Formulation of Entomopathogenic Nematodes - American Chemical

Chapter 13

Formulation of Entomopathogenic Nematodes R. Georgis, D . B . Dunlop, and P. S. Grewal

Downloaded by UNIV OF SYDNEY on March 11, 2013 | http://pubs.acs.org Publication Date: May 5, 1995 | doi: 10.1021/bk-1995-0595.ch013

biosys, 1057 East Meadow Circle, Palo Alto, C A 94303

Entomopathogenic nematodes in the genera Steinernema and Heterorhabditis are commercially available for the control of soil-inhabiting insects. Stable formulations have been achieved by immobilizing and/or partially desiccating infective stage (IJ) nematodes. These formulations allowed introduction of nematode products with acceptable shelf-life into various market segments. A breakthrough in nematode formulation was accomplished with the development of a unique water dispersible granular formulation that allows nematodes to enter into a hydrobiotic state extending nematode survival and pathogenicity for up to 6 months at 4-25°C and up to 8 wks at 30°C. This formulation is easy and quick to apply and is well suited for a wide variety of agricultural and horticultur applications.

Entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) are attractive alternatives to chemical pesticides. Desirable attributes such as ease of mass-production, efficacy comparable to most insecticides in favorable habitats, and safety to non-target organisms have invoked commercial interest in these parasites. Significant progress achieved in the last 5 years in liquid culture and application and formulation technology has strengthened the position of nematodebased products in the marketplace (2,3). Marketing and/or research agreements between nematode producers and agrochemical companies and distributors have given nematode products a world wide recognition (Table I). In the U S A , Japan, Canada and Western Europe, successful market introduction was only achieved after steinernematid-based products were proven comparable with chemical insecticides based on cost and ease of application. Since the technology of liquid fermentation and formulation stability of 0097-6156/95/0595-0197$12.00/0 © 1995 American Chemical Society

In Biorational Pest Control Agents; Hall, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

198

BIORATIONAL PEST CONTROL AGENTS

heterorhabditids lags behind, their cost is 1-3 times higher than steinernematids and chemical insecticides (3). The last 5 years also had its share of disappointments. The development efforts to establish efficacious data against com rootworms (Diabrotica spp.), root maggots (Delia spp.) and wireworms (Elateridae) were unsuccessful (3).


Table I. Major Products and Formulations of Steinernema and Heterorhabditis-Based Products Formulation

Nematode Species

Product

Company/Disaibto

Alginate gel

S. carpocapsae

Exhibit

Ciba-Geigy, USA, W. Europe Dr. R* Maag, Switzerland Celaflor, Germany Pan Britanica Ind., United Kingdom Rhcne-Roufenc, My

Sanoplant Boden-Nutzlinge BioSafe

Clay

H. bacteriophora H. megidis S. feltiae S. scapterisci

Flowable gel

S. carpocapsae S. feltiae

Otinem Nemasys-H Larvanem Nemasys Entonem ProAct

Ecogen, USA A.G.C., UK Koppert, Holland A.G.C., UK Koppert, Holland BioControl, USA

BioVector BioSafe Stealth Exhibit

biosys, USA SDS Biotech, Japan Ciba-Geigy, Canada Ciba-Geigy, USA, W. Europe Amycel, USA

Magnet Water dispersible granule

S. carpocapsae

Vector T&L BioFlea Halt Interrupt Defend BioSafe Vector PCO

S. riobravis

Vector MC

Lesco, USA Farnam, USA Farnam, USA PittmarhMoc^USA biosys, USA V.W. & Rogers, USA Lesco, USA

A l l life stages of these nematodes occur in the insect host, except the infective third-stage juvenile (IJ). The infective juveniles locate and penetrate into the insects, and release mutualistic bacteria Xenorhabdus or Photorhabdus into the hemocoel. The bacteria multiply killing the insects within 24 - 48 hours. The nematodes feed on the bacteria and host tissues, reproduce, and produce Us after two or three generations. The IJs leave the cadavers hosts host after the depletion of their nutrients, seeking new insects. Depending on the nematode species and the insect host, the life cycle is completed in 6 - 14 days at 20-28°C. The duration and the life cycle of the nematodes in the liquid culture is similar to that


13. GEORGIS ET AL.

Formulation of Entomopathogenic Nematodes

199

in insects. At present, effective production of steinernematids has been achieved in 15,000-80,000 liter fermenters with a yield capacity as high as 150,000 IJs per cm . 3

Formulation


Formulation of nematodes serves two main purposes. One it extends shelf-life of the product, and second, it enables easy transport, delivery and application. Limited shelf-life of infective stage nematodes is a major obstacle in expanding their commercial potential. Factors affecting storage stability of nematodes are least understood. Holding large volumes of nematode suspensions in tanks has met with numerous problems including contamination. Theoretically, shelf-life of the non-feeding infective juveniles would be a function of stored energy and rate of its utilization. Lipid is a major energy reserve for non-feeding infective stages (8). We found that initial lipid level of nematodes have a direct impact on shelf life. The rate of utilization of stored energy depends upon many factors such as temperature, environmental stress, and activity. Behavior of nematodes during storage in water suspensions differs among species. 5. carpocapsae and S. scapterisci are less active during storage, but 5. glaseri, 5. feltiae, and H. bacteriophora are highly active. This behavior has a direct influence on energy burn-rate, and therefore, impacts nematode shelf-life (9). However, we have also observed that nematode batches with lower lipid content were generally more pathogenic,suggesting that trade-offs may exist. A large number of formulations have been developed for nematodes. In most of these formulations, the nematode movement is restricted to preserve stored energy (Table II). Generally, handling, shipping and application of large quantities of product is suitable in the high and medium value crops such as mushrooms, berries, artichokes, citrus, mint, and turfgrass. However, more stable formulations are needed for the nematodes to become commercially competitive with chemical insecticides in most of these markets and in low-value traditional agricultural markets such as cotton and corn. In this regard, recently a breakthrough in nematode formulation was achieved with the development of water dispersible granular formulation that allows the nematodes to enter partially into an anhydrobiotic state extending nematode survival and pathogenicity for up to 6 months at 4-25°C and up to 8 wks at 30°C (Table II). This formulation is scaleable and is easy to apply without any time consuming preparation steps (Table HI). The formulation is suited for a wide variety of consumer, agricultural and horticultural applications. The successful market acceptance of the nematode-based formulations will depend greatly on their consistent performance under field conditions. Therefore, it is important to maintain nematode quality throughout all stages of product development. The first step in standardization is aimed at obtaining reliable and consistent nematode production. Inoculum batches from in vivo cultures are produced from stocks of nematode strains that are stored by cryopreservation (8) to minimize variation in nematode pathogenicity between various production lots (3). Subsequent steps are focused on maintaining the viability and pathogenicity In Biorational Pest Control Agents; Hall, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.


2 0 0

of the nematodes immediately after the nematodes are harvested from the fermenter until the product is applied by the end-user. To assure this process, L T (the time needed to kill 50% of test insects) performance standards and optimum lipid contents of infective stage nematodes have been determined and are used to measure product stability. To assure stable products, nematodes are stored in large aerated tanks and are formulated within 1-3 months of the completion of production (Table IV). 5 0

Table II. Description and Storage of Major Steinernema and Heterorhabditis-Based Products 1


Formulation and Description

Product Storage 20-25°C 4-10°C

2

Alginate gel 20X10 nematodes (in 0.5 liter container) or 250X10 (in 4 liter container) trapped into a gel matrix and coated on a mesh screen 6

6

3-5 mo

6 mo

3

Clay

6

60X10 nematodes spread on 80 g clay

0

3 mo

3

Flowable gel Up to IX10 nematodes suspended in a gel matrix enclosed 4-6 wk in a special film (18cm ). Usually 4 to 6filmsare enclosed in 20 cm container Water dispersible granule 100X10 nematodes (formulated in 350 g) or 250X10 nematodes 6 mo (formulated in 680 g) of granule material (each approximately 5 mm diameter) enclosed in 700 ml and 1200 ml container, respectively 9

3 mo

2

3

3

6

1

2

3

6

6 mo

Based on product labels. Form of appliction is aqueous spray after dissolution of the alginate gel with sodium citrate. Form of application is aqueous spray after placement of the formulation in the spray water.

Table III. Characteristics of Steinernema carpocavsae - Water Dispersible Granular Formulation Character Data 8

Stability

Ease of Use

Product Size/Coverage

up to 6 months 4-25°C up to 8 wks at 30°C 6 days at 36°C 2 days at 38°C Dissolve quickly in water Compatible with most agrochemicals Compatible with most commercial sprayers Adequate for use in various market segments 350 gm (100X10 nematodes) treats up to 500 m Size comparable to chemical products Minimal product/packaging disposal requirements Competitive. For example BioSafe (Solaris Ortho) costs $2.99compared to $1.80-2.50/50m for Dursban (DowElanco); and Exhibit (Ciba) compared to $0.86 and $0.65-1.30/100m for Triumph (Ciba) and Oftanol (Miles), respectively. 6

Cost 3.39/50m $1.36/100m for 2

2

a

2

2

Nematode viability over 90% with stable pathogenicity In Biorational Pest Control Agents; Hall, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

2

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Another aspect of product assurance is the timing of production according to the market need. Most of the production is accomplished from January to March for products needed from May to August. However, for August to December markets, nematodes are produced from March to June. Certainly, such considerations are dependent on the nematode species, formulation type, storage requirements, market forecast and the distribution channels.


Application Technology Steinernematids and heterorhabditids have been proven efficacious against various soil inhabiting insects (Table V). These nematodes differ in virulence to specific hosts, tolerance to adverse environmental conditions, ability to seek out hosts, and behavior in the soil (5,6). Based on these characteristics, efforts made in recent years have led to matching optimal strain or species in a particular habitat against a particular insect species. Many factors affect our ability to place quantities of nematodes on or in close proximity to the target host in order to produce optimal results at the lowest possible cost To compensate for the impact of abiotic and biotic factors on nematode efficacy and persistence, the inundative application of a high concentration of a specific nematode species (approximately 2.5X10 - 7.5X10 infectives/ha) is needed (Table VI). Certainly careful considerations to optimal strain, irrigation requirements, timing of application, and method of application are needed to achieve predictable control (4). 9

9

Table IV. Storage and Shipping Requirements for Steinernematid-Based Products Steps in product development and distribution

Storage Parameters Condition

Period

Post-harvest

4-10°C

1-3 months

Formulation

4-8°C

3-6 months

Shipping

Non-refrigeration

2-15 days

Distributor and/or end user (Product Storage)

4-10°C 20-25°C

3-6 months See Table II

As with chemical insecticides, spraying nematodes as a curative treatment directly onto the soil surface is the most commonly used application method. This method is quick, simple, and provides good coverage. In some situations, nematodes have demonstrated control potential when applied at planting time. In certain soil environments where the target stage does not appear until 4-6 wk after planting (e.g., corn rootworm, Diahrotica spp.), insect damage was not prevented by nematodes applied at planting. However, efficacy improved when nematodes were applied 4 wk after planting (10). In Biorational Pest Control Agents; Hall, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

202


When the duration of nematode persistence is less than the time period during which a pest can damage a crop, multiple nematode applications at intervals of two or more weeks may be necessary to achieve the desired level of control This is especially important for insects such as mole crickets (Scapteriscus spp.) and fungus gnats (Sciaridae) that have multiple overlapping generations. The number of applications needed is the subject of current research.


Table V. Major Pests for Commercially Available Steinernema and Heterorhabditis species in Western Europe, Japan, and North America Segment

Nematode Species*

Artichoke

Common Name

Scientific Name

Sc

Artichoke plume moth

Platyptilia cardiuidactyla

Citrus

Sc, Sr

Blue green weevil Sugarcane rootstalk borer

Pachnaeus litus Diaprepes abbreviatus

Cranberry and berries

Sc,Hb,Hm Sc Sc Sc,Hb,Hm

Black vine weevil Cranberry girdler Crown borers Strawberry root weevil

Otiorhynchus sulcatus Chrysoteuchia topiaria Sesiidae 0. ovatus

Fruit Trees

Sc

Stem borers

Sesiidae

Greenhouse and Nursery Plants

Sc,Hb,Hm Sf Sc Sc, Hb, Hm

Black vine weevil Sciarid flies Stem borers Strawberry root weevil

0. sulcatus Sciaridae Sesiidae 0. ovatus

Mint

Sc

Mushroom

Sf

Cutworms Mintfleabeetle Mint root borer Black vine weevil Sciarid fly

Noctuidae Longitarsus waterhousei Fumibotys fumalis 0. sulcatus Lycoriella spp.

Sugar beet

Sc

Sugar beet weevil

Cleonus mendikus

Turf

Sc

Armyworm Billbugs Black cutworm Bluegrass webworm European crane fly Japanese lawn cutworm Mole crickets White grubs

Pseudaletia unipuncta Sphenophorus spp. A. ipsilon Parapediasia teterrella Tipula paludosa Spodoptera depravata Scapteriscus spp. Scarabaeidae

Sr, Ss Sg,Hb Vegetable and field crops

Sc

Cutworms Cucumber beeties Flea beetles

Noctuidae Chrysomelidae Chrysomelidae

Pet/Vet

Sc

Cat flea

Ctenocephalides felis

* Sc = & carpcapsae, Sr = S. riobravis, Sg = S. glaseri, Ss = S. scapterisci, Sf = S. feltiae, Hb = H. bacteriophora, Hm = K megidis In Biorational Pest Control Agents; Hall, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

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Nematodes should be applied to moist soil. Post-application irrigation and continuous moderate soil moisture are essential for nematode movement, persistence, and pathogenicity (2,4), and for nematodes to achieve insect control to a level comparable to standard insecticides (3). Although nematodes are recommended to be applied during early morning or evening to avoid the effects of ultraviolet radiation and temperature extremes, in many situations nematodes can be applied at any time of the day as long as post-application irrigation is employed within 30 minutes (3,4). Low temperature limits the pathogenicity of steinernematids and heterorhabditids, either by its influence on the activity of the nematode, the bacterial symbiont, or both. In the field, soil temperatures below 12-14°C resulted in unsuccessful insect control (3). As with most soil pesticides, a spray volume of 750 - 1890 liter/ha is usually required for most nematode species to reach the depth occupied by the target insect. Nematodes can be applied to the target zone with almost any commercially available spray equipment (Table VI). These include small pressurized sprayers, mist blowers, electrostatic sprayers, as well as aerial application via helicopters. In addition, nematodes are commonly applied using drip and sprinkler irrigation systems. Pressures of up to 1068 kPa have no detrimental effect on nematodes. The nematodes can pass easily through sprayer screens with openings as small as 100 microns in diameter. Table VI. Primary Equipment Systems Used to Apply Nematode-Based Products System

Segment

Treatment Site Dosage

Overhead Irrigation Helicopter Drip Irrigation

Berries

Broadcast Broadcast Furrow

^XlO^XlO'/ha 7.5X10 /ha 2.5X10 -5.0X10 /ha

Microjet Irrigation

Citrus

Spotted

2.0X10 /tree

Ground Sprayers

Turfgrass

Broadcast

2.5Xl(?7ha

Ground Sprayers

Mint

Furrow

2.5X10 /ha

Hose-end Sprayer

Mushrooms

Broadcast

0.5X10 -1.0X10 /m

Sprinkler Irrigation Ground and Hand Sprayers

Greenhouse and Broadcast Nursery Plants Pot

9

9

9

6

9

6

6

2

9

7.5X10 /ha 2X10*-5X10V4 liter pot

Numerous field data generated over the last decade showed that nematodes can protect crops from insect damage, thus, are not slow biological control agents. Insects such as white grubs (Scarabaeidae) and root weevils (Curculionidae) are controlled successfully within 2-4 wk post-application, whereas, 3-7 d is generally sufficient for the control of lepidopterans.


204



The compatibility of steinernematids and heterorhabditids with various chemical pesticides is a major concern when considering their inclusion in integrated pest management systems. Nematodes can be mixed safely with commercial preparations of Bacillus thuringiensis, pyrethroids, and various pesticides and fertilizers. Some pesticides can adversely affect nematodes, however they still can be used together if nematodes are applied before the pesticide or vice versa, thus allowing time for the pesticide to become absorbed or degraded to a level non-toxic to the nematode. To date, several successful attempts have been made to further increase nematode efficacy when employed in conjunction with chemical and microbial agents. (2,7).

Conclusion The quality of commercial nematode products is critical if insect-parasitic nematodes are to realize their full potential as biological insecticides. The stability and ease of use of dispersible granular formulation and the excellent quality of nematodes grown in liquid culture are significant steps towards this goal. A l l commercial formulations including water dispersible granular have been developed to maintain product stability during storage and transportation, and they are applied as a spray in water against the target pest. Granular, capsules and bait pellets (1,2) that can be applied by aircraft and standard granular applicators that protect and (or) release nematodes in the soil are also desirable and worth further investigation. Steinernematid and heterorhabditid species and strains differ in virulence, tolerance to adverse environments and behavior in soil. Therefore, future markets will require the introduction of various species and stable formulation to optimize and to expand the market potential of nematode-based products. Additionally, it is important to emphasize that the increased use of nematode products will require a change in the attitudes and behavior of the technical advisers and the end users. For example, managers will need to focus more attention on monitoring of pest populations and timing applications. The key to successful marketing and acceptance of nematode products will depend largely on how well producers and distributors response to consumer needs, anticipate and react to the changing environment, and develop quality products to solve their problem. Companies involved in commercialization of these nematodes are focusing on increasing their market shares as well as attempting to introduce nematode products against insects of large volume - low value markets such as corn and cotton. To achieve this goal, research is directed towards optimizing the production process, improving the application technology (e.g., timing and method of application), and utilizing more virulent nematode species/strains (through genetic manipulation or natural isolation) that may strengthen the position of the nematode-based products in the marketplace (2,7). Genetic engineering may be the solution for the development of an optimum formulation by inducing the nematodes to enter into a full anhydrobiotic state.


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LITERATURE CITED 1. 2.


3. 4. 5. 6. 7. 8. 9. 10. RECEIVED

Connick, W.J., Jr., Nickle, W.R. and Vinyard, B.J. J. Nematol. 1993, 25, 198-203. Gaugler, R. and Kaya, K.K. Entomopathogenic nematodes in biological control. C R C Press. Boca Raton, Florida, 1990. Georgis, R. Biocont. Sci. and Tech. 1991, 2„ 83-99. Georgis, R. and Gaugler, R. J. Econ. Entomol. 1991, 84, 713-720. Grewal, P.S., Lewis, E.E., Gaugler, R. and Campbell, J.F. Parasitology. 1994, 108, 207-215. Grewal, P.S., Selvan, S. and Gaugler, R. J. Therm. Biol. 1994, (in press). Kaya, H.K. and Gaugler, R. Annu. Rev. Entomol. 1993, 38, 181-206. Popiel, I. and Vasquez, E.M. J. Nematol. 1991, 23, 432-437. Selvan, S., Gaugler, R. and Campbell, J.F. J. Econ. Entomol. 1993, 86, 353-360. Wright, R.J., Witkowski, J.F., Echtenkamp, G. and Georgis, R. J. Econ. Entomol. 1993, 86, 1348-1354. January 31, 1995


Formulation of Entomopathogenic Nematodes - American Chemical

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