Final Report for SW04-113
We evaluated the influence of fertility practices and cover-cropping on the use of insect-attacking, entomopathogenic nematodes (EPNs) as biocontrol agents against the Colorado potato beetle. In two field experiments we released laboratory-reared EPNs into potato field plots treated with either organic or conventional soil amendments (manure and chemical fertilizer respectively). These experiments demonstrated that manure application reduced EPN infection rates on potato beetles, compared to beetle infection rates in plots receiving chemical fertilizer. Factors such as soil pH, predation, and interference may be limiting survival of EPNs in plots fertilized with manure. Survey data show that organic and conventional potato fields have robust endemic EPN populations. Thus conservation of the EPNs already resident in fields may be an important component of a potato beetle integrated pest management program. A series of field cage experiments evaluated the impact of both insect predator and insect pathogen communities on potato beetle control. We found decreasing pest survival with increasing natural enemy diversity. Subsequent analyses demonstrated that improved beetle suppression was driven by the pairing of predators with insect pathogens. In two other large-scale field trials, conducted in each of two field seasons, we found that the EPN species Steinernema feltiae significantly reduced populations of plant-attacking root knot nematodes while also controlling at least one generation of Colorado potato beetle larvae.
1. Investigate inoculative releases of entomopathogenic fungi and nematodes for beetle control.
2. Determine whether cover crops affect densities of pest & beneficial nematodes and insects.
3. Examine the combination of entomopathogens, predators, and cover crops for beetle control.
4. Investigate the economics of adoption of the techniques we are examining.
5. Distribute cultures of entomopathogens, and information on how they interact with cover crops, to conventional and organic potato growers.
Potatoes are a major component of agriculture in the Pacific Northwest. For example, in Washington potatoes are the #3 dollar-value crop with annual production on 170,000 acres worth over $450 million. Two beetles are devastating pests – the Colorado potato beetle (“CPB”), Decemlineata leptinotarsa, and wireworms, Limonius spp. CPB can completely defoliate plants, destroying a crop (Hare 1980), while wireworms damage tubers in the ground (Jannson and Seal 1994, Horton and Landolt 2002). Because insects are so damaging, conventional growers rely on “calendar sprays” of broad-spectrum pesticides, as frequently as every 10 days, making potatoes among the most intensively sprayed crops in the region (Ruffle and Miller 2002).
Organic potato production is relatively new in the irrigated inland northwest. Growth in acreage has been rapid, from <200 acres in the early 1990s (A. McErlich, personal communication) to several thousand acres in 2003, but organic growers have few control options for CPB and wireworms beyond a limited range of neem, spinosad and Bt treatments. While pesticides can effectively control the CPB, these chemical treatments are expensive. Currently no effective chemical control is available to organic growers for the control of wireworms. Wireworms are a particularly grave problem in organic crops planted in former CRP land, or following corn, because grasses are important alternative hosts for wireworms. Recently, organic growers have sometimes sustained near-total crop losses due to beetle damage. In a recent informal survey we conducted, all organic growers identified insects as the number one threat to their crops (weeds are controlled by frequent tillage), slowing the speed of adoption of organic potato production.
Both beetle pests spend a substantial portion of their life cycle underground – potato beetles pupate in the soil, while wireworms spend the entire juvenile stage underground – and so fungal pathogens and soil dwelling nematodes (“entomopathogenic nematodes”) are ideally placed to attack these pests (Lacey 2002). The fungus Beauveria bassiana has been the most studied microbial control agent for CPB. Control of CPB ranging from poor to excellent has been reported for the fungus (Hajek et al., 1987; Poprawski et al., 1997; Lacey et al., 1999; Poprawski and Wraight, 2000; Wraight and Ramos, 2002). Beauveria bassiana offers the advantage of recycling in host cadavers and persisting in the soil beneath potato plants thereby affecting the survival of subterranean stages of the beetle. Several studies have documented a variety of factors that influence the activity of this fungus against CPB in more humid conditions and rain fed crops (Poprawski et al., 1997; Jaros-Su et al., 1999; Inglis et al., 2000; Martin et al., 2000; Todorova et al., 2000; Furlong and Groden, 2001; Wraight and Ramos, 2002), but relatively few have been conducted in Pacific Northwest, especially under organic farming conditions. Entomopathogenic nematodes have also been proposed as control agents of CPB (Toba et al., 1983; Cantelo and Nickle, 1992; Nickle et al., 1994; Berry et al., 1997; Stewart et al., 1998). Berry et al. (1997) detected nematode activity in soil in their Hermiston, OR, research plots up to 14 weeks following nematode application. Relatively little research on the effect of entomopathogenic nematodes on wireworms has been conducted. Toba et al. (1983) attempted unsuccessfully to infect larvae of Limonius californicus with entomopathogenic nematodes. However, recent studies conducted with several nematode species on the Pacific wireworm, L. canus, at the USDA Yakima Agricultural Research Laboratory, reveal potential for control using steinernematid nematodes (L. Lacey, unpublished data). In addition the fungus Metarhizium anisopliae has been reported from wireworms (Zakaruk, 1981) and is currently under development for control of these beetles in British Columbia and Alberta, Canada, and in Washington State (L. Lacey, personal communication). Attractive baits developed by Horton and Landolt (2002) for L. canus, offer potential for enhancing control by drawing wireworms to entomopathogen release sites. In many cases entomopathogenic nematodes can be applied in combination with synthetic pesticides without the worms being harmed (Georgis 1990). This compatibility between chemical and biological control should allow both organic and conventional growers to take advantage of insect control by entomopathogens.
Recently, mustard cover crops (“green manures”) have been increasingly adopted by both conventional and organic potato growers in the Columbia Basin of Washington state, such that these cover crops are now used on ca. 10% of conventional acreage, and most organic acreage, of potatoes in the state (McGuire 2003). These mustards are planted in the fall before a field is to be planted with potatoes, and then cut and tilled into the soil either in the late fall or early spring. Mustard cover crops help prevent soil erosion, increase organic matter in the soil, and suppress plant-parasitic nematodes (McGuire 2003). While the impact of these manures on free-living and plant-parasitic nematodes is particularly well documented (Riga et al. in press, Collins et al. in review), their impact on pest and beneficial insects, and on entomopathogenic nematodes, is unknown. Green manures might augment densities of beneficial insects by providing cover and alternative prey in the fall when fields would otherwise be fallow (Bugg and Pickett1998), and by adding organic matter to the soil should increase densities of detritivorous insects that are also alternative prey for beneficials (Wise et al. 1999). Green manures might also suppress soil-dwelling insect pests, if they have the same suppressive effect on insects that they have on plant-parasitic nemotodes (Riga et al. in press). Further, by improving soil quality, green manures might make subsequent potato crops more vigorous and thus more resistant to insect feeding injury (McGuire 2003). On the other hand, green manures might make pest problems worse if they are toxic to important soil-dwelling beneficials such as entomopathogenic nematodes and carabid and staphylinid beetles.
The objective of our proposal was to examine several techniques to augment and establish populations of entomopathogenic nematodes in potato fields. Furthermore, we examined whether mustard cover crops, which are already being used by our grower-cooperators to control plant parasitic nematodes, will interfere with the establishment of beneficial fungi, nematodes and predatory insects, or indeed whether cover cropping and these biocontrol agents will work together to control beetle pests. Our goal was to provide growers with a more complete picture of the advantages and disadvantages of using biocontrol agents on their own and in combination with cover crops. All of our research was overseen and directed by the Organic Potato Grower Advisory Group (“OPGAP”), all of who, despite the group’s name, farm both organic and conventional potatoes. The versatility of our grower cooperators allowed us to investigate novel pest control techniques under both conventional and organic management.
Soil fertility effects on insect pathogens
To examine the augmentative release of entomopathogenic nematodes in potato fields under organic versus conventional fertility, we conducted a series of field experiments at the Washington State University Othello Research Station in the summers of 2005, 2006, and 2007. Potato (cv. Russet Ranger) seed pieces were straight planted into approximately 0.25 acre of land in sandy loam soil. Each plot measured 4.27m x 4.27m with a total of 48 plots in 2005, 24 plots in 2006, and 6 plots in 2007. The plots contained 5 rows of potatoes with 2 fallow rows as a buffer to adjacent plots, with 4.27m of bare ground separating plots within rows. The treatments were laid out in a fully factorial design with a total of eight replicates per treatment combination in 2005, and six replicates in 2006. In 2005 the six treatment combinations consisted of crosses between organic or conventional soil amendments with three entomopathogenic nematode rates (no EPNs, low EPNs, and high EPNs). In 2006 there were four treatment combinations consisting of the presence/absence of EPN application on plots receiving either organic or conventional soil fertilizers. In 2007 only two treatments were created, organic and conventional fertility management, without an EPN application. Organic soil amended plots were prepared by a preplant shovel application of a chicken (28.12kg/plot) and cow manure (47.17kg/plot) mixture (later incorporated into the soil). Conventional (chemical) fertilized plots were pre-plant strip applied at a rate of 362.87kg/acre. Only one application of fertilizer (organic or conventional) was applied at the start of the season. Two weeks after the fertilizer was incorporated the potatoes were straight planted into the area. Throughout the season the potato crop was irrigated using sprinklers every other day for three hours per day.
In 2005, entomopathogenic nematodes were applied at two rates (low and high), twice during the season. The initial application, 20 June 2005, targeted the first generation of the Colorado potato beetle, when the last instar larvae (first life stage to be associated with the soil) were at their highest density. The final application, 5 July 2005, occurred two weeks prior to the peak of the second generation. In 2006, a ‘high’ rate of entomopathogenic nematodes was applied a single time during the season, before the first peak of the first generation of potato beetles. In all experiments receiving EPN applications, a mixture of two species -- Steinernema carpocapsae and Heterorhabditis marelatus -- was applied. The low rate consisted of ~100,000,000 IJs/acre, while the high rate consisted of ~1,000,000,000 IJs/acre. The nematodes were reared in the laboratory by collecting them from infected wax worm cadavers on sponges, using a modified white trap method: a Petri dish was snuggly fitted with a moistened sponge (1cm thick) where a donut shape (2.5 cm wide) transparency paper and moistened filter paper respectively lay centered on the sponge. The infected wax worms were laid on top of the filter paper with the head facing the center of the sponge, creating a ring. The nematodes were extracted from the sponges a week after emergence from the wax worms, the day of the field application, by immediately placing the nematodes into a backpack sprayer (Field King 5-gallon Deluxe Sprayer). The nematodes were applied in the morning of each application date and all plots were irrigated with sprinklers one hour prior to application and then at least one hour after application. No other fertilizers or pesticides were added throughout the duration of the experiment.
The wax worm (obtained from Sunshine Mealworm, Silverton, OR), Galleria mellonella, is highly susceptible to entomopathogens and was used as a sentinel host in field bioassays. Many studies evaluate nematode infection by gathering soil samples from the field, placing the soil in Petri dishes, then placing wax worms on the soil surface within a Petri dish. Instead, in our work we placed waxworms directly into field plots to sample for EPNs. Host infection, station samples taken on the morning of each sampling period beginning one week after the fertilizer was incorporated into the soil. Groups of five larvae were placed in mesh sacks (window screen closed and sealed with a twist tie). Five sacks (sub-samples) were placed in each plot and buried at a depth of 10-15 cm, a depth similar to that at which Colorado potato beetles pupate, for 48 hrs. Sacks were then collected and larvae were placed into White traps (5 larvae/Petri dish) where larvae were monitored for nematode infection over 2 weeks. In 2005, Colorado potato beetle larvae were used as the sentinel host during peaks when larval populations were at their highest. The data generated using this sampling method was % wax worm or potato beetle infected, per plot and sample date. All data were analyzed using SYSTAT 9. A repeated measures ANOVA was used to analyze the interaction between soil fertility and nematode application over the field season for 2005 and 2006. Control plots were used to get a measure of naturally occurring nematode infection and were not used in the analyses. The data at time 0 was also not used as it was a measure of nematode infection before applying nematodes to make any adjustments with resident EPNs. In 2006 and 2007, organic and conventional plots were sampled to examine differences in soil properties including pH, moisture, and microbial activity, factors that may decrease EPN efficacy.
Predator and pathogen biodiversity experiments
To examine the impacts of conservation biological control, a series of field experiments were conducted at the Washington State University Othello Research Station in the summers of 2006 and 2007. The potato system consists of several resident, naturally occurring predators and pathogens. Aboveground natural enemies include generalist predators such as the convergent lady beetle (Hippodamia convergens), an active forager on plant foliage and a well-known potato beetle egg predator; the damsel bug (Nabis spp.), a sit-and-wait predator of stationary eggs and various mobile larval stages in the plant canopy; and a predatory ground beetle, Pterostichus melanarius, a nocturnal predator of potato beetle larvae moving on the soil surface. These predators are functionally diverse because of their stage-specific attack preferences, hunting styles and location in the foliage, all of which might lead to complementarity and thus more efficient resource exploitation. Belowground, several entomopathogens occur that attack the mobile pre-pupal potato beetles as they move through the soil, the quiescent pupal stage, and the teneral adults that burrow through to the soil surface after pupation. Steinernema and Heterorhabditis are entomopathogenic nematode genera known to infect potato beetle stages belowground. In addition, Beauvaria bassiana is a fungal pathogen commonly used to control potato beetles in the soil. Similar to the aboveground predators, the pathogens found belowground are also functionally diverse. Each entomopathogen attacks potato beetles differently: while B. bassiana infects insects crawling at or near the soil surface, Steinernema carpocapsae is an ambusher (sitting and waiting for the host to come in contact) and Heterorhabditis marelatus is a cruiser (using chemoreceptors to actively search for its host) each occurring at different soil depths.
Cages were constructed with plastic tubs as the base (68 liter, Sterilite) to isolate a plot area and catch pupating potato beetles. A frame was constructed out of PVC pipe (1.27 cm thick, 0.61 m x 0.46 m x 0.30 m) and fitted with nylon netting to contain insects, which was then placed atop each tub. The containers were placed into the ground, filled with soil, and 2 potato plants were transplanted into each tub. In 2006, five potato beetle egg clusters (10 eggs per cluster) were haphazardly attached to leaves and 40 larvae (20 first and second instar and 20 third and fourth instar) were placed on leaves. Potato beetle eggs and larvae were hand picked from an adjacent field and added into each cage immediately. The potato beetles were given 24 hours to establish, after which predators were released by treatment. Aboveground predators were field collected using a D-vac suction sampler and hand picked while pathogens were reared or purchased. In this study, natural enemy species richness (0, 1, 2, and 5 species) of three aboveground predators (Hippodamia convergens, Nabis spp., Pterostichus melanarius) and three belowground pathogens (S. carpocapsae, H. marelatus, and B. bassiana) was varied using a substitutive design. Total abundance was kept constant with increasing species richness level at a rate of ten predator individuals or entomopathogen equivalent (25,000 EPNs/m2 and approximately 4.3x109 spores/m2) for all diversity treatments. The responses measured were the number of potato beetle adults and pupae surviving after 28 days, as well as the final biomass of plant material (to examine whether predation of potato beetles by predators led to increased plant growth). Also, predators were collected at the experiments’ end to determine if intraguild predation was a factor. In 2007, ten potato beetle egg clusters (15 eggs per cluster) and 60 larvae were used (50 first and second instar and 10 third and fourth instar). In this study, functional natural enemy groups (predators and pathogens) were manipulated creating four treatments (predators only, pathogens only, both predators and pathogens, and no natural enemies). The predator only treatment consisted of all three aboveground predators, the pathogen only treatment consisted of all three belowground pathogens, and the both treatment consisted of all six species varied using a substitutive design.
Every week for four weeks a destructive sample was conducted collecting all life stages of the potato beetle, plant biomass, and predator/pathogen survival. These data were analyzed using SYSTAT 9 for ANOVA and SigmaPlot for fitted lines. Tukey’s posthoc test was used for comparisons where appropriate.
Impact of mustard cover crops on EPNs
In two large-scale field plot experiments, the first conducted in 2006 and the second in 2007, two species of EPNs were investigated for control of CPB and M. chitwoodi: Steinernema feltiae, and Steinernema riobrave - both are mid-soil hunters that reside and search for prey in close proximity to the larvae of CPB (during the season around 5cm deep in the soil). The mustard species Brassica carrinata, in defatted seed meal form, was incorporated into the soil 15 days prior to planting potatoes. Two EPN treatments were sprayed onto soil at low and high rates (3 billion/acre versus 6 billion/acre, respectively) on the day of planting. Combinations of EPN species and mustard meal followed the same protocol, with mustard incorporated 15 days prior to planting and EPN sprayed onto soil on the day of planting. Mid-season, another application of EPN at 2 billion/acre was applied onto soil to target the CPB 4th instar larvae. Before EPN application, 2 buckets were filled with soil and 10 CPB beetle 4th instar larvae were placed into each bucket for each plot. The buckets were also sprayed during application, and then covered with perforated lids. Two days later, buckets were unearthed; CPB infection rate by EPN was assessed three days later after infection had established. The experiment also included a no-treatment control and a Mocap comparison standard. The experimental design was a randomized complete block design to account for variation across the field.
Soil fertility effects on insect pathogens (Figure 1)
In 2005, there was a significant main effect of nematode rate on host infection (F=107.418, P<0.0001). This difference illustrates that the low rate of nematode application was significantly different from the high rate of nematode application. The low rate of application elicited a low host infection response compared to the high rate of application, which that showed significantly higher host infection. More importantly, there was a main effect of soil fertility on nematode infection (F=5.779, P=0.017). Surprisingly, regardless of nematode application rate, plots amended with organic fertilizer had a significantly lower infection response than did plots amended with chemical fertilizer.
In 2006 there was a statistically significant main effect of soil fertility (F=8.366, P=0.006) and time (F=39.37, P<0.0001), as was seen in 2005, indicating that organic soil fertility had a negative effect on host infection. In addition, there was a statistically significant interaction between soil fertility and time (F=3.75, P=0.010), showing a change in infection rate between the two soil fertility types from the beginning of the season to the end of the season. The beginning of the season showed decreased infection rates in organic soil fertility and no differences between soil fertility treatments later in the season. Using the potato beetle as a sentinel host, we found a significant main effect of nematode rate on host infection (F=143.464, P<0.0001) 24h after EPN application. The low rate of nematode application rendered significantly lower host infection than the high rate of nematode application. In addition, there was a main effect of soil fertility on host infection (F=6.5451, P=0.014). Similarly, organic soil amended plots had significantly lower infection of potato beetles than conventional soil amended plots. The differences between treatments were diminished approximately one month after EPN application.
We hypothesized that organic soil fertility, because of improved soil attributes from organic soil amendments, would be more conducive to EPNs than conventional fertility. Although our data contradict what was hypothesized, we have been able to eliminate some factors that may decrease the efficacy of EPN augmentation in organic systems. Currently, we suspect that predation and/or interference may be a factor suppressing infection in organic plots. Soil samples were collected during all 3 field seasons, and a dehydrogenase enzyme activity measurement was conducted to examine soil microbial activity. Preliminary data show that more soil microbial activity is occurring in organic plots compared to conventional plots. This suggests that there may be a more diverse suite of soil microbes in soils receiving organic fertilizer, such competitors or predators of EPNs, that may be reducing EPN success in organic soil. We have recovered from organic soils competitors such as Beauvaria bassiana, another entomopathogen, and predators, such as Arthrobotys, the nematode trapping fungus. In addition, these soil samples show no significant differences in soil pH and soil moisture, and thus these factors seem unlikely to explain poorer EPN survivorship in organic soil.
Predator and pathogen biodiversity experiments (Figure 2)
We found decreasing potato beetle survival with increasing natural enemy species richness (R2=0.45, P=0.0001). In addition, plant biomass increased with increasing natural enemy species richness (R2=0.14, P=0.047). All combinations of predator with pathogen reduced herbivore survival more than even the most effective single species in monoculture, suggesting that these groups complement one another. Importantly, potato beetle control improved only when at least one predator and pathogen was present (F=9.69, P<0.0001). Thus, the pairing of predators with pathogens was key rather than natural enemy species diversity per se, suggesting that conservation of both functional groups is important. Finally, intraguild predation was not important between predator and pathogen groups (across species richness F=0.44, P=0.65, between predators and pathogens F=0.57, P=0.46). Examining the presence of predator-pathogen groups by time there is a significant time by treatment effect (F=8.715, P<0.0001).
Impact of mustard cover crops on EPNs
Soil samples were taken before treatments were applied, mid-season, and end of season to assess the presence of M. chitwoodi in the soil. The population of M. chitwoodi in the soil before treatments was high, with on average 25- 200 juveniles per 250 cc soil. The economic threshold for M. chitwoodi is 1 juvenile per 250 cc soil, so the starting population was extremely high. The mid-season soil samples showed a significant decrease in M. chitwoodi populations in both the high and low Steinernema feltiae treatments. compared to the control treatment. The mustard treatment reduced M. chitwoodi populations by mid-season; however, soil samples at the end of the season did not show any M. chitwoodi population decrease from the mid-season. The CPB 4th instar larvae were 95-100% infected by both species of EPN. In conclusion, the EPN species S. feltiae significantly reduced M. chitwoodi populations while also controlling at least one generation of CPB larvae in the field, with a high rate of CPB infection.
First, we have learned that augmentation of entomopathogens, applied as a bio-insecticide, will be less effective when crops are under organic fertility management (using manure rather than chemical fertilizer). Rather, high-organic matter fields house robust populations of naturally occurring insect pathogens, which instead might be conserved to improve pest control. We also found that the combination of pest-attacking predators and pathogens is more effective for potato beetle control than are either natural enemy class alone. Thus, both predators and pathogens should be targeted for conservation. Finally, EPN applications can be an effective strategy for controlling root-knot nematodes, although the use of mustard green manures may reduce the effectiveness of EPN applications. With our research results now in hand, we are continuing our outreach efforts to get this information to growers.
Educational & Outreach Activities
The preparation of publications is the focus of our work right now. We anticipate at least 6 peer-reviewed journal articles will result from this WSARE project. One manuscript will describe our experiments looking at the impact of organic versus chemical fertility on EPNs; an additional two manuscripts will describe synergism between predators and pathogens as potato beetle biocontrol agents; while a series of 2-3 manuscripts will describe various aspects of mustard green manure effects on EPNs in the laboratory and in the field. We will send preprints to WSARE of each manuscript as it is completed and accepted for publication, which we anticipate will happen over the next calendar year.
We have collected the data necessary for an economic analysis, and as the final year's field data are tabulated and analysed we will look more at the economics of the techniques we have examined. All of our field experiments were more difficult to complete than we had imagined, and thus examination of our pest management schemes at a commercially-realistic scale remains a goal for future work in this system.
The key challenge of this project will be achieving farmer adoption of what we have learned. We have found as a result of our work that the soil foodweb in potato fields is incredibly complex, with different species interacting with one another in incredibly complex ways. We have a few messages that we are working to extend to growers:
1. Both the predators and pathogens that attack Colorado potato beetle are important for biological control, and should be conserved in tandem.
2. Using insect pathogens as a biopesticide may be more difficult under organic crop management, because soils rich in organic matter house diverse communities of competitors and predators of the insect pathogens.
3. The use of mustard green manures, while helpful in controlling plant parasitic nematodes, may come at the cost of harming beneficial insect-attacking nematodes.
Areas needing additional study
Our greatest lesson from this project is that the soil foodweb in potatoes, and no doubt many other crops, is incredibly diverse and complex. We see great need for future research examining how management practices targeted at one component of the soil foodweb also impact, both directly and indirectly, other species in the soil. Further challenges will be assessing the complex economic impacts of these direct and indirect effects, and then relating what is learned to growers in a form that is easy to understand and useful.