Using Entomopathogenic Nematodes for Crop Insect Pest Control

Using Entomopathogenic Nematodes for Crop Insect Pest Control Using Entomopathogenic Nematodes for Crop Insect Pest Control Introduction the region, the information in this publication can be successfully applied throughout the Pacific Northwest. Nematodes are non-segmented, elongated roundworms that are colorless, without appendages, and usually microscopic. There are non-beneficial and beneficial nematodes. Non-beneficial nematodes are also called “plant parasitic nematodes” and cause damage to crops and other types of plants. Beneficial nematodes attack soilborne insect pests, yet are not harmful to humans, animals, plants, or earthworms, and can therefore be used as biological control organisms (Denno et al. 2008). Beneficial nematodes that cause disease within an insect are referred to as “entomopathogenic” and have the ability to kill insects (Figure 1). Life Cycle of Entomopathogenic Nematodes The life cycle of most nematodes includes an egg stage, four juvenile stages, and an adult stage. The third juvenile stage of entomopathogenic nematodes is referred to as the “infective juvenile” or “dauer” stage and is the only free-living stage (Figure 2). The infective juvenile is capable of surviving in the soil, where it locates, attacks, and infects a pest insect (Poinar 1990). Under optimal conditions, it takes 3–7 days for steinernematids and heterorhabditids to complete one life cycle inside a host from egg to egg. Emergence of infective juveniles from the host requires about 6–11 days for steinernematids and 12–14 days for heterorhabditids (Kaya and Koppenhöfer 1999). Figure 3 is a diagram of the life cycle of entomopathogenic nematodes from host infection to emergence from the host. Entomopathogenic nematodes from the families Steinernematidae and Heterorhabditidae have proven to be the most effective as biological control organisms (Kaya and Gaugler 1993). They are soil-inhabiting organisms and can be used effectively to control soilborne insect pests, but are generally not effective when applied to control insects in the leaf canopy. When considered as a group of nearly 30 species, each with its own suite of preferred hosts, entomopathogenic nematodes can be used to control a wide range of insect pests, including a variety of caterpillars, cutworms, crown borers, grubs, corn root worm, cranefly, thrips, fungus gnat, and beetles. Entomopathogenic nematodes have been released extensively in crop fields with negligible effects on nontarget insects and are regarded as exceptionally safe to the environment. The keys to success with entomopathogenic nematodes are (1) understanding their life cycles and functions; (2) matching the correct nematode species with the pest species; (3) applying them during appropriate environmental conditions (soil temperature, soil moisture, sunlight); and (5) applying them only with compatible pesticides. Because entomopathogenic nematodes are living organisms, they require careful handling to survive shipment and storage as well as appropriate environmental conditions to survive in the soil after application. Figure 1. Tens of thousands of infective juveniles of the entomopathogenic nematode Steinernema carpocapsae spilling out of a Galleria wax moth larva in search of new hosts (photo provided by Randy Gaugler). How Entomopathogenic Nematodes Control Insect Pests Many species of entomopathogenic nematodes occur naturally in the Pacific Northwest, including Heterorhabditis marelata, which has been shown to control some insect crop pests (Berry et al. 1997). However, most entomopathogenic nematodes used for pest management are not native to the region. It is unknown whether these introduced species will overwinter in the Pacific Northwest and successfully colonize insect hosts in subsequent years. While much of the research regarding entomopathogenic nematodes has been conducted outside of An understanding of host-finding strategies will help you properly match entomopathogenic nematode species to pest insects to ensure infection and control (Gaugler 1999). Only entomopathogenic nematodes in the infective juvenile stage will survive in the soil and find and penetrate insect pests. Infective juvenile entomopathogenic nematodes locate their hosts in soil 1 Ambushing. Entomopathogenic nematodes that use the ambushing strategy tend to remain stationary at or near the soil surface and locate host insects by direct contact (Campbell et al. 1996). An ambusher searches by standing on its tail so that most of its body is in the air, referred to as “nictation.” The nictating nematode attaches to and attacks passing insect hosts. Ambusher entomopathogenic nematodes most effectively control insect pests that are highly mobile at the soil surface, such as cutworms, armyworms, and mole crickets. Figure 2. An infective juvenile entomopathogenic nematode (photo provided by Randy Gaugler). Cruising. Entomopathogenic nematodes that use the cruising strategy are highly mobile and able to move throughout the soil profile. Cruisers locate their host by sensing carbon dioxide or other volatiles released by the host. Cruiser entomopathogenic nematodes are most effective against sedentary and slow-moving insect pests at various soil depths, such as white grubs and root weevils. Infection Generally, several entomopathogenic nematodes will infect a single insect host. Infective juvenile nematodes penetrate the insect’s body cavity either through natural body openings (such as the mouth, anus, genital pore, or breathing pore; Figure 4) or by breaking the outer cuticle of the insect; heterorhabditids do this using a dorsal “tooth” or hook. Once inside the body cavity of by means of two strategies—ambushing and cruising (Gaugler et al. 1989). Ambusher species include Steinernema carpocapsae and S. scapterisici; cruisers include Heterorhabditis bacteriophora and S. glaseri. S. riobrave and S. feltiae do a bit of both ambushing and cruising (Campbell and Gaugler 1997). Figure 3. Diagram of the generalized life cycle of entomopathogenic nematodes (image provided by David Shapiro-Ilan). 2 the host, the infective juveniles release bacteria that live symbiotically within the entomopathogenic nematode’s gut but do not harm the nematode. The nematode-bacterium relationship is highly specific: only Xenorhabdus spp. bacteria co-exist with steinernematids, and only Photorhabdus bacteria co-exist with heterorhabditids. Once released into the host, the bacteria multiply quickly and under optimal conditions cause the host to die within 24 to 48 hours. particular pest more effectively than another species. Therefore, the insect pest must be identified before choosing the entomopathogenic nematode species most appropriate for biological control. Table 1 is a general guide of available entomopathogenic nematodes and the pests they have been shown to control. Table 2 provides a description of each species of commercially available entomopathogenic nematode. Obtaining Entomopathogenic Nematodes Entomopathogenic nematodes feed on both bacteria they release and host insect tissue. After a few days inside the host, entomopathogenic nematodes mature to the adult stage. These adult entomopathogenic nematodes produce hundreds of thousands of new juveniles that may undergo several life cycles within a single host (Figure 5). When the host has been consumed, the infective juveniles, armed with a fresh supply of bacteria, emerge from the empty shell of the host, move into the soil, and begin the search for a new host. A protective exterior cuticle surrounds the infective juvenile, protecting it from the environment and predators. Under ideal conditions, steinernematids emerge 6–11 days after initial infection and heterorhabditids emerge 12–14 days after initial infection (Kaya and Koppenhöfer 1999). The duration of infective juvenile survival in soil is unknown because they can become prey to invertebrates and microorganisms. Perhaps the biggest challenge to the use of entomopathogenic nematodes as effective biological control organisms is the variable quantity and quality of nematodes in commercial products (Gaugler et al. 2000). Entomopathogenic nematodes are cultured on a large scale in laboratories and are available from many commercial suppliers in North America and Europe. In past assessments of cottage industry commercial products, most contained lower numbers of entomopathogenic nematodes than the suppliers claimed. In addition, in some cases the species of entomopathogenic nematodes in the product were mixed and therefore inconsistent with the product label. The industry has made progress, however, in increasing the quality of its products. See Table 3 for lists of commercial suppliers compiled by university and state government professionals. Entomopathogenic nematodes can also be purchased through gardening mail-order catalogs and at some local agricultural and nursery supply stores. Using Entomopathogenic Nematodes Worldwide, over 80 species of entomopathogenic nematodes have been identified and 11 commercialized (Kaya and Koppenhöfer 1999). The different species of entomopathogenic nematodes vary in the range of insects they attack, environmental needs, and stability in commercial products (Gaugler 1999). A given species of entomopathogenic nematode may also control a Shelf Life In general, entomopathogenic nematodes do not have a long shelf life. Many microbial insecticides, including Bacillus thuringiensis, have a resting stage facilitating longterm storage. The infective juvenile entomopathogenic Figure 4. Infective juvenile entomopathogenic nematodes searching for an entry portal into a host (photo provided by Randy Gaugler). Figure 5. Hundreds of thousands of new entomopathogenic nematodes within a host insect (photo provided by Randy Gaugler). 3 Table 1. Current use of steinernema and heterorhabditis nematodes as biological control organisms1 (Shapiro-Ilan and Gaugler 2010). Crop(s) Targeted Pest Common Name Pest Scientific Name Efficacious Nematodes2 Artichokes Artichoke plume moth Platyptilia carduidactyla Sc Vegetables Armyworm Lepidoptera: Noctuidae Sc, Sf, Sr Ornamentals Banana moth Opogona sachari Hb, Sc Bananas Banana root borer Cosmopolites sordidus Sc, Sf, Sg Turf Billbug Sphenophorus spp. (Coleoptera: Curculionidae) Hb,Sc Turf, vegetables Black cutworm Agrotis ipsilon Sc Berries, ornamentals Black vine weevil Otiorhynchus sulcatus Hb, Hd, Hm, Hmeg, Sc, Sg Fruit trees, ornamentals Borer Synanthedon spp. and other sesiids Hb, Sc, Sf Home yard, turf Cat flea Ctenocephalides felis Sc Citrus, ornamentals Citrus root weevil Pachnaeus spp. (Coleoptera: Curculionidae) Sr, Hb Pome fruit Codling moth Cydia pomonella Sc, Sf Vegetables Corn earworm Helicoverpa zea Sc, Sf, Sr Vegetables Corn rootworm Diabrotica spp. Hb, Sc Cranberries Cranberry girdler Chrysoteuchia topiaria Sc Turf Crane fly Diptera: Tipulidae Sc Citrus, ornamentals Diaprepes root weevil Diaprepes abbreviatus Hb, Sr Mushrooms Fungus gnat Diptera: Sciaridae Sf, Hb Grapes Grape root borer Vitacea polistiformis Hz, Hb Iris Iris borer Macronoctua onusta Hb, Sc Forest plantings Large pine weevil Hylobius albietis Hd, Sc Vegetables, ornamentals Leafminer Liriomyza spp. (Diptera: Agromyzidae) Sc, Sf Turf Mole cricket Scapteriscus spp. Sc, Sr, Sscap Nut and fruit trees Navel orangeworm Amyelois transitella Sc Fruit trees Plum curculio Conotrachelus nenuphar Sr Turf, ornamentals Scarab grub3 Coleoptera: Scarabaeidae Hb, Sc, Sg, Ss, Hz Ornamentals Shore fly Scatella spp. Sc, Sf Berries Strawberry root weevil Otiorhynchus ovatus Hm Bee hives Small hive beetle Aethina tumida Hi, Sr Sweet potato Sweetpotato weevil Cylas formicarius Hb, Sc, Sf Nematodes listed provided at least 75% suppression of these pests in field or greenhouse experiments. 2 Nematode species are abbreviated as follows: Hb = Heterorhabditis bacteriophora, Hd = H. downesi, Hi = H. indica, Hm = H. marelata, Hmeg = H. megidis, Hz = H. zealandica, Sc = Steinernema carpocapsae, Sf = S. feltiae, Sg = S. glaseri, Sk = S. kushidai, Sr = S. riobrave, Sscap = S. scapterisci, Ss = S. scarabaei. 3 Efficacy against various pest species within this group varies among nematode species. 1 4 Table 2. Characteristics of seven species of entomopathogenic nematodes that are commercially available in the United States (Gaugler 1999 and Berry 2000). Steinernema carpocapsae. An ambusher type, it is most effective against highly mobile surface insects such as webworms, cutworms, armyworms, girdlers, and wood borers. Most effective at soil surface temperatures between 70°F and 85°F. Can be formulated in a partially desiccated state in clay granules to provide several months of room-temperature shelf life. S. feltiae. Combines ambusher and cruiser strategies and attacks immature fly larvae (dipterous insects), including mushroom flies, fungus gnats, and crane flies. Maintains infectivity at low soil temperatures, even below 50°F. Has relatively low stability in formulation and a short shelf life. S. glaseri. A large cruiser type that attacks white grubs and other beetle larvae, particularly scarabs. Expensive and difficult to produce and manage due to its tendency to lose its bacterial symbiont. Highly active and robust infective juveniles are difficult to contain within formulations. S. riobrave. Combines ambusher and cruiser strategies, and attacks corn earworms, citrus root weevils, pink bollworms, and mole crickets. Isolated from the Rio Grande Valley of Texas. Maintains infectivity at soil temperatures above 95°F and in semi-arid conditions. Heterorhabditis bacteriophora. A cruiser-type nematode that attacks caterpillar and beetle larvae, including root weevils, particularly black vine weevil. Most effective in warm temperatures (above 68°F). Infective juveniles persist only a few days in the soil. Has poor stability in formulation and a short shelf life. H. marelata. A cruiser-type nematode that attacks beetle larvae, including white grubs and root weevils. Isolated from the Oregon coastal region. Active at cool soil temperatures (50–55°F). H. megidis. A large cruiser-type nematode that has been effective in controlling black vine weevil larvae. Has not been widely researched or tested for insect control. Isolated in Ohio, researched and developed in Europe, and now available in the United States. Tends to have poor formulation stability and a short shelf life. vermiculite or liquid suspension will not be discernible from the carrier material. The container should have a mild odor; if there is a strong smell, like ammonia, then it is likely the nematodes are dead. If the formulation is a sponge or vermiculite, remove a tiny portion of the product with tweezers and place in a teaspoon of cool water (approximately 60ºF) for six hours. If the formulation is a liquid suspension, swirl the liquid to ensure distribution of the nematodes and remove a small droplet (about 0.05 ml). Table 3. Recommended guides to commercial suppliers of entomopathogenic nematodes. Commercial Sources of Insect Parasitic Nematodes. 2000. Compiled by P. Grewal and K. Power. Ohio State University. http://www2.oardc.ohio-state.edu/nematodes/nematode_ suppliers.htm Commercial Producers and Suppliers of Nematodes. 2010. Compiled by D.I. Shapiro-Ilan and R. Gaugler. Cornell University. http://www.biocontrol.entomology.cornell.edu/pathogens/ nematodes.html Place the soaked nematode sample (from the sponge or vermiculite) or the droplet from the liquid suspension on a slide or in a small, clear glass bowl. View the samples with a hand lens (15X) or microscope. Live entomopathogenic nematodes will be mobile and have a bend to their shape. S. carpocapsae has a resting “J” shape and will move only when prodded with a pin or needle (Figure 2). All other nematodes will move in an “S” pattern (Lewis 1999). If the nematodes are straight and not moving, it is likely they are dead. A mortality rate of 10% is typical. If more than 20% of the nematodes are dead, inform your supplier immediately. Vendors of Beneficial Organisms in North America. 2010. Compiled by J. White and D. Johnson. University of Kentucky College of Agriculture. http://www.ca.uky.edu/entomology/entfacts/ef125.asp Suppliers of Beneficial Organisms in North America. 1997. Compiled by C.D. Hunter. California Environmental Protection Agency, Department of Pesticide Regulation, Environmental Monitoring and Pest Management Branch. http://www.cdpr.ca.gov/docs/pestmgt/ipminov/bensup.pdf nematode stage is not a resting stage; juveniles are metabolically active and use energy reserves while in formulation (Lewis 1999). For this reason, it is advisable to order entomopathogenic nematodes only 3–4 days prior to application. Entomopathogenic nematodes should be shipped by overnight delivery in their infective juvenile stage and used within 1–2 days after arrival. Entomopathogenic nematodes should be stored in their shipment containers under refrigeration until ready for use. The storage life of entomopathogenic nematodes is species- and formulation-dependent. Specific storage instructions will be included with the entomopathogenic nematode shipment and should be carefully followed. Table 4 is a summary of storage times for entomopathogenic nematodes in different formulations. Storing nematodes under refrigeration will increase their shelf life, but their infectivity will still decrease the longer they are in storage. When the Examine the entomopathogenic nematodes upon receipt to make sure they arrived alive. The shipment container should not feel warm or hot. Open the container and check the color and odor of the nematodes. To the naked eye, the nematodes on a sponge formulation will appear as a light tan or gray paste, while nematodes in 5 Table 4. Expected storability of some commercially available formulations of entomopathogenic nematodes (adapted from Grewal 1999b). Storage Duration Formulation Nematode Species Room Temperature Refrigerated Liquid concentrate S. carpocapsae S. riobrave 5–6 days 3–4 days 12–15 days 7–9 days Sponge S. carpocapsae H. bacteriophora 0 days 0 days 2–3 months 1–2 months Vermiculite S. feltiae H. megidis 0 days 0 days 4–5 months 2–3 months Alginate gels S. carpocapsae S. feltiae 3–4 months 1/2–1 month 6–9 months 4–5 months Flowable gels S. carpocapsae S. glaseri 1-1 1/2 months 0 days 3–5 months 1–1 1/2 months Water dispersible granules S. carpocapsae S. feltiae S. riobrave 4–5 months 11/2–2 months 2–3 months 9–12 months 5–7 months 4–5 months Wettable powder S. carpocapsae S. feltiae H. megidis 2 1/2–3 1/2 months 2–3 months 2–3 months 6–8 months 5–6 months 4–5 months Nematode wool H. bacteriophora 21 days unknown storage life has expired, expect 70–100% mortality of the nematodes (Grewal 2000). Entomopathogenic nematodes should be applied late in the day or on a cool, overcast day when light and temperatures are low. Apply them to moist soil following either a rainfall or irrigation, and lightly irrigate afterward. This washes them into the soil and decreases soil surface temperatures. Do not over-irrigate, as saturated soil will impede nematode activity due to lack of oxygen. Soil Conditions Entomopathogenic nematodes can die if they are applied to soils that are too dry, too hot, or too cold, or if they are exposed to ultraviolet (UV) light from the sun. Nematodes live in the water-filled spaces, or pores, between soil particles. They need water to move and successfully locate a host, and oxygen to survive. Heavy clay soils hold water well, but may contain too little oxygen, and the small pore space may restrict nematode movement. Sandy soils must be irrigated to maintain the water-filled pores. If applying entomopathogenic nematodes after an extended dry period, break the crust of the dry soil with a rake or harrow and irrigate the soil before the application, preferably to a depth of 4–6 inches. Remove plant debris before the application, as it will prevent the nematodes from reaching the soil surface. Preparing for Application Entomopathogenic nematodes should be prepared for field application no earlier than one hour ahead of time. If nematodes are in a liquid suspension, shake the shipment container well and pour the liquid into the application container (e.g., tank, backpack sprayer, or watering can). Rinse the shipment container twice with cool water (approximately 60°F), and pour the rinse water into the application container. If nematodes are on a sponge, soak the sponge in one gallon of cool water for 10 minutes, then pour the water into the application container. Rinse the sponge several times, pouring the rinse water into the application container after each rinse. If nematodes are in vermiculite, add the vermiculite-nematode mixture directly to water in the application container and stir until dispersed. Once the nematodes have been mixed with water, agitate the mixture every five minutes to keep the nematodes in suspension and supplied with oxygen. Soil temperatures between 77°F and 82°F are ideal for applying all entomopathogenic nematode species. In general, soil temperatures greater than 85°F can decrease the efficacy of some nematode species, while soil temperatures less than 50°F can immobilize others at the soil surface, causing them to be exposed to UV light that can kill them. The range of soil temperatures that nematode species can survive and infect host insects does vary, however. For example, S. feltiae can be effective at 57°F, while S. riobrave can be effective at 95°F. Application Equipment Read the product label for specific application instructions. Entomopathogenic nematodes that are formulated 6 with vermiculite may be best applied as a granular product. Other formulations can be applied using standard liquid pesticide, fertilizer, and irrigation equipment with pressures of up to 300 PSI. Electrostatic, fan, pressurized, and mist sprayers can be used. If tanks are agitated through excessive sparging (recirculation of the spray mix), or if the temperature in the tank rises above 86°F, the nematodes will be damaged. Irrigation systems may also be used for applying most species; however, high-pressure recycling pumping systems are not good delivery systems (Shetlar 1999). An excellent overview of sprayer equipment is provided in the Private Applicator Pesticide Education Manual, EM020 (Ramsay et al. 2009). may be applied. Research at the University of Florida has demonstrated that a rate of up to 200 million nematodes per acre applied in a band provided effective control of root weevil in citrus orchards (Duncan et al. 1999). More research is needed to determine specific rate responses for each species of entomopathogenic nematode in various cropping systems to control specific pests. Calibrate equipment to ensure appropriate application rates. An excellent overview of sprayer calibration is provided in the Private Applicator Pesticide Education Manual, EM020 (Ramsay et al. 2009). Evaluating nematode applications It can be difficult to be sure if the entomopathogenic nematodes reached the soil and the target pests, as it is very laborious to recover the cadavers of the insects they have killed. There are two simple tests that can be used to assess the efficacy of all entomopathogenic nematode species. Both tests employ Galleria mellonella waxworms (Berry 2007), which are the caterpillar stage of a waxmoth species that are extremely susceptible to entomopathogenic nematode infection. Galleria waxworms are readily available at fishing bait and pet supply stores. Remove all screens smaller than 50-mesh from your spray or irrigation equipment to allow nematodes to pass through the system. Check spray nozzle orifices for clogging during application. Direct spray nozzles at the soil to maximize the number of nematodes being applied directly to the soil. A large spray volume is ideal. Volumes of 2–6 gallons of water per 1,000 square feet (87–260 gallons per acre) are recommended on most nematode labels (Shetlar 1999). The water in the spray will wash the nematodes from plant surfaces into the soil. Lower volume spray applications of 0.5–1.0 gallon per 1,000 square feet (20–45 gallons per acre) can be used if the area or field is irrigated prior to and immediately following entomopathogenic nematode application. Overhead irrigation following nematode application will wash the nematodes from plant surfaces into the soil. If the spray droplets are allowed to dry prior to this irrigation, the nematodes will be exposed to UV light and die while still on the plant surfaces. Applying nematodes during a rainfall will also ensure that nematodes reach the soil surface. For the first test, place 2–3 Galleria waxworms in a tea strainer and bury the strainer 4 inches deep in the soil. You can bury the waxworms either just before you apply the entomopathogenic nematodes or anytime afterwards. It is best to place several baited strainers in the area where nematodes are being applied—either 3–4 strainers for a garden area or approximately 10 strainers per acre. Remove the strainers from the soil after 2 days, rinse the waxworms with distilled water, and store them on moistened filter paper or thick paper towel in a dark location at room temperature. Check the waxworms regularly over the next 7–10 days to look for nematode infection. Infected waxworms will usually change color; Steinernematid-infected waxworms will turn yellow, tan, or brown, while heterorhabditid-infected waxworms will turn pink or purple. If the waxworms turn black, they were likely killed by other means. Use equipment that is clean and free of pesticide residues. Also, do not mix entomopathogenic nematodes with nitrogen fertilizers, particularly urea (Grewal 2000). Although there is evidence that nematodes are tolerant to many herbicides and fungicides, they are sensitive to certain insecticides and nematicides. Refer to the nematode product label for specific listings of chemicals that are lethal to entomopathogenic nematodes. To check that live nematodes are being applied to the soil, set pans or containers on the soil surface prior to application. Immediately after application, use a hand lens (15X) or a microscope to check the liquid in the pans or containers for live, moving nematodes. For the second test, collect entomopathogenic nematode-treated soil from the treated area at least one day after nematodes have been applied. Collect at least 10 soil samples from a garden area and 20 soil samples per acre. Each soil sample should be approximately ¼ cup from a depth of 4 inches. Mix the soil together, place ¼ cup into a wax cup, and place a Galleria waxworm on top of the soil. Evaluate 2–3 wax cups for a garden area or approximately 10 wax cups per acre. Place the cups in a dark area at room temperature for 2 days. Rinse, store, and evaluate the waxworms as described above. Application Rates Before applying any biological control, including entomopathogenic nematodes, read the product label for specific application instructions. A broadcast application rate of 1 billion nematodes per acre is generally recommended to control most soil insects. For smaller areas, the recommended application rate is 250,000 nematodes per square meter. If nematodes are banded (applied in a band beside the crop row), a lower rate - 7