Interactions Among Organic Fertility, Mustard Green Manures, and Insect Biocontrol by Entomopathogenic Nematodes

2006 Annual Report for SW04-113

Project Type: Research and Education
Funds awarded in 2004: $138,922.00
Projected End Date: 12/31/2007
Region: Western
State: Washington
Principal Investigator:
Ekaterini Riga
Washington State University
William Snyder
Washington State University

Interactions Among Organic Fertility, Mustard Green Manures, and Insect Biocontrol by Entomopathogenic Nematodes


We are evaluating the influence of fertility practices and cover-cropping on the use of insect-attacking, entomopathogenic nematodes (EPNs) as biocontrol agents against Colorado potato beetles. A field experiment augmenting EPNs into organic and conventional soil amendments (manure and chemical fertilizer respectively) showed 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 field cage experiment 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. Importantly, this positive result on beetle suppression was driven by the pairing of predators with insect pathogens. In other field trials 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.

Objectives/Performance Targets

Our project has 5 objectives:

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 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.


We have made progress on several of the objectives to date. In support of Objective 1, the augmentative release of entomopathogenic nematodes within two fertilized systems was conducted, in field experiments at the Washington State University Othello Research Station in the summers of 2005 and 2006. 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 and 24 plots in 2006. The plots contained 5 rows of potatoes with 2 fallow rows as a buffer to adjacent plots, and 4.27m 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 organic versus conventional soil amendments, crossed 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, crossed with organic versus conventional fertilizer application. Organic soil amended plots were prepared by a pre-plant shovel application of a chicken (28.12kg/plot) and cow (47.17kg/plot) manure mixture, later incorporated into the soil. Conventional (chemical) fertilizer was pre-plant strip applied at a rate of 362.87kg/acre. Only one application of fertilizer (organic and 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 last instar larvae (first life stage to be associated with the soil) were at their highest density. The final application, 5 July 2005, occurred several weeks prior to the peak of the second generation of potato beetles. In 2006, a high rate of entomopathogenic nematodes was applied a single time during the season, before the first peak of the first generation potato beetles. A mixture of two insect-attacking nematode species, Steinernema carpocapsae and Heterorhabditis marelatus, was applied at both the low rate (containing ~100,000,000 IJs/acre) and high rate (containing ~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 shaped (2.5 cm wide) transparency paper and moistened filter paper 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, on the day of application to the field plots, 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. This study deviates from this common practice by instead measuring infection rates in the field. Host infection was estimated with host infection 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, similar to Colorado potato beetle pupation depth, 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 associated with the soil were at their highest. The response gathered was % wax worm or potato beetle infected. 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, in case resident nematodes were present (densities of resident nematodes were always low).

In 2005, there was a significant main effect of nematode rate on host infection (F=107.418, P<0.0001, Fig. 1). 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 in turn 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 receiving organic fertilizer (manure) had a significantly lower infection response than was recorded in plots treated with chemical fertilizer. Approximately one month from the final EPN application the differences between soil fertility treatments were nonexistent. 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 (Fig. 1), indicating that manure again 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), indicating a divergence in infection rates between the two soil fertility types through the season: the beginning of the season showed decreased infection rates in organic soil fertility, but no differences between fertilizer treatments later in the season. Using the potato beetle as a sentinel host, similar to the wax worm, there was a significant main effect of nematode application rate on host infection (F=143.464, P<0.0001) 24h after EPN application (Fig. 2). The low rate of nematode application rendered significantly lower host infection than did 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), because plots treated with manure again showed lower infection rates than did chemical fertilizer plots.

In our proposal, we had originally hypothesized that organic soil fertility, because of improved soil attributes from organic soil amendments, would be more conducive to EPN survivorship than would plots treated with chemical fertilizer. Clearly, our data contradict what was hypothesized, as insect-attacking nematodes performed more poorly in plots treated with manure. Several factors may limit the effectiveness of EPN augmentation in plots treated with manure, including lower soil pH, and predation by or interference with other soil fauna encouraged by the addition of organic matter within manure. Currently, we suspect that predation and/or interference may be a factor suppressing infection in organic plots. Several soil samples were collected in 2005 and 2006 and a dehydrogenase enzyme activity measurement was conducted to examine soil microbial activity. Preliminary data from 2005 showed that more soil microbial activity was occurring in plots treated with manure, compared to conventional plots. This suggests that there is a more diverse suite of soil organisms, perhaps including other entomopathogens and predators of nematodes. In other soil samples we have observed the presence of competitors such as Beauvaria bassiana, another entomopathogen, and Arthrobotys, the nematode trapping fungus, a nematode predator. Our investigation of any biological factors limiting augmentation using EPNs is just beginning, however.

To examine the impacts of combining insect predator and nematode conservation for biological control, a field experiment was conducted at the Washington State University Othello Research Station in the summer of 2006. 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 attack on potato beetles when several predator species are together. 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), with each nematode species 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. 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. 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 enemy 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 4.3×109 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. These data were analyzed using SYSTAT 9 for ANOVA and SigmaPlot for fitted lines. Tukey’s posthoc test was used for comparisons where appropriate.

These data show that there was decreasing potato beetle survival with increasing natural enemy species richness (R2=0.45, P=0.0001; Fig. 3). In addition, plant biomass increased with increasing natural enemy species richness (R2=0.14, P=0.047; Fig. 3). 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; Fig. 4). Thus, the pairing of predators with pathogens was key rather than natural enemy species diversity per se suggesting that conservation of these 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; Fig. 5).

In support of Objective 3, we examined control of potato beetles and plant parasitic nematodes using EPN application. Meloidogyne chitwoodi, a root knot nematode, and the Colorado potato beetle (CPB) cause severe damage to potato crops. Potato crops are a major component of Washington agriculture- in 2005 the crop value was estimated at 459.7 million (USDA-NASS). M. chitwoodi infected fields are rejected if only 5-15% potatoes are culled. CPB is heavily sprayed with 6-8 foliar pesticide applications in a four-month season. Conventional control has relied upon toxic pesticide and fumigants- both of which raise human and environmental health concerns. Additionally, most fumigants are on the list to be banned to satisfy the requirements of the Food Quality Protection Act. This project proposes an integrated pest management approach using mustard amendments in conjunction with entomopathogenic nematodes as biocontrol agents to target both M. chitwoodi and CPB. M. chitwoodi and CPB have vastly different lifecycles, but CPB larvae and M. chitwoodi can be targeted in the same physical space in the soil by entomopathogenic (EPN) nematodes. EPN infect and kill larval stage insects, and have also been shown to reduce root knot nematode populations. Mustard is a green manure crop that can be rotated between potato seasons- mustard crops or mustard meals (defatted mustard seed) are tilled under and the chemicals released have proven nematicidal properties. However, the effect of these chemicals on EPN is uncertain. Mustard amendment combined with EPN was investigated for control of M. chitwoodi and CPB.

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). Secondly, a species of mustard, Brassica carrinata in defatted seed meal form was incorporated into the soil 15 days prior to planting potatoes. The two EPN treatments were sprayed onto soil at low and high rates (3 billion/acre, 6 billion/acre) 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 were 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 and 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 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.

Remaining Work

In 2007, three certified organic and 3 conventional potato fields will be used to further investigate why fields treated with manure are so resistant to application of insect-attacking nematodes. Each production field will have six (10m2) designated plots scattered throughout. Three plots will be treated with EPNs while the other three plots will be untreated. Host infection will be estimated as previously described. Two independent trials will be run where EPNs will be applied to six different plots in the same field at the beginning, and then again later, in the season. In addition to the field plots, soil will be collected from certified organic and conventional potato fields and will be thoroughly mixed in a bucket, so that an aliquot for each field type is available. From each aliquot the appropriate treatment will be prepared. There will be six treatments with organic or conventional soil placed into large Petri dishes. This will be crossed with the presence/absence of resident microbes. To prepare the soil used in the absent microbe treatment, the soil will be sterilized using an autoclave. The present microbe treatments will be prepared in two ways; first, resident microbes from soil will act as the present microbe treatment and will not be sterilized, while sterilized soil where microbes are incorporated after sterilization will be identified as sterile present microbe treatment. The soil will be packed into large Petri dishes according to the respective treatment and half will be treated with EPNs. Two days after EPN application the live host will be added (10 larvae/dish) and will be retrieved after two days. Mortality and infection will be recorded.

Impacts and Contributions/Outcomes

We are generating information on how soil fertility management, cover-cropping, and augmentation of entomopathogenic nematodes can be used in combination to control potato beetles and other pests. We have begun educating growers about these findings, in extension presentations.


Hal Collins
Soil Microbiology, USDA/ARS, WSU-Prosser
Soil Microbiology, USDA/ARS, WSU-Prosser
24106 Bunn Road
Prosser,, WA 99350-8694
Office Phone: 5097869250
Andrew McGuire
Cooperative Extension Grant Co.
Cooperative Extension Grant Co.
PO Box 37, 35 C St NW
Ephrata, WA 98823
Office Phone: 5097542011
Herbert Hinman
Extension Economist
Dept. of Ag. & Res. Economics, Wash. State Univ
121D Hulbert Hall
Pullman, WA 99164
Office Phone: 5093352855
Terry Miller
Northwest Biocontrol Insectary & Quarantine
Pullman, WA 99164-6382
Office Phone: 5093355815
Dennis Johnson
Dept. of Plant Pathology
Johnson 317
Pullman, WA 99164
Office Phone: 5093353753
Ricardo Ramirez
Dept. of Entomology, Washington State Univ.
FSHN 166
Pullman, WA 99164
Office Phone: 5093353724
Dan Nordquist
Administrative Contact
Office of Grant and Res. Dev., Washington State Un
Pullman, WA 99164-9661
Office Phone: 5093353140
Lerry Lacey
Yakima USDA/ARS Agricultural Research Laboratory
5230 Konnowac Pass Road
Wapato, WA 98951
Office Phone: 5094546550
Donna Henderson

Graduate Student
Department of Plant pathology
Pullman, WA 99164