Biorational Pest Control Agents - American Chemical Society

Chapter 15

Commercial Development of Entomopathogenic Fungi Formulation and Delivery David W. Miller

Downloaded by UNIV OF SYDNEY on May 3, 2015 | http://pubs.acs.org Publication Date: May 5, 1995 | doi: 10.1021/bk-1995-0595.ch015

Research and Development, EcoScience Corporation, 377 Plantation Street, Worcester, MA 01605

To be effective in the control of insect pests, insect pathogens must come into contact with the target insect, either by ingestion or external contact leading to cuticle penetration. This distinguishes pathogens from chemical pesticides, where the method of exposure is less critical and can be reliant on indirect means such as translocation leading to systemic action or activity via the vapor phase. This requirement for direct contact places unique and strong demands on the formulation and application methods for microbial pesticides. In fact, the operational concept of delivery is much more useful than that of application. The microbial active ingredient must be placed or brought into contact with the target, delivered to it, as it were. The principle commercially relevant genera of entomopathogenic fungi, Metarhizium and Beauveria, are contact pesticides. The infective fungal conidia penetrate the insect cuticle to initiate the infection process, leading to insect death. The development of pest control products based on fungal conidia has been hampered by problems with shelf-life, formulation, delivery and cost effectiveness. 0) These problems largely remain in spite of the over 100 years these agents have been in use. EcoScience has begun to develop tools which will form the technical core for a variety of fungal-based insect control products. To achieve this, the microbial product development pathway has been conceptualized as a series of discrete steps, specifically tailored to the active ingredients with which we deal. This perspective has already yielded registered and nearly-registered microbial insecticide products. The Microbial Product Development Pathway The pathway illustrated in Figure 1 captures the steps in the development of microbial products in a way which gives insight to their unique aspects and 0097-6156/95/0595-0213$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.

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requirements. Of particular note is the fact that these are living organisms which must, of course, remain alive through production and into and through the product distribution pipeline. A shelf-life of perhaps several years under frequently unfavorable conditions is required. Focusing on those pathway elements which must impact shelf life; that is, stabilization, formulation, and packaging, makes clear the unique challenge of developing microbial products. We have found that these technical challenges are initially most successfully and productively handled as individual steps. At the same time though, they must be seen to be of a piece in providing a finished product of acceptable commercial characteristics. To identify and employ the conditions which keep an organism viable and healthy, that is, stabilized, for a long period of time requires an understanding of the physiological demands of the organism and providing for them through appropriate means. We have purposefully placed the stabilization step ahead of formulation and packaging because this maintains the maximum process flexibility. Stabilization, therefore, is best viewed as a process option. For example, organisms which are most effectively stabilized under dry conditions are best dried before any formulation components are added. Once dry, a considerable number of formulation options will then exist (or remain). Formulation in the figure refers to the modifications made to the active ingredient, already stabilized as discussed above, and subsequently formulated into a form where it can be most easily delivered for its intended pest control purpose. Formats might include dusts, wettable powders, granules or liquids. Again, addressing the microorganism's requirements for long-term stability can either narrow the formulation options or increase them. Packaging is an historically unappreciated component of microbial pesticide product development. In part, this reflects the lack of appreciation for the role of the physiology of the active ingredient in the product development effort. Now, as will be seen below, this is being addressed, and packaging provides the means to maintain conditions which provide for these physiological requirements, yielding products with considerable shelf life. Packaging is our friend and, with stabilization and formulation, provides a set of tools which lead to commercially acceptable products. How the technical elements captured in the product development pathway come together will be illustrated with two examples from the product portfolio of EcoScience. Bio-Path® Cockroach Infection Chamber Cockroaches are a human pest of legendary proportions familiar to most everyone. (2) The german cockroach, Blatella germanica, is the predominant pest species. Large amounts of chemical insecticides are used in the attempt to control roach populations with the attendant concerns of resistance on the part of both the insect and the consumer. It is chiefly a forager and will investigate narrow spaces for In Biorational Pest Control Agents; Hall, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.


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Commercial Development of Entomopathogenic Fungi

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food and shelter, preferring dark, humid spaces. The fact that insect pathogenic fungi are contact insecticides would seem to make them ideal agents for a microbial pesticide directed at cockroaches. We have pursued this opportunity and developed a product now fully registered for sale by the U.S. Environmental Protection Agency, the Bio-Path® Cockroach Infection Chamber for the control of german cockroaches. The chamber is a plastic device similar in size and appearance to a chemical insect bait station but very different in its mode of action. The chamber (see Figure 2) contains 4 entrances equally placed around its perimeter. Located on the inside of the top section is a layer of the fungus Metarhizium anisopliae with the fungal conidia facing down into the chamber. Ramps lead up from the entrances so that entering roaches are brought into contact with the fungus layer; that is, the chamber serves as the formulation to deliver the active ingredient to the target. Roaches are infested with the fungus and leave soon after entering, taking fungal inoculum with them. A roach directly contaminated with the fungus will die within two weeks. Roaches so infested play a key role in the further spread of the fungus to their colleagues. Through the Horizontal Transfer™ effect, an infested roach, coming into contact with its nestmates, spreads the fungus to them. This leads to an amplification of control beyond just those insects which entered the chamber. This phenomenon is, of course, another method of delivery and illustrates how microbial pesticides offer not only environmental benefits but, as a new class of actives, provide unique features. The Bio-Path product has been through many tests in both the laboratory and the field. In the field, it is providing performance comparable to chemical bait stations (see Figure 3). Currently, the product is being handled by professional pest control operators and has just been introduced to the retail marketplace. It is being produced through solid state fermentation and assembled in a modern facility capable of producing several million chambers per year. A consumer-oriented product needs a significant shelf-life; assume, two years at room temperature. This has been a major commercial limitation for most microbial pesticides, including fungi. To achieve this goal, our efforts have been directed at determining the requirements of the fungus - those both environmental and physiological. The fungus in the Bio-Path chamber consists of the conidia attached to the vegetative mycelia. These remain attached to the solid agar substrate and constitute an actively metabolizing culture, although the implications of this were not initially obvious to us, especially as regards the spores. Under the assumption that packaging could provide a key to maintaining the appropriate environmental conditions to stabilize the fungus, we examined several standard, commercial packaging materials for their impact and influence on long-term fungal viability. Packaging materials can be either opaque or of varying degrees of transparency to gases. Aluminum foil packaging of, in general, 0.007 in. thickness is a complete


216


STEPS IN THE DEVELOPMENT OF A MICROBIAL PESTICIDE

ASSAY STRAIN


BIOMASS PRODUCTION STABILIZE FORMULATE

PACKAGE SPRAY DELIVERY* (APPLICATION)

ECOSCIENCE

Figure 1. The microbial product development pathway.



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2

4

6

8

217

12

Week Post-treatment

•

Raid Max

•

MaxR)rce/Combat •

Bo-Rath ESC

• Racebo

Figure 3. This figure illustrates the % reduction achieved in a German cockroach population through the use of several commercially available treatments. Each treatment represents about fifteen apartments. In each case, an average number of roaches is determined for the treatment group through a one week pre-count of each of the apartments. The specific treatments are then introduced, and the apartments are monitored at the times indicated. The % reduction indicates the current average roach population for each treatment relative to its pre-count. The treatments are: Combat (active ingredient: hydramethylnon), Raid Max (active ingredient: sulfluramid), Bio-Path, and placebo (empty Bio-Path chambers).


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barrier to gases of importance to living organisms: oxygen, carbon dioxide and water vapor. On the other hand, polyethylene film allows the relatively free passage of oxygen and carbon dioxide but is a significant barrier to water vapor, with the degree of these properties depending on film thickness and density. We sealed chambers containing fully-developed Metarhizium cultures into pouches of either 0.007 in. aluminum foil or low density polyethylene (LDPE) of 0.008 in thickness. These were then incubated for several months at 25°C or 37°C before analyzing fungal conidia viability. The interior of the pouches was also monitored for atmospheric composition. As can be seen in Table 1, the viability of the fungus suffered dramatically in the foil pouches. This was correlated with a total loss of oxygen and a great increase in carbon dioxide. The fungus in LDPE, however, survived well with an elevated carbon dioxide level but a good oxygen level. These results and others similar indicate the importance of oxygen to (and removal of C 0 from) the fungus in storage. They also indicate the actively respiring state of the fungus culture and the importance of the packaging choice in maintaining fungal viability. A similar experiment indicated that in the presence of oxygen, high relative humidity was essential for long-term fungal viability providing another reason to select LDPE packaging. 2

A two-year test of the completed packaging concept demonstrated high viability and efficacy of Metarhizium in the Bio-Path chamber for the full period. To summarize this formulation and delivery concept: The fungus Metarhizium anisopliae has been formulated into a chamber designed to maximize delivery of the pathogen to the cockroaches which enter it. Roaches leave the chamber and deliver the fungus to yet additional roaches. The product, manufactured through solid state fermentation, is packaged with a system which, by addressing the physiological requirements of the fungus, provides a commercially relevant shelflife of two years. The patented chamber delivery concept has been shown to be of utility against a variety of insect pests. Fungal Conidia Sprays The insect pathogenic fungi, Metarhizium and Beauveria, have a very broad host range measured as either breadth of recorded natural species infections or by laboratory bioassay. (3) Nonetheless, most efforts to employ them as insecticide active ingredients have been based on the use of a particular isolate against a particular insect pest, usually an isolate found associated with the pest in some natural infection. There are two problems with this: One, technology developed for this unique situation doesn't really carry to other systems, requiring continual re-invention and, as a result, two, the potential for a broad spectrum, environmentally friendly, natural insect control agent is not realized. Our approach has been to develop a set of technologies which create a fungal product that can be thought of and used as a broad spectrum insecticide, having the performance and handling characteristics of classical chemical products while


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Table 1. Stability of fungal conidia under different storage conditions. Conditions within Foil Pouch at Three Months Temperature %o

2

%C0

2

% Viability*

22°C-A 22°C-B

0.3 o

23.4 22.5

15.00 11-15

30°C-A 30°C-B

o o

23.7 22.0

0.00 0.00


Conditions within LDPE Pouch at Three Months

22°C-A 22°C-B

8.5 8.8

3.0 2.5

92.1 90.9

30°C-A 30°C-B

8.7 4.5

3.0 4.2

86.0 87.8

* % viability is determined as the number of germinating conidia versus total conidia after 13 hours on potato dextrose agar media at 28°C.

maintaining the environmentally favorable characteristics of a product based on a naturally occurring microorganism. In the development of such a product, the key issues to address are: Manufacturing, shelf-life under unfavorable conditions, ease of application and efficacy. This abbreviated discussion will touch on those issues related to formulation and delivery. As the above discussion on the Bio-Path product revealed, a respiring culture in a high humidity environment requires atmospheric conditions comparable to a natural environment. However, a more standard format for a broad spectrum, wide applicability insect control product is as a dry formulation. Such a product could be more easily distributed and stored. When needed, it could, for example, be re-suspended and delivered as a spray for insect control. There are some intriguing possibilities here: A dry product is light and compact, a dry product provides a variety of formulation options, and, for a microorganism, decreasing the water content holds out the possibility of improved stability, particularly at higher temperature. As has been in published under the Patent Cooperation Treaty in number W93/24013, this goal has been achieved for Metarhizium. In particular, it was found that storage under dry conditions yields a product with exceptional stability characteristics. It was also found that removal of the oxygen further improved stability, presumably protecting the biological machinery from oxidative effects more pronounced at elevated temperatures.


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Formulations composed of dry fungal conidia, amended with bulking agents and appropriate surfactants and then correctly packaged, provide exceptional products, having good shelf stability, ease of use and efficacy. We have such products now in extensive field trials, where substantially similar formulations, used as aqueous suspensions and sharing a common fungal species, are showing good activity against insects as diverse as termites, aphids, and whiteflies. Current storage data indicates at least one year of good fungal viability at 37°C. The concept we have tried to establish in this presentation is the importance of addressing the requirements of both the fungus and the insect target in developing products with desirable (and commercially required) product characteristics. Not surprisingly, our principal areas of focus then are formulation and delivery, taking advantage of and addressing the unique characteristics of fungi. Acknowledgments David W. Miller, Ph.D. acknowledges the major contributions of the EcoScience research staff to the work described in this paper. Thank you to Ms. Robin Gay for administrative assistance. Literature Cited (1) Fungi in Biological Control Systems, Burge, M . N . , Ed., Manchester University Press: Manchester, U K . 1988 (2) Understanding and Controlling the German Cockroach, Rust, M . K . , Owens, J.M., Reierson, D.A., Eds. Oxford University Press: New York, N Y . 1995. (3) Goettel, M.S., et al. In Safety of Microbial Insecticides; Laird, M . , et al, Eds. CRC Press, Inc.: Boca Raton, Florida. 1990. pp. 209-231. RECEIVED

March 13, 1995


Biorational Pest Control Agents - American Chemical Society

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