Production Formulation and Application

Production of entomopathogenic fungi has not advanced greatly beyond the use of simple grains as substrates for the Deuteromycete fungi, such as Metarhizium and Beauveria. For many other entomopathogenic fungi, especially among the Entomophthorales, growth in culture is difficult or has yet to be achieved. Both liquid and solid substrates have been substantially investigated (Burgess 1998). Two-stage systems, where both liquid and solid substrates are used, have occasionally proved successful. For example, fermentation to produce hyphae to use as starter cultures is now a widespread practice. There are number of advantages to using liquid cultures as starter cultures: (a) the competitive ability of the fungus is enhanced, reducing the risk of contamination from other microbes, (b) growth is more rapid in the early stages, (c) the liquid culture can be screened for contamination prior to use, and (d) the liquid ensures even coverage of the solid substrate (Jenkins et al. 1998). Liquid starter cultures are commonly used to begin solid substrate production. However, experience with M. anisopliae in our laboratory is probably typical of many other laboratories, where inoculation of rice grains with fermenter broth of M. anisopliae hyphal bodies gave no improvement in production over the use of conidia from plate cultures (Glare et al. unpublished data). Production on grains is generally in the range of 108-1010conidia/g of dry substrate [e.g., Feng et al. (1994)], taking between 2 and 3 weeks to reach maturity at optimal temperatures. Interestingly, Metarhizium and Beauveria sporulate better when the substrate is relatively poor in nutrient content. When grains were supplemented with sugars and yeast additives, less conidia per gram of substrate was obtained than with grains alone (Nelson et al. 1996). Similarly, in Brazil, M. anisopliae has been found to produce conidial yields of 5-15 times higher using rice bran/rice husk substrate mixtures than yields usually obtained for rice grains, with viabilities of higher than 85% (Dorta et al. 1990).

The production of Green Muscle™ M. anisopliae for locusts in Africa used a two-stage production system with fermenter production of inoculum used to inoculate rice (Cherry et al. 1999). The process requires relatively low capital investment, but has high labor costs. As with production of most fungi, high variability in yield was reported between batches, and this variability was only partly accounted for by temperature and duration of incubation (Cherry et al. 1999).

A method that showed some promise in the 1980s was the preparation of dried mycelium. Hyphal bodies were harvested by filtration, washed with water to remove culture medium residue, and then coated with a sugar solution before drying. This method was used with M. anisopliae and B. bassiana (Pereira and Roberts 1990). They found that conidial production was similar to other methods after storage for up to 4.5 months at 4°C and could be superior to other methods with respect to storage at room temperature, however no products at present use this technology.

The Emerald Bio production plant (previously Mycotech) in Butte, Montana, represents the technological end of the production of entomopathogenic fungi. Largely utilized for the production of B. bassiana, it is a "state of the art" dedicated facility, with in-line sterilization and large temperature controlled growth facilities. The actual production method is a trade secret, but is based on fermented starter cultures and solid substrate growth and sporulation. This highly technical facility contrasts with the numerous low technology "factories" producing fungi for insect control in China and much of Latin America.

Compatibility between production, formulation, and application techniques is vital for the successful use of microbial biopesticides. The LUBILOSA program for locust control used Metarhizium in oil formulations and ULV spraying, which required lipophilic conidia for easy suspension in oils (Jenkins et al. 1998). While production of submerged conidia was seen as having many advantages, the resulting conidia were hydrophilic and lost viability quickly. Therefore, production on grains remains the standard with the locust products. For many years, approaches to the use of entomopathogenic fungi involved point release ("classical biological control") or simple application of conidia, formulated in water with wetting agents. However, appropriate formulation can advance entomopathogenic fungi from curiosity to effective biocontrol agents. It has been an area that has benefited from the application of biotechnology. Formulation has been important in terms of improved survival during storage, persistence in the field (such as UV and desiccation tolerance), and ease of application.

The LUBILOSA program, where M. anisopliae var. acridium was developed into a biopesticide for locust control in Africa, is an excellent example of formulation overcoming environmental constraints. As locusts live in hot, dry climates and M. anisopliae conidia require high humidity to germinate, it seems impossible that an entomopathogenic fungus could successfully control the pest. However, formulating Meta-rhizium conidia in nonevaporative diluents such as oils allowed the conidia to attach and germinate on susceptible locusts. M. anisopliae oil formulations are especially useful at low relative humidities (Bateman 1997). There have been several interesting studies on formulating hyphal material from members of the Entomophthorales. These fungi, because of the fragile nature of the mycelium and conidia, pose a much greater formulation problem than most of the Deuteromycetes, which has contributed to their lack of commercial success. McCabe and Soper (1985) patented a process of drying the mycelium of Zoophthora radicans and coating it with sugar, as a method for long-term storage. More recently, Shah et al. (1998) demonstrated algination as a method for formulating Erynia neoaphidis mycelium. An important area of formulation and production is the drying of conidia of entomopathogenic fungi. Moore et al. (1996) have shown that survival of conidia of M. anisopliae was highest at low (< 5%) relative humidity, therefore, this is an important aspect of producing a stable product.

Use of appropriate application techniques that are suited for the application of biopesticide to the target pest is an obvious, but often neglected aspect of biopesticide use. Advances in chemical pesticide applications have slowly filtered through to use with biopesticides, such as ultra-low volume (ULV) application of M. anisopliae for locust control (Lomer et al. 2001). Nonevaporative diluents such as oil are required to take advantage of ULV spraying. Rotary atomizers have been used for low volume oil formulations and ULVs for less than 5 l/ha. Electrostatically-charged ULV sprayers have been investigated for better coverage on leaf undersides (Sopp et al. 1989). Generally, application of fungal-based biopesticides has been with conventional equipment and research has focused on spray coverage, droplet size, and placement (i.e., penetration to the underside of leaves). Hydraulic spray systems have been used to apply water-based formulations on crops, air-blast and air-assist technologies are primarily used for low volume applications in fields and orchards. The best success has been with large numbers of droplets with high spore content per droplet (Goettel et al. 2000).

Introducing large amounts of fungal inoculum into the soil and securing an even spread remains a problem. Many methods have been tested for application of fungal containing granules or conidia on grains to soil, including using seed drills for subsurface application, and hand application. The problems of spread of conidia after application to soil has lead to the Melolontha and researchers are developing an area wide approach based on augmentative applications of Beauveria brongniartii for long term suppression of pest populations (Hajek et al. 2001).

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