2006-04-24

Bio-pesticide
production

1
Eva Nordberg-Karlsson Maria Karlsson, B01
Department of Biotechnology, LTH

2
Introduction
Artificially produced pesticides are today an important component in agriculture, but the
demand for biological alternatives is increasing, especially in the ecological agriculture.
Microbial control agents can be effective and serve as alternatives to broad-spectrum
chemical pesticides.

Biopesticides could be divided into three major classes: Microbial pesticides, Plant-
incorporated protectants and biochemical pesticides. This work will focus on microbial
pesticides. Organisms used as pesticides include bacteria, viruses, fungi, protozoa and
nematodes.

Many applications require large scale fermentor production of microbial inoculants,

followed by formulation steps for long term stability and ease of environmental
dissemination. Presently, lack of knowledge about fermentation/formulation
technologies and consequence analyses for novel microorganisms, seriously prevents
their implementation to solve environmental problems and to contribute to growth of
novel biotechnology industries. The costs of the production are generally high for
biocontrol products, and there may be problems of timely supply and storage. To improve
the use of biological agents the production costs need to be decreased.

Biopesticides have many advantages and one is that they are often pest specific and thus
harmless to nontarget organisms including humans. The disadvantages are mostly linked
with their persistence, speed of kill, specificity and cost relative to conventional chemical
pesticides.

Future developments for commercially use demand that the biopesticide is easy to use,
reliable, economical, and lastly it should also contribute to the sustainability of the whole
system.

3
Introduction..........................................................................................................................2
Microbial pesticides.............................................................................................................4
Bacteria............................................................................................................................4
Bacillus thuringiensis...................................................................................................4
Other bacteria...............................................................................................................5
Viruses.............................................................................................................................5
Other viruses................................................................................................................5
Fungi................................................................................................................................5
Protozoa...........................................................................................................................7
Plant-incorporated protectants.............................................................................................7
Biochemical pesticides........................................................................................................7
Production............................................................................................................................8
Bacteria............................................................................................................................8
Virus.................................................................................................................................8
Fungi................................................................................................................................8
Nematodes.......................................................................................................................8
Fermentation parameters.............................................................................................9
Costs of production......................................................................................................9
Regulation..........................................................................................................................10
Research.........................................................................................................................10
The use of biopesticides.....................................................................................................11
Advantages........................................................................................................................12
Disadvantages....................................................................................................................12
Future developments..........................................................................................................12
Discussion..........................................................................................................................13
References:........................................................................................................................14

4
Microbial pesticides
Bacteria
Bacteria have been used in the biological control of insect pests since the early 20th
century, but very few entomopathogenic bacteria have been developed into commercially
availably biopesticides.1 Most of the pathogenic bacteria are found in the families
Bacillacea, Pseudomonadaceae, Enterobactiaceae, Streptococcaceae and Micrococaceae.
Members of the family Bacillaceae are gram positive motile or non-motile rods
that produce an endospore. Bacillus is a major genera in the family as well as
Clostridium. Members are separated mainly by their oxygen requirements with most
members of Bacillus being aerobic and Clostridium being anaerobic.

Bacillus thuringiensis
A species of the Bacillacea, Bacillus thuringiensis (B.t.), has been the most widely used
and successful microbial pesticide ever registered. The sales of B.t. since its registration
in 1961 account for the majority of all microbial pesticide sales worldwide. B.t. has been
developed into over 100 products which are used, collectively, against over 1000 pest
species. There are several important subspecies of B.t. that have registered and marketed
as separate products on different insect pests. Two examples are Bacillus thuringiensis
subspecies israelensis, and Bacillus thuringiensis subspecies Tenebrionis.2 Each strain of
B.t. produces a different mix of proteins and specifically kills one or a few related species
of insect larvae. While some B.t.’s control moth larvae found on plants, other B.t.’s are
specific for larvae of flies and mosquitoes. The target insect species are determined by
whether the particular B.t. produces a protein that can bind to a larval gut receptor,
thereby causing the insect larvae to starve killing the insect within 1-2 days.3 Another
effect of this is that the gut damage prevents the insect from feeding. Since the toxin
produces the damaging effect, B.t. is effective against a wide range of insects. The lethal
generalized infection caused by the toxin generally does not spread to new hosts.4

B.t. is often classed as an insecticide of biological origin, rather than a true biological
control agent, because of its toxin-mediated action and lack of spread in the insect
population. The similarity of B.t. to chemical insecticides has negative consequences:
unusually for a biological insecticide, resistance to B.t. has been observed in target
species. For example, the diamondback moth, which has proved very capable of adapting
to resist chemical, has also developed resistance to some strains of B.t.5

Other bacteria
While B.t. strains are the most frequently used of all biopesticides, few other bacterial
species have been developed commercially. Two species of bacteria have currently been
1
Hokkanen 2003
2
Lindsey Flexner 1999
3
Wheeler 2002
4
Harper 2000
5
Harper 2000

5
registered as microbial insecticides: Bacillus sphaericus and Serratia entomophila. B.
sphaericus which is a strict aerobic spore former that is registered for mosquito control.
The other one is a gram-negative, anaerobic, rod-shaped bacteria in the family
Enterobacteriaceae, called S. entomophila and it does not form spores.6

Viruses
A variety of viruses are in use as biological controls, with many more under development.
Baculoviruses are members of the family Baculoviridae, a large group of highly specific
viruses of which larvae of butterflies and moths are the main hostess. The majority of
baculoviruses used as biological control agents are in the genus Nucleopolyhedrovirus.
All baculoviruses form thick protein shells around the protein–DNA nucleocapsid that is
the infectious core of the virus. This complex, called occlusion bodies, is then surrounded
by a carbohydrate-rich protection coat. When the baculoviruses is being eaten by insect
larvae, the cells of the insect’s gut are infected and the viruses start to grow. A
nonoccluded (‘budded’) form of the virus is formed and the virus is spread throughout the
body of the insect, killing it in 4-5 days. A new generation of virus is then released from
the liquefied mass that results.

Resistance to viral insecticides in susceptible forms of the host insect is very rare. Many
insects acquire increasing resistance as they age, but this reflect changes in the maturing
insect body and is not transmitted to the next generation of larvae. Adult forms are often
very highly resistant.7

Other viruses
Other viruses infecting insects are the entomopoxoviruses (family Poxviridae) and the
cytoplasmic polyhedrosis viruses (family Reoviridae). However, neither of these has been
developed as a commercial bio-pesticide.8

Fungi
Some hundred species of entomopathogenic fungi have been reported, but only a few of
these have been or are currently being developed for insect control.9 The potentially
hazardous effects of a fungal pathogen depend on its origin (native or exotic), on the way
it will be used (classical inundation, conservation), and on the area it will be used.10 They
all belong to the division Eumycota which is divided into Mastigomycotina,
Zygomycotina, Ascomycotina, and Deuteromycotina.

There are numerous biotic and abiotic constraints on the ability of fungi to infect their
hostess. These include desiccation, UV light, host behavior, temperature, pathogen vigor
and age, etc. In most species of entomapathogenic fungi, access to the host is through the
cuticle and may involve complex biochemical interactions between the host and the
fungus before germination, penetration, growth, and reproduction of the fungus can
6
Lindsey Flexner 1999
7
Harper 2000
8
Harper 2000
9
Lacey, 2001
10
Hokkanen 2003

6
occur. The fungi Imperfecti (Deuteromyces: hyphomycetes), have simpler life cycles and
lack sexual reproduction, and many have considerably broader insect host ranges.11 All of
the fungi that have been registered as insecticide have been mitosporitic fungi with the
exception of the water fungus Lagenidium giganteum. 12

Lagenidium giganteum is a fungal insecticide widely used for control of mosquitoes. The
cells of L. giganteum release spores that can move independently and are capable of
seeking out a suitable host for the next stage of their life cycle. Specific chemical signals
on the outer layer of the mosquito trigger the spores to burrow in and grow throughout
the body. After a few days of starvation for the host the cells will release a fresh
generation of spores. Even if there is no host available, the infection can remain active for
years. Two spores can combine, leading to the formation of a highly stable form of spore
that can last even in very dry conditions. 13

Negative effects on nontarget insects have been registered. One example is Beauveria
bassiana, a fungi pesticide used to control locusts, grasshoppers and crickets which also
can infect lady beetles (ladybirds). Lady beetles are often used as a biological control
agent themselves and harm of such an insect is not desirable. 14

Nematode
Entomopathogenic nematodes have several characters that qualify them as commercially
valuable biocontrol agents. They are highly effective, and in many cases surpass the
control results achieved with chemical compounds. Nematodes are mobile and can
persistent at the site of release. The recycle inside the host insect causes sustainable
effects on the pest population.15

The major focus of research and development has been on nematode species in 7
families, Mermithidae, Tetradonematidae, Allantonematidae, Phaenopsitylenchidae,
Sphaerulariidae, Steinernematidae, and Heterorhabditidae, because of their potential as
biological control agents of insects. Only species within the genera Steinernematidae and
Heterorhabditidae have gained major importance, while the activity of these species has
been documented against a broad range of insect pest. 16 They have a close symbiotic
association with bacteria of the genera Xenorhabdus associated with the steinernematids
and Photorhabdus associated with the heterorhabditids. The bacteria belong to the
Enterobacteriaceae. Heterorhabditis are unable to kill an insect without the presence of
its symbiotic bacteria, the Photorhabdus, whereas some Steinernematidae also produce
toxins that contribute to the pathogenicity of the symbiotic complex.17 The bacteria
proliferate and produce suitable conditions for nematode reproduction. During a free-
living stage the nematode will invade the insect and start to feed on the symbiotic cells.
The nematodes will be developed to adults and produce offsprings, as long as abundant
11
Lacey, 2001
12
Lindsey Flexner 1999
13
Harper 2000
14
Harper 2000
15
Hokkanen 2003
16
Lacey, 2001
17
Hokkanen 2003

7
nutrients are available. The bacteria will be transferred into the host and survive only due
to relation with the nematode. Probably are the bacteria unable to compete with other soil
bacteria. Inside the host the bacteria will multiply and produce toxin.18 The
nematode/bacterial complex is highly virulent, killing its host within 48 h through the
action of the bacteria.19 When the nutrients are consumed the offspring will develop to the
free-living stage, where it is adapted to survive under adverse environmental conditions
in the soil, and leave the insect cadaver in search for other hosts.

Protozoa
Protozoans acting as biopesticides are an extremely diverse group and generally slow
acting. They can be particularly effective at reducing the fitness and fecundity of insects,
which make them important in natural biological regulation. Most protozoan infections
cause sluggishness, irregular or slowed growth, resulting in reduced feeding, vigour,
fecundity, and longevity.20 Their biology is quite complex: they reproduce only in living
host and many of them need intermediate host. A few of them have been moderately
effective as control agents. One group containing many potential insect pathogens is the
Microsporidians. There is one commercially available control agent, Nosema locustae,
targeted against crickets and grasshoppers.21

Plant-incorporated protectants
Plant-incorporated protectants (PIPs) are pesticidal substances that plants produce from
genetic material that has been added to the plant. For example, a gene from the B.t.
pesticidal protein can be used and introduced into the plant’s own genetic material. Then
the plant, instead of the bacterium, manufactures the substance that destroys the pest.

Biochemical pesticides
Biochemical pesticides are naturally occurring substances that control pests by nontoxic
mechanisms. Conventional pesticides, by contrast, are generally synthetic materials that
directly kill or inactivate the pest. Biochemical pesticides include substances, such as
insect sex pheromones, which interfere with mating, as well as various scented plant
extracts that attract insect pests to traps.22

Production
Many applications require large scale fermentor production of microbial inoculants,
followed by formulation steps for long term stability and ease of environmental
dissemination. Safe use requires careful consequence analyses, often followed
by a registration procedure. Presently, lack of knowledge about fermentation/formulation

18
Hokkanen 2003
19
Lacey, 2001
20
Lindsey Flexner 1999
21
Harper 2000
22
Wheeler 2002

8
technologies and consequence analyses for novel microorganisms, seriously prevents
their implementation to solve environmental problems and to contribute to growth of
novel biotechnology industries. 23

Bacteria
Production of B.t. is done in a large capacity industrial scale bioreactors. The scale of
production and media cost make it the most inexpensive of all the microbial insecticides
to produce. However, it is still expensive compared to many synthetic insecticides. In
addition, media must be optimized for each new strain put into commercial production.
Fermentation optimization has the potential to greatly influence cost of production and
ultimately commercial competitiveness. Recombinant technology may be a powerful tool
for improving the commercial production of B.t. 24

Virus
In total, virus insecticides account for only 1% of biopesticides used worldwide. The low
level of commercial use of baculovirus insecticides is in part due to their relatively slow
action, but also the production cost. Viruses can be prepared in cultured cells, permitting
the use of high-efficiency reactor systems. However, both insect cells and the viruses that
infect them are still providing difficulty to grow in such systems. Therefore viruses have
to be grown in insect larvae, a both time-consuming and expensive method keeping the
production costs high.25

Fungi
Commercial production of L. giganteum has difficulties, while it has a narrow host range
and distinct sterol and nutritional requirements. A fungus more amenable to mass
production is the Deutermycetes and is also less expensive. They have a broader host
range and are able to grow and sporulate on many generalized media. They can adapt to a
wide variety of growing conditions. 26

Nematodes
The nematodes can be mass produced, formulated, and easily applied as biopesticides.
They have been exempted from registration in many countries and are amenable to
genetic selection.27 In order to provide markets with large amounts of material,
production is preferably done in liquid culture, because scale-up and downstream
processing of large culture volumes can be mechanized thus reducing the production cost.
The nematodes are resistant to shear forces and can thus be applied with conventional
spraying equipment.28 In general, both steinernematids and heterorhabditids tend to do
best against soil-inhabiting insects and borers. There has been limited success when
applying nematodes to foliage, aquatic habitats, or on pests inhabiting manure.29
23
www.mistra.org
24
Lindsey Flexner 1999
25
Lindsey Flexner 1999
26
Harper 2000
27
Lacey, 2001
28
Hokkanen 2003
29
Lindsey Flexner 1999

9
The potential of nematodes used as biocontrol agents may expand by increasing search
capacity, virulence, and resistance to environmental extremes, among other attributes.
Recently a heat-shock protein have inserted into H. bacteriophora by the use of
molecular techniques, resulting in a transgenic nematodes that were prominent better than
the wild types at surviving high-temperature stress. The high costs associated with
production and formulation of nematodes in comparison to the costs of chemical
pesticides and other biopesticides restrict their use to high value niche markets and
sensitive areas where chemicals cannot be used. 30

Fermentation parameters
Fermentation parameters, such as nutrients, aeration, temperature and pH, are available
in literature for some microbial species. However, this information is based
towards yeast and some bacteria (E. coli, Bacillus spp., certain lactic acid bacteria)
because of their current use in food and biotechnology industries. Less is known about
optimum growth parameters for fermentation of non-conventional microorganisms.
Knowledge about formulation strategies such as preconditioning of the microorganisms
and use of additives that promote survival, storage stability and ease of use exists mainly
in the biotechnology industries and is not generally accessible. 31

Costs of production
Costs of production are generally high for biocontrol products, and there may be
problems of timely supply and storage. To improve the use of biological agents the
production costs need to be decreased. Increased automation and better understanding of
the patterns of growth could be the solution. With some biological control agents there
are factors that are inherently expensive. For example, baculovirus insecticides must at
present be produced in live insects, which is both time consuming and expensive.
Growing a virus in insects can also cause other problems. Increasing the speed with
which biological agents kill is highly desirable for their use in pest control. However, this
has obvious negative effects on productivity if the agent must be grown in insects, since
the consequently reduced yield will increase costs.

A very significant factor in reducing the cost of such insecticides would be the
availability of high-capacity (bioreactor) cell culture systems for their production. This is
the focus of a great deal of work but is unlikely to become widespread in the near
future.32

Regulation
For registration of products containing microorganisms, the need for safety
documentation regarding the microorganism and the product containing the organism
differs depending on the intended use.33 The time needed for registration differ in
different areas. The Environmental Protection Agency (EPA) is in charge of biocontrol
agent regulation in the USA. The EPA needs on an average 2 years to register microbial
30
Hokkanen 2003
31
www.mistra.org
32
Harper 2000
33
www.mistra.org

10
biocontrol agents, in the EU authorities need about 7 years for the same products.
National registration, which takes at least another two years, has to follow. 34 The reason
for this is that in EU there is no separate regulatory system for biopesticides. All
pesticides must go through the same registration process.

In the EU biological pesticides are defined as biocidal products. The European Parliament
and the Council adopted the Directive 98/8/EC in 1998 concerning the placing of biocidal
products on the EU market (Biocidal Products Directive, BPD). The background for this
Directive is a need for harmonization of the legislation of the Member States regarding
this type of substances.35

Sweden: A biocidal product has to be approved before it can be placed on the Swedish
market. As a member in the EU, Sweden has implemented the Biocidal Products
Directive. When the transition period for the Directive has come to an end on 13
May 2010, all biocidal products that are placed on the Swedish market must be
authorised in accordance with the Directive. Until then, all member states are allowed to
continue to apply their current systems or practice of placing biocidal products on the
market. Kemikalieinspektionen is the authority that approves and registers all biological
pesticides before it can be used commercially.36

The US regulatory system has several routes: conventional registration (for traditional
synthetic, chemistry-based agrochemicals); biopesticide registration (for natural
products); and minimum risk pesticides (Environmental Protection Agency, EPA). The
US system differentiates between products formulated from natural chemicals, biological
organisms and synthetic chemicals. Before a pesticide can be marketed and used in the
United States, the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) requires
that EPA evaluate the proposed pesticide to assure that its use will not pose unreasonable
risks of harm to human health and the environment. 37 EPA’s definition of biopesticide
includes many naturally derived materials suck as plant oils and baking soda in addition
to living and nonliving biological agents.38

Research
REBECA – Regulation of Biological Control Agents is an EU Policy support action to
review possible risk of biocontrol agents, compare regulation in the EU and the USA and
to propose alternative, less bureaucratic and more efficient regulation procedures
maintaining the same level of safety but accelerating market access and lowering
registration costs.39

International: The Organisation for Economic Co-operation and Development (OECD) is

an intergovernmental organization composed of 30 industrialised countries in North
America, Europe and the Pacific. The OECD Pesticide Registration for Biological
34
http://www.rebeca-net.de/
35
http://ecb.jrc.it/biocides/
36
Jordbruksverket, 2002
37
Marks 2006
38
Charudattan 2002
39
http://www.rebeca-net.de/

11
Pesticides helps governments work together to assess pesticide risks. The assessment of
risks is important because it provides the basis for governments' decisions whether to
approve, or "register," new biological pesticides and whether to renew the registration of
old ones. By working together, governments can evaluate a biological pesticide's risks
more quickly and thoroughly. This speeds up the process of approving safer new
pesticides and stopping use of riskier ones.40

The use of biopesticides

Strategies for the use of entomopathogens for insect control are basically the same as that
for other biological control agents. They may be used to augment naturally occurring
pathogens (augmentation), conserved or activated in nature (conservation), introduced
into pest populations as classical biological control agents to become established and
exert long–term regulation of the pest (inoculative release), or used inundatively for rapid
short-term control (inundative release).41 Inoculative introduction is nowadays highly
limited due to strict regulations. Most successful inoculative agents are persistent in the
environment or host and are able to be transmitted within and between host populations
and generations.42

Bio-pesticides are to a great degree influenced by the environment. For instance,

entomophagous nematodes are only able to survive below the soil surface or in other
sheltered microenvironments where they are protected from the harmful effects of
ultraviolet radiation and desiccation.43

Advantages
Control of pest insects with chemical pesticides has generated several problems including
insecticide resistance, outbreaks of secondary pests normally held in check by natural
enemies, safety risks for humans and domestic animals, contamination of ground water,
decrease in biodiversity, and other environmental concerns. Biopesticides have many
potential advantages compared to chemical biopesticides. They are often pest specific and
thus harmless for humans and other nontarget organisms. The host specificity of bio-
pesticide might suggest that they can be better targeted at pests and pathogens than broad
spectrum chemicals. Other advantages include reduction of pesticide residues in,
preservation of other natural enemies, and increased biodiversity in managed ecosystems.
It is unusual of development of resistance to the microbial control agent (except from
B.t.).44 Biopesticides are effective when used in small quantities and have short residual
activity, but require much more knowledge for growers to use them with the same
effricacy as with conventional pesticides.45

40
www.oecd.org
41
Lacey, 2001
42
Babic 2004
43
1. Hokkanen 2003
44
Lacey, 2001
45
Wheeler 2002

12
Disadvantages
Potential adverse effects of microbial pesticides include displacement of nontarget
microorganisms and allergenic, toxic, and pathogenic effects on humans and other
nontarget organisms. However, microbial agents typically have narrow host ranges and
occur naturally in the environment to which they are applied. Such factors reduce the
likelihood of harm should a microorganism be used for pest control.46 The narrow
specificity may also represent disadvantages when mixtures of pests and diseases need to
be controlled. It may be difficult to control all the pests and diseases on a crop with
biocontrol agents, and it may at the same time be impossible to use a strategy that
combines chemicals and bio-pesticides because of incompatibility. Other disadvantages
are mostly linked with their persistence, speed of kill, and production cost of biopesticide
relative to conventional chemical pesticides.47 There is a risk that introduced organism
would become a pest itself by shifting to other target species or outcompeting natural
species.48

Future developments
Biopesticides used commercially must be compatible and complementary to other
components of the system, it must be easy to use, reliable, economical, and lastly it
should contribute to the sustainability of the whole system. Further improvements of
natural enemies’ activity could be achieved with genetic modifications. Several scientific,
regulatory and political issues will have to be resolved before genetically modified
natural enemies can be used practically. Furthermore genetically modified organisms
cannot be used on biological farms at all.49

Increased utilization in future will require:

 Increased pathogen virulence and speed of kill.
 Improved pathogen performance under challenging environmental conditions
(cool weather, dry conditions, etc.)
 Greater efficiency in their production
 Improvements in formulations that enable ease of application, increased
environmental persistence, and longer shelf life
 Better understanding of how they will fir into integrated systems and their
interaction with the environment and other integrated pest management (IPM)
 Greater appreciation of their environmental advantages
 Acceptance by growers and the general public50

Survival can be improved by adding substances that provide better protection against
environmental and mechanical stresses. These additives can be compatible substances
that the microorganisms themselves produce, such as disaccharides, amino acids and

46
http://www.oecd.org/dataoecd/4/23/28888446.pdf
47
Lacey, 2001
48
Babic 2004
49
Babic 2004
50
Lacey, 2001

13
amino acid derivates. Each new microorganism will very likely require special
solutions.51

Discussion
The use of biopesticides could be an efficient and ecological method to decrease the
legal, environmental, and public hazards of chemicals. The reduced usage of chemical
pesticides will significantly contribute to the conservation of natural sources and results
in diminish of environmental pollution, thus preserving biodiversity. Reduction in
pesticide production will decrease risks for workers in chemical industry and
environmental risks due to transport and storage of chemical pesticides. Further
improvements and investigations of the benefits and risks of biological control are
required for increased utilizing of this pest control method.

To register a new biological product and get it out on the market take up to 7 years in EU.
This time consuming process could be a bottle for the biotechnology industries due to the
lack of income from the product. A matter of course is the utmost importance in having
precise and complete investigations of possible risks before the biopesticide is available
on the market. A separate classification for biopesticides could lead to improvement in
the EU regulatory system and reduce the time needed for registration of a new product.

Future developments of biopesticides include the requirement of making the microbial

more efficient against pests and sustainability in harsh environments. Nevertheless is the
importance of greater production efficiency. Costs of production are generally high for
biocontrol products, and there may be problems of timely supply and storage. To improve
the use of biological agents the production costs need to be decreased. Increased
automation and better understanding of the patterns of growth could be the solution.

51
www.mistra.org

14
References:
Babic, J., Alternative and biological methods of pest and weed control in agriculture,
Nova Gorica (2004)

Benbrook C. The challenges of organic soybean production in the Riverina, Organic

News, http://www.tocal.nsw.edu.au/reader/news-organic/org-news-vol-2-issue-3.pdf?
MIvalObj=25801&doctype=document&MItypeObj=application/pdf&name=/org-news-
vol-2-issue-3.pdf, Volume 2 Issue 3 March-April, ISSN 1449-325X 2005

Charudattan, R., Chandramohan, S., Wyss, G., Biological control, Wheeler, W.,
Pesticides in agriculture and the environment, USA (2002)

Harper, D., Biological control by microorganisms, Greenfeild, Bedfordshire, United

Kingdom (2000)

1. Hokkanen, H., Lynch, J., Biological Control, Cambridge University Press (2003)

2. Hokkanen, H., Hajek A., Environmental Impacts of Microbial Insecticides, Dordrecht,

the Netherlands (2003)

Jordbruksverket, Biologisk bekämpning av skadedjur, Kalmar, Sweden (2002)

Lacey, L.A., Frutos, R., Kaya, H.K. Vail P., Insect pathogens as biological control agents:
do they have a future, Biological control 21, 230-248 (2001)

Lindsey Flexner J., Belnavis D., Biological and biotechnological control of insect pests,
Florida, USA (1999)

Marks, D., The regulation of Bugoil: Experiences from North America and the EU,
Preston, UK (2006)

Wheeler, W., Role of research and regulation in 50 years of pest management in

agriculture, Pensacola, USA (2002)

Websites:

www.oecd.org

www.mistra.org, DOM – Domesticering av mikroorganismer, årsrapport 2003, 13/3 2006

http://ecb.jrc.it/biocides/

15