Parasitic nematodes are widely used as biological control agents, for example to control insect and slug pests in agriculture and horticulture. They are typically mass produced in fermenters, formulated onto inert carriers, and used as pesticides. In conventional formulations, the nematodes are non-feeding infective larvae which rely on their intrinsic energy reserves for survival. Accordingly, it is generally necessary to inactivate the nematodes for storage and use.

In typical commercial products, nematodes are inactivated for storage and shipping by being partially dehydrated onto an inert carrier, such as clay or vermiculite. However, nematode survival in such products is generally poor, the products are difficult to store and handle, and are not suitable for all types of nematode. In addition, when the formulation is mixed with water and applied through a sprayer, lumps of carrier can block the spray nozzles. Also, these formulations can also be difficult to use with larger nematodes such as Steinernema glaseri.

In nature, nematodes live on the surface of soil particles in a film of water on the particle surface. However, when soil is dry, or is flooded so the nematodes cannot adhere to the film of water, the nematodes become inactive. Thus, liquid formulations offer another means by which nematodes may be inactivated for storage and use. However, liquid formulations have not been widely used because nematodes tend to sink in

aqueous solutions, and die due to lack of oxygen.

An object of the present invention is to provide a nematode containing formulation which seeks to overcome the above described disadvantages of conventional nemetode containing formulations.

According to the present invention there is provided a nematode containing formulation, which comprises nematodes in a colloidal dispersion, wherein the colloidal dispersion has a density substantially equal to the density of the nematodes.

Thus, the nematode containing formulation of the present invention has a neutral density, i. e. the density of the colloidal dispersion is substantially equal to the density of the nematodes. The nematodes in the colloidal dispersion float and remain substantially evenly distributed throughout the dispersion.

The present inventors have found that nematodes can survive for longer periods, they respire less, and their lipid reserves deplete more slowly in the formulation of the present invention, compared to when formulated in water under aerated conditions. Nematodes typically have relative densities of approximately 1.03-1. 08, and solutions in this density range can be achieved by dissolving large amounts of salts in water.

However, such solutions are unsuitable for storing nematodes because they exert extremely strong osmotic pressures. The colloidal dispersion used in the present invention has a density which is substantially equal to that of the nematodes, but causes negligible osmotic pressure.

Any suitable nematode may be used in the formulation of the

present invention, for example any nematodes of the genera Steinernema, Neosteinernema, Heterorhabditis, or Phasmarhabdiditis.

The nematode concentration in commercially available nematode formulations varies, and may, for example, be from 10, 000 to 250,000 nematodes per ml, for example 20,000 to 100,000 nematodes per ml.

The term"colloidal dispersion"is used herein as would be conventionally understood by a person skilled in the art.

Thus, this term refers to a dispersion of very small insoluble particles of a material in a fluid, the particles being larger than individual molecules of the material, but small enough so as to remain suspended in the fluid without settling.

Typical particle sizes range from several nm to several mm, the lower particle size boundary being determined by the molecular size of the material in question, and the upper particle size boundary being determined by the particle size at which external forces such as gravity become more influential on the particles than the Brownian motion of the particles within the fluid.

Colloidal dispersions may be charged, and thus interact with long range forces, i. e. the particles repel each other, which can stabilize the dispersion by helping to prevent agglomeration. For example, with colloidal silica particles in water, hydroxide ions from ionized water bond to the surface of the silica particles, which accordingly become negatively charged. A counter-ion, for example sodium or ammonium ions, may be added to the colloidal dispersion to neutralize the charged colloidal particles. The properties of a colloidal dispersion can be affected by changes in pH.

Any suitable colloidal dispersion may be used in the present invention, for example silver halide, gold sol, polymer latex, and silica dispersions. However, colloidal silica (i. e. silicon dioxide) is a particularly preferred colloidal dispersion for use in the formulation of the present invention.

Colloidal silica is well known in the art, and is widely available commercially. For example, a suitable colloidal silica for use in the present invention is Ludox (RTM) (Du Pont).

Colloidal silica may be cationic, anionic or non-ionic.

However, in typical commercially available colloidal silica, the silica particles are anionic, having a stabilizing counter-ion, for example sodium ions (from, for example, sodium monoxide) or ammonium ions (from, for example, ammonium hydroxide). For cationic colloidal silica, the counter-ion is negatively charged, for example chloride ions (from, for example, hydrogen chloride).

The average diameter of silica particles in commercially available colloidal silica is typically from 1 to 100nm, in particular 10 to 50nm, for example 5 to 25nm.

The silica content of commercially available colloidal silica is typically 5 to 50 % wt, for example 25 to 40 % wt. However, in the formulation of the present invention, the density of the colloidal dispersion is substantially equal to the density of the nematodes, typically from 1.03 to 1.08. Accordingly, colloidal silica having a relatively low silica content is most suitable for use in the present invention, for example a silica content of from 5 to 20 % wt, preferably 5 to 15% wt.

Where a counter-ion is present in the colloidal silica, the weight ratio of silica to counter-ion provider (for example, sodium monoxide) may be in the range of 50 to 500, for example 100 to 250.

As referred to above, the properties of colloidal silica are affected by pH. Typical commercially available colloidal silica has an alkaline pH, in particular a pH of 8 or higher, for example from 8 to 10. In formulations in which the colloidal dispersion is alkaline, pH can be adjusted so as to be substantially neutral, i. e. to replicate the pH of soil water.

Colloidal silica typically has an appearance ranging from clear to opalescent to milky, according to the particle size of the silica. At lower particle sizes (for example, below 10nm), the colloidal silica may be clear, at intermediate particle sizes (for example, 10 to 50nm), the colloidal silica may be opalescent, and at higher particle sizes (for example, above 50nm) the colloidal silica may be milky.

Preferred colloidal dispersions for use in the present invention are free flowing, having a low viscosity (for example, 1 to 50 cp at 25°C), and are non-biodegradable.

Preferred colloidal dispersions allow for incorporation of biocides to prevent fungal growth, and are environmentally substantially harmless.

Embodiments of the present invention will now be described in detail with reference to the accompanying drawings, in which: Figures 1A and 1B show the survival of H. megidis and S. feltiae respectively over 16 weeks when stored in aerated

quarter strength Ringer's solution and Ludox (RTM) solutions of various densities at 4°C and 15°C respectively ; Figures 2A and 2B show the dose response relationship between mortality of T. mollitor larvae and nematode dose for H. megidis stored at 4°C, and S. feltiae stored at 15°C respectively, following storage of the nematodes for 16 weeks in aerated quarter strength Ringer's solution and Ludox (RTM) solutions of varying densities prior to bioassay at 4°C ; and Figure 3 shows nematode respiration measured by CO2 output per day by S. feltiae in quarter strength Ringer's solution and Ludox (RTM) 1085, and by H. megidis in quarter strength Ringer's solution and Ludox (RTM) 1035 stored over a period of 15 days.

Example 1) Materials and methods a) Nematode strains Commercially produced strains of Steinernema feltiae and Heterorhabditis megidis having densities of 1085 and 1035 respectively, formulated on clay, were obtained from Koppert UK. The nematodes were extracted from the clay by centrifuging in a 48% w/v sucrose solution then washed on a 53 mm sieve. The density of nematodes was determined by density gradient centrifugation in a solution of 90 % (v/v) Percoll (Amersham Pharmacia Biotech Piscataway, NJ) and 10% (v/v) 1.5 M NaCl. Density was estimated using Density Marker Beads (Amersham Pharmacia Biotech, Piscataway, NJ).

b) Nematode survival Preparations of Ludox HS-30 (RTM) (DuPont Ltd), a commercially available colloidal silica, were made at three densities for each nematode species (1035,1040 and 1045 g/l for H. megidis, and 1075,1080 and 1085 g/l for S. feltiae). Ringer's solution tablets were added at quarter strength to simulate the osmotic pressure of soil water, and the pH was adjusted to 7.0 by addition of 4M H2SO4 Nematodes were stored in 250 ml Erlenmeyer flasks containing 200 ml solution and 2 million nematodes (10,000 nematodes ml-1). Nematode survival in these solutions was compared with aerated quarter strength Ringer's (QSR) solution, by preparing similar flasks and storing on a flask shaker. Replicate flasks of each H. megidis preparation were incubated at 4°C, and replicate flasks of each S. feltiae preparation were incubated at 15°C. Nematode survival was recorded weekly for a period of sixteen weeks by taking two replicate 0.5 ml sub-samples, diluting and counting nematodes microscopically. c) Nematode infectivity Nematodes were bioassayed against Tenebrio mollitor after sixteen weeks. Instar larvae of T. mollitor were obtained from Live Foods Direct, (Sheffield, UK). Twenty-five larvae were kept in 9 cm petri dishes containing 12 g of peat moss.

Petri dishes were treated with 1000,3000, 10,000, 30,000 or 100,000 of the nematode larvae suspended in 8 ml of water.

One such series of doses was prepared from each replicate flask in the storage experiment (four replicates in total).

Plates were incubated at 15°C and larval mortality was recorded after one week.

d) Measurement of respiration In a separate experiment, respiration of S. feltiae and H. megidis was recorded during storage in neutral density Ludox (RTM) (density for S. feltiae = 1085 g/l ; density for H. megidis = 1035 g/1), and QSR solution (static). Fifty thousand nematodes were suspended in 10 ml of test medium in 50 ml Erlenmeyer flasks fitted with self sealing rubber caps (Suba-Seal). The headspace above the liquid was estimated gravimetrically prior to the start of the experiment. Six replicate flasks of each treatment were incubated at 15°C.

Three flasks were sampled after 5 days and the remaining three were sampled after 15 days. On each sampling occasion a 5 ml sample of headspace gas was taken through the seal, and diluted with 10 ml nitrogen. The amount of CO2 in the headspace was estimated using a gas chromatograph, calibrated using standards ranging from 350 ppm to 5000 ppm. After gas sampling, nematodes were counted so that respiration could be expressed as mg CO2per 1000 nematodes per day.

2) Results a) Survival-H. mevidis Both time and storage medium had significant effects (P<0.001), and the two factors interacted significantly (P<0.001). For all treatments, nematode numbers fell sharply for the first three weeks. However, nematode survival remained at a similar level until the end of the experiment for H. megidis stored in Ludox (RTM) having a density of 1035 g/l, whereas nematode survival continued to fall in all other treatments (Fig 1A).

b) Survival-S. feltiae Both time and storage medium interacted significantly (P<0.001). The rate of decline in nematode numbers was much faster in the aerated QSR solution than in all Ludox (RTM) treatments (Fig 1B). c) Nematode infectivity-H. mevidis There were significant (P<0.001) differences in virulence between nematodes stored in the different media. Virulence was highest in nematodes stored in Ludox (RTM) having a density of 1035 g/l (log LD50 = 4. 236 0.0357), nematodes stored in Ludox (RTM) having densities of 1045 and 1040 g/l had intermediate virulence (log LDso = 5.106 0.0528 and log LD50 = 4.9806 0.0486 respectively), whereas nematodes stored in QSR were least pathogenic (log LD50 = 5. 8486 0.0832) (Fig. 2A). This indicates that the nematodes that survived best (in Ludox (RTM) having a density of 1035 g/1) were the most pathogenic. d) Nematode infectivity-S. feltiae Nematodes from Ludox (RTM) having a density of 1085 and 1080 g/1 were most pathogenic (log LD50 = 4.3308 0. 07 and log LD50 = 4.559 0. 0657 respectively), with nematodes stored in Ludox (RTM) having a density of 1075 g/l being slightly less virulent (log LD50 = 4.944 0.0762) (Fig. 2B). e) Nematode respiration At both sampling dates, the CO2 in the headspace was measured and then the numbers of surviving nematodes was recorded in order to calculate CO2 production per nematode per day. This statistic was not significantly different between the two dates so the two data sets were combined. For S. feltiae, nematode respiration was significantly (P = 0.01) higher when

stored in QSR compared with Ludox (RTM), with nematodes stored in QSR daily producing more than three times the amount of carbon dioxide. For H. megidis, nematodes stored in QSR daily produced approximately twice the amount of CO2 (Fig. 3).

As shown by the results discussed above, both nematode species showed improved survival in Ludox (RTM) compared to aerated QSR. For all treatments, nematode numbers fell during the first few weeks, following which the rate of decline slowed.

The nematodes had been formulated and stored in a partially dried state prior to being used in the tests, and survival might improve by using freshly produced nematodes.

Nematodes suspended in Ludox (RTM) respired less than in the QSR formulation, indicating that the basal metabolism has slowed.

Nematodes suspended in Ludox (RTM) were more virulent at the end of the storage period than those suspended in aerated QSR.

The colloidal dispersion used in the formulation of the present invention must have the appropriate density, i. e. a density substantially equal to the density of the nematodes.

For example, H. megidis suspended in Ludox (RTM) having a density of 1035 g/1 at 4°C have improved survival and virulence compared to other Ludox (RTM) preparations or aerated QSR.

It will be understood that the embodiment illustrated shows an application of the invention only for the purposes of illustration. In practice the invention may be applied to many different configurations, the detailed embodiments being straightforward for those skilled in the art to implement.