Formulation and Storage
Formulation Development

The first attempts at formulating entomopathogenic nematodes were initiated in 1979, but at best, shelf-life as about 1 month. Infective juveniles carried on moist substrates such as sponge, vermiculite and peat require continuous refrigeration to maintain their viability (Georgis, 1990). Immobilization of the nematodes in a matrix increased shelf life, but desiccation of the nematodes, which reduces energy utilization, has been the most effective means of extending their shelf-life. As more basic studies on the nematodes were conducted, distinct biochemical, behavioural and morphological differences among steinemematid and heterorhabditid species were documented. For example, an important physiological adaptation is to have an appropriate lipid composition that enables the nematodes to regulate membrane function and to adjust for environmental extremes (Yamaoka et al., 1978). The relatively high lipid content of these nematodes suggests that they are adapted to survive prolonged periods of environmental stress (Selvan et al., 1993b ).The selection of formulation type, ingredient, packaging size and storage conditions cannot be undertaken unless the oxygen, moisture and temperature requirements for each species are defined. Thus, a better understanding of the biochemistry, physiology and behaviour of the infective nematodes will lead to the development of formulations that are more stable and easier to use. Clearly, the evolution from moist to partially desiccated formulations points to the advances that have been made in understanding the nematodes. Nematode metabolism is temperature-dependent, with warm temperatures increasing the rate of lipid reserve used but decreasing the time that the infective juveniles remain viable and pathogenic.

Differential water absorption by different species of entomopathogenic nematodes appears to affect their desiccation tolerance. A slow rate of water loss or'ut1take is essential for nematode survival during desiccation and rehydration. This maintains the structural integrity of the membranes (Preston and Bird 1987). Steinernematids have better desiccation survival capabilities because they. can regulate the slow drying and rehydration compared to heterorhabditids (Selvan et al., 1993a). Entomopathogenic nematodes may adopt various energy utilization strategies to maximize their surviva1 in the environment. Some nematode species are active foragers or cruisers (e.g. H. bacteriophora, H.megidis, S. glaseri and expend a significant amount of energy to find their host; others are sit-and-wait foragers or ambushers (S. carpocapsae, S. scapterisci) and do not expend much energy. In terms of energy expended, Baldwin (1964) proposed that oxidation of 1 mg of lipid, protein, or carbohydrate yielded 39.3, 23.7 and 17.4 J energy, respectively. Considering these values, estimated energy content of a single infective juvenile of H. bacteriophora, H. megidis, S. glaseri, S. feltiae, S. carpocapsae, and S. scapterisci was 0.038, 0.042,0.123,0.065, 0.030 and 0.029, respectively (Selvan et al., 1993a). Even though infective juveniles of S. carpocapsae and H.bacteriophora contained similar amounts of energy, S. carpocapsae lived longer (16-week ambusher) than H. bacteriophora (7-week cruiser) when these nematodes were held at 25 C in water (Selvan et al., 1993a). In another study, Selvan et al. (1993b) demonstrated that S. glaseri (cruiser) contained three times the energy of H. bacteriophora and survived for 24 weeks (Klein, 1990), whereas H. baceriophora (cruiser) survived only 3 weeks.

Active movement of the infective juveniles that results in expended energy may be responsible for the differential mortality. H. bacteriophora infective juveniles were infective in water (Gaugler and Campbell, 1991), whereas S. carpocapsae remained inactive with a typical J-shape posture. These behvioural differences may explain why S.carpocapsae based products have a room temperature shelf-life of 5 months, while heterorhabditid based products require continuous refrigeration for equivalent shelf-life (Georgis et al 1995). The infective juveniles of S. glaseri have a high oxygen consumption rate because of their continuous movement and large size. Consequently, products based on this nematode have a limited room temperature shelf- life compared to S. carpocapsae based products.

The foraging strategy of the nematodes dictates whether a given formulation can be used) (Table 9.2). In laboratory bioassays, both S. carpocapsae and S. scapterisci nictate (nematodes stand on their tails and wave) 50-80% r of the time, whereas H. bacteriophora and S. glaseri spend 70-90% of their time moving on a sand/agar plate. When searching for a host in nature, S. glaseri moves through soil, whereas S. carpocapsae often remains stationary on the soil surface (Kaya and Gaugler, 1993). Because of their tendency to be stationary, S. carpocapsae infective juveniles can be immobilized in the 20% calcium alginate formulation that can be dissolved within 30 min with sodium citrate (Table 9.3). In contrast, S. glaseri and H. bacteriophora are active foragers and can be immobilized only in a 35% calcium alginate formulation, which requires twice the amount of time to dissolve. At 20% calcium alginate, S. glaseri and H. bacteriophora actively migrate out of the alginate within 1-2 weeks, resulting in their death.

Desiccated Nematodes

A significant advance in formulation was made with the development of the water - dispersible granule (WDG) in which the infective juveniles are partially desiccated during the formulation process (Tables 9.1 and 9.4). The infective juveniles are encased in 10-20-mm diameter granules consisting of silica, Clays, cellulose compounds, lignins and starches (Table 9.5). Nematode droplets, each with approximately 40000 infective juveniles, are mixed with the formulation ingredients on rotating pans, creating l0-20-mm granules. Each granule contains a soft center of the nematode suspension surrounded by the dry ingredients. The moisture level in each granule can be adjusted, depending on the nematode species, at 35-45%. This can be done by calculating the residence time and the amount of formulation ingredients that are delivered to the pans to create the desirable hardness of the granules. Depending on the nematode species, granules collected from the pans are then exposed to optimum temperatures resulting in partial desiccation by the gradual absorption of water from the nematode.

The desired shelf life can be met as long as the moisture level of >90% RH can be maintained for the partially desiccated nematodes in the granules. Prior to formulation, a fungicide is added to the nematode suspension to restrict contaminants in the granules to a low.

Table 1. Comparison between two formulations of S. carpocapsae, based on 250 x 106nematodes per container
Characteristics Water dispersible granule Calcium alginate
Product description 250 x 106 nematodes in 680 9g Granules (40000 nematodes per Granule) mesh screen 250 x 106 nematodes trapped
Nematode status Partially desiccated Immobilized
Container size 1.2 I 4.0 1
Shelf-life* 5-6 months at 4-25°C, 2 months at 30°C, 6 days at 36°C 3-4 months at 4-25°C, 0.5 months at 30°C, 1.0 at 36°C
Ease of use Dissolve immediately in water, Ready to use, easy to measure 20-30 min preparation steps, once diluted the entire product must be used
Product size: coverage Comparable to chemical products and adequate for use in various markets Adequate only in greenhouse and home-garden markets
Disposal Minimal product disposal Burden to dispose of screens
Usage range various nematode species are compatible with WDG Suitable for few nematode species
Cost Low for ingredients and manufacture higher than WDG
Note: *>90~o nematode viability and uncbanged pathogenicity from the day of formulation.

Table 2. Major ingredients of water-dispersible granule formlulation
Ingredients Function
Diatomaceous earth Absorbent
Hydroxyethyl cellulose Binder
Amorphous silica. Absorbent
Fumed hydrophobic silica Absorbent
Lignosulphonate Dispersant and binder
Modified starch Binder
Attapulgite clay Absorbent
Fungicide Control contaminants
Wetting agent Facilitate mixing in water