Early in 1996, the IITA pilot production plant was upgraded by the installation of a large autoclave, increased liquid fermentation (shaker) capacity, larger laminar flow hood, purchase of extra plastic tubs, air conditioners, a dehumidifier and a large freezer. The physical structure of the plant was modified with the separation of the two main incubator spaces to provide one at high humidity and one at low humidity. A cold, dry room for spore drying and packaging was installed (5 – 10°C, 45%RH).

A consultant, Dr. A. Arnold visited on two occasions, August and November 1996; he designed and installed a ventilation system, connected the high-power dehumidifier to the spore drier and cold room, and upgraded the spore separator (Mermelbat) (Bateman and Jenkins (9)).

Five local staff (PG-1) worked in the plant from early 1997 onward, under the supervision of the local supervisor (PG-6).

The basics of the production system are described in Jenkins et al., (1998) (2) and a detailed description of the plant and operating procedures in Heviefo et al., (9). Details of the economic assessment are given in LUBILOSA (1997 (7)) and Cherry et al., (1999).

Spores are stored on agar slopes in small bottles; spores are scraped from the surface and made up into a solution in Tween 80. This is used to inoculate the liquid medium, consisting of brewer's yeast and sucrose. After three days' growth, the liquid medium is mixed with autoclaved rice in plastic bags; these bags are kept, folded over, in the plastic tubs and incubated in the first incubation room at ambient humidity and 28oC for 8 days. The plastic bags are then cut away and the bowls stacked in the second incubation room at about 60% RH and 25°C for 10 days. This is referred to as the conditioning period, and appears to be essential to obtaining spores that will store well.

Spores are separated from the substrate in a rotating drum; a strong air-stream carries the spores to a cyclone extractor, nick-named the Mermelbat. This separates particles larger and smaller than the actual conidia and breaks up chains of conidia.

From the Mermelbat, the spores are transferred to a spore drying device in the cold room, where their moisture content is reduced to <5% over a period of 3 to 4 days. The spores are then packed into aluminium-polythene foil sachets and stored in the freezer.

Conidia were produced continuously during 1997 and 1998. Figures 1 - 3 show the amounts produced; detailed characterisation of factors underlying yield variability is given by Cherry et al. (1999).

Control of the level of contaminating micro-organisms is essential to obtaining a good quality, safe, effective product; even levels as low as 1 in 10⁶ of contaminants such as Aspergillus can change an essentially safe and tested product into one with high risks. Contamination checks are carried out visually on the inoculum slope; by plating out the liquid medium, by plating out the solid substrate after inoculation, and by visual inspection of the growing bowls. At the end of the production process, a final inspection step ensures the quality of the final product.

The best yields obtained on rice are about 35g/kg. Other substrates, such as cotton seed and oil palm seed waste have given yields equivalent to 90g/kg in laboratory scale experiments, but this was only possible using substrate quantities of 50-100g.

Commercial producers have opted to use different substrates according to local availability, the details of which are confidential.

The national programme in Madagascar, funded under a USAID grant, is the only programme continuing with a small-container style production system, and LUBILOSA is contracted to assist with expanding this facility.

Most work advising on spore production has been carried out with LUBILOSA's two commercial producers, NPP in France and BCP in South Africa. Dr. N. Jenkins has regularly visited both plants to assist in optimising productivity and minimising contamination.

Licensing agreements with the two commercial producers were completed in November 1998 with BCP and March 1999 with NPP. Transfer of intellectual property rights was a complex issue, which is discussed further under section 7 Removing constraints. Definition of the criteria for selecting these two companies and a description of the process are described in the Implementation Plan (Dent, 1997 (8))

The 'Mermelbat' spore extractor described by Bateman et al IN PREP and installed at IITA has continued to function satisfactorily, and spore batches meet specification. The basic principles established during the design phase of this device have been used to design the larger scale cyclones currently being installed and tested by BCP and NPP.

A smaller-scale device, the 'mini-Mermelbat' is currently being designed for use in small-scale plants, and is available for sale or transfer to NARES.

Details of a public-domain oil-flowable formulation will be published (Bateman et al. (9)), while confidential, high-specification version has been developed for NPP and BCP.

The oil-flowable formulation was field tested in Niger in 1997 and in South Africa in January 1998. Some slight modifications were made on the basis of the test results.

Aqueous formulations suitable for application as EC with knapsack sprayers have long been requested by farmers. Trial formulations containing spores in Codacide at 10% and 1%, and in Tween at similar concentrations were field tested by a student Sebastian Olichon at IITA and in a NARES trial in the Gambia.

One of the principal issues raised by locust control officers using Metarhizium operationally is that of knowing whether locust bands have already been treated or not. We have addressed this issue by recommending the addition of ultra-violet tracers. Lunar Yellow provides a cheaper alternative to Lumogen (about 5% of the cost); however, it is more difficult to formulate, and a test in Niger in 1998 failed to keep the tracer in suspension. Further laboratory trials are planned in Ascot.

Preliminary collaborative work with the University of Reading indicated a close correlation between published work on seed storage and the storage properties of fungus spores (Hong et al., 1997). This was further explored in some detailed experimental work (Hong et al., 1998). Results indicate that spores may be stored for several years at low temperatures, and many months in the range 30 to 40°C. In particular, the moisture content of the spores is the main factor determining their longevity, and the data have been drawn up into a basic model permitting a prediction of storage times. For optimal storage, the moisture content must be below 5%; however, spores are highly hygroscopic. They are able to reabsorb moisture from the atmosphere very quickly, hence the need to carry out the spore packaging in a cold dry room. Conversely, too rapid re-hydration is detrimental to the spore viability (Moore, Langewald and Obognon, 1997). These storage results represent a major breakthrough and will have wide significance beyond LUBILOSA

A model has been developed based on these results (Figure 4), and this is currently being validated against field data being collected in pesticide stores along the supply route from Cotonou to eastern Niger.

Further refinement of this work showed the importance of the 'conditioning' or pre-drying period (Moore et al, 1996; Hong et al., (9)), and refinements of the model are needed to incorporate the effects of methods of drying, and nutrient contents (spore quality). The model is available at the LUBILOSA web site.

We tested the use of time-temperature integrating indicators (TT1). These are currently used in shipping food and vaccines to indicate product freshness. The use of these indicators, which cost only a few cents each, would provide a good assurance of the viability of spores (Lomer et al. (9)).

Moore et al (1996) showed the importance of 'conditioning' spores for several days, preferably at reduced humidity. This requirement has been built into the production plant; before extraction, the spore-laden substrate is kept for 10 - 12 days in an air-conditioned room. After extraction, the spores are further dried for 3 to 4 days in a dehumidifier until a moisture content of <5% is reached. The effects of these manipulations on spore longevity have been incorporated into an improved spore storage model (Hong et al., 1999).

It is likely that under operational conditions, spores will sometimes be stored badly, and spray operations will be conducted using spores of reduced viability. Bioassays at Ascot have examined the efficacy of such spores. These bioassays indicated that conidial batches with less than 70% viability may have reduced virulence, but due to the nature of bioassays it is difficult to prove this statistically. In the interests of ensuring efficacy of the product in the field LUBILOSA does not recommend application of conidia with viability of less than 70%.

We are also investigating the use of time-temperature integrating indicators (TTI). These devices cost a few cents each, and can be stuck to the outside of spore packets to indicate if the package has been exposed to temperatures likely to degrade the spores (Lomer et al., 1999 (9)).

Published data indicates that certain lipids can be detrimental and inhibit spore germination, or conversely enhance germination. Various cuticular lipids were studied by a PhD student, S. Jarrold at the university Bath. No practically useful effects were discovered (Jarrold, 1998; (7)).

A book by H. D. Burges (Formulation of microbial pesticides, beneficial micro-organisms, nematodes and seed protectants, publ. Kluwer Academic Press, 1998) provide a complete list of currently known UV protectants. Trials conducted in Mali in 1994 indicated no significant increase in spore longevity with the addition of oxybenzone (Shah et al., 1998). Furthermore, current field data on spore survival in the field indicates excellent survival. Research effort in this area was therefore reduced.

Publication by Fargues et al (1996) gives an indication of the range of UV tolerance of different isolates. However, strain selection bioassays have not indicated that a change of isolate would be worthwhile.

Experiments were carried out to investigate the possible role of copper in enhancing spores resistance to UV. No difference was detected between the spores produced in the presence of copper sulphate and the control treatment.

Further work on strain characterisation is proposed with P. Bridge of CABI-IMI in order to develop a field characterisation method. The method by which the genus Metarhizium has been reclassified is DNA sequencing, which is not readily available in Africa.

Field research showed that the principal constraint to the success of mycopesticides in the field is the body temperature of the locust. Previously, bioassays had been conducted at a constant temperature of 30°C; a bioassay was developed with fluctuating temperatures of 12/12 20/35°C to simulate actual grasshopper body temperatures. To consider as a replacement, a new strain would have to give an improvement of at least 30%, e.g. bringing the AST down from 9 to 7 days with 90% overall mortality. We had also hoped to find a single strain which will kill both Zonocerus and other locusts and grasshoppers

Bioassays carried out at Cotonou and Ascot late in 1996 using the fluctuating temperatures designed indicated only minor variations in performance, insufficient to justify a change of strains (Jenkins et al., 1997 (2)).

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