 kPa (17%), with no mortality at -3000 kPa.

S. scarabaei persistence was also excellent and was not affected by soil water potential (Figure 4A). Averaged across all soil water potentials, S. scarabaei recovery initially increased but declined thereafter and was significantly lower on day 140 than all other days. H. bacteriophora significantly declined much more quickly at all soil water potentials (Figure 4B), but declined the fastest at -10 kPa, somewhat slower at -100 kPa, much slower at -1000 kPa, and particularly -3000 kPa.

CONCLUSIONS

Our observations further illuminate the excellent potential of S. scarabaei for short-term and long-term suppression of white grub populations. In addition to its high virulence against a wide range of white grub species, it also showed high virulence across a wide range of substrate types. While S. scarabaei infectivity tended to decline from the coarser sandy soils to the finer clay soils, significant mortality was observed in greenhouse pot experiments even in the finest soils. Even in clay loam, S. scarabaei was only somewhat less infective in laboratory and greenhouse experiments. Only in highly acidic sand (pH 3.9) and highly organic potting mix did S. scarabaei infectivity decline significantly, though it still caused significant mortality.

In comparison, H. bacteriophora showed similar infectivity from the coarser to the finer soils, was also negatively affected in acidic sand, but was the most infective in the potting mix. Both S. scarabaei and H. bacteriophora were most infective at moderate soil moisture and less in saturated soil and drier soil. However, the infectivity range of S. scarabaei extended further into the dry range, with significant mortality even in dry soil (-3000 kPa), where H. bacteriophora did not cause infections. S. scarabaei also showed excellent persistence over all substrate types and soil moisture levels, whereas H. bacteriophora generally had shorter persistence, with more useful persist-ence levels only in the drier soils, where it becomes inactive.

The present study indicates that long-term white grub suppression should be achievable over a wide range of soil conditions. The major problem still to overcome in the commercialization of S. scarabaei is the development of effective mass production technology, which has proven to be difficult and may require more in-depth studies on S. scarabaei's nutritional requirements.

ACKNOWLEDGMENTS

We appreciate the technical assistance of Matthew Resnick, Sonya Kasper, Zachary Egen, and Jessica Tourangeau. This research was supported, in part, by grants from the USGA's Turfgrass and Environmental Research Program and the Rutgers Center for Turfgrass Science.

Editor's Note: This complete report can be found at USGA Turfgrass and Environmental Research Online: http://usgatero.msu.edu/v05/n19.pdf.

Albrecht M. Koppenhöfer, Ph.D., is associate professor and extension specialist and Eugene M. Fuzy is senior laboratory technician, Turfgrass Entomology, Depart-ment of Entomology, Rutgers University, New Brunswick, N.J.