TIBTECH - MAY 1986
death. About 100 insects are suscept-
Production and use of
biological pest control
agents
George
Biological pest control agents are gaining prominence for the control of
insect pests in agriculture and forestry. The shift from chemical control
has been due to environmental concerns and recent innovations in
biotechnology. Production and use of biological insect control agents is
the challenge of the future for pest management.
Biological pest control agents are
gaining in prominence for the control
of insect pests in agriculture and
forestry 1-3. The growth of interest in
biological pest control agents occur-
red in part because of public
awareness of the dangers of in-
discriminate use of chemical pesti-
cides, especially after the publication
of Silent Spring by Rachel Carson, in
1962. Additionally, research pub-
lished in the last two decades on the
relationship between pesticides and
mutation, cancer, human disease,
damage to wildlife, and toxicity to
plants has pointed towards a shift
away from chemical control of pests.
In the last decade developments in
biotechnology are generating interest
and the means necessary for displace-
ment of synthetic chemical insecti-
cides which currently dominate the
marketplace 4'5. Various estimates
indicate that biological pest control
agents, which currently represent 1%
of the annual $10 × 10 9 worldwide
market for insecticides will increase
to become 10% or more by the end of
the decade. The potential for wide-
spread use of biological pest control
agents~ is timely because of their
relative host specificity and ecologic-
ally non-disruptive nature.
Biological pest control agents are
naturally occurring organisms, in-
cluding viruses, bacteria, fungi,
protozoa and nematodes. Viruses
G. G. KhachatouriansisattheDepartment
of Applied Microbiology and Food
Science, and Bioinsecticide Laboratory,
College of Agriculture, University of
Saskatchewan, Saskatoon, S7N OWO
Canada,
(~ 1986, Elsevier Science Publishers 13.V., Amsterdam
G. K h a c h a t o u r i a n s
have an interesting ecological specifi-
city and unique location within the
biological world. For example, the
insect viruses which are members of
the family Baculoviridae (BV) are
responsible for diseases of insects and
mites 6 and do not contain structural
or biochemical similarities to viruses
of the vertebrates or higher plants. A
substantial portion of the bacteria and
fungi are facultative pathogens of
insects affecting the host by a number
of mechanisms such as invasion of
tissues and/or production of toxins.
The bacteria can be divided into three
ecological types: (1) those which
reproduce within the susceptible host
system; (2) those which do not
reproduce, necessitating repeat appli-
cation; and (3) those of either type (1)
or (2), depending on the circum-
stances 7. Because of these unique
properties a number of these micro-
organisms have been commercially
produced and are replacing existing
chemical pesticides.
Production and use ofbioinsecticides
Viruses
There are about 650 viruses that
have been isolated from insects.
About 540 were obtained from
lepidopteran, 90 from hymenopteran
and 20 from orthopteran, coleopteran
and dipteran insects. During the
1970s the first commercial viral
pesticide was registered. Since then
the potential of these agents has been
realized (Table 1) and tremendous use
of viruses such as baculoviruses, has
been made for control of insect
populations a. Insect virus infections
of susceptible larvae result in patho-
logical symptoms and, ultimately,
0166- 9430/86/$02.00
ible to baculoviruses. Certain baculo-
viruses produce granulosis disease. In
general, the mass production of many
of the insect viruses involves incu-
bation of infected insects, and har-
vesting of diseased larvae or adult
insects. The subsequent purification
of the viral preparation is time
consuming and labor-intensive, the
expense of which is added to the cost
of viral insecticide production in-
creasing the production costs 4-5-
fold. The obstacles to commercial
production and use are numerous 8
and include: (1) the development of
efficient processes for large scale
production; (2) marketability of the
products especially where the select-
ive host ranges are required; (3) de-
velopment of procedures for schedul-
ing and monitoring of field appli-
cations; and (4) stabilization of the
product under field conditions, e.g.
sunlight and dessiccation.
Bacteria
Many bacteria cause disease and
death in insects. Four groups have
been recognized based on their
pathogenicity to insects: (1) obligate
pathogens (e.g. Bacillus popilliae, B.
larvae); (2) crystalliferous spore-
forming bacilli (e.g.B. thuringiensis);
(3) facultative pathogens (e.g.
B. sphaericus, Pseudomonas aerugin-
osa, Achromobacter spp.); and (4)
potential pathogens (e.g. Serratia
marcescens) 9.
The best example of bacterial
insecticides and one that has been
investigated for almost 80 years is that
of the members of the genus Bacillus.
Four species are considered as
primary insecticides: B. thuringien-
sis, B. popilliae, B. moritai and
B. sphaericus. The greatest commer-
cially used species are B. thuringien-
sis which is cultured & vitro, and
B. popilliae, which is grown in rive
on the Japanese beetle (Popilliae
japonica). B. thuringiensis produces
entomocidal toxins, ~- and [5-exotox-
ins and 6-endotoxin. The latter, also
called insecticidal crystal protein
(ICP); is synthesized during sporu-
lation, and accounts for the commer-
cial value of B. thuringiensis as a
biological pest control agent 4. Oral
administration of ICP to lepidopteran
larvae causes cellular damage to the
TIBTECH - MAY 1986

- Table I
Viruses in commercial and experimental production a
Virus Target Product Manufacturer/Country
Heliothis NPV Heliothiszea Elcar Zoecan (Sandoz) Corp./USA
(corn earworm)
Heliothis virescens Biotrol-VHZ Nutrile Products Inc./USA
(tobacco budworm)
Lymantria dispar N PV Lymantria dispar Gypchek US Forest Service
(gypsy moth) Virin-ENSh USSR
Neodiprion sertifer N PV Neodiprion sertifer Virin-Diprion USSR
(European pine sawfly) NPV Kemira Oy Co./Finland
Orgyia pseudotsugata N PV Orgyia pseudotsugata Biocontrol-1 US Forest Service
(Douglas fir tussock moth)
Dendrolimus spectabilis Dendrolimus spectabilis CPV Japan
CPV (pine caterpillar)
aAdapted from Refs 2 and 6.

gut epithelium and cessation of turers for Bacillus products world- insect populations 13'14. For the com-
feeding. With sub-lethal doses an wide are shown in Table 2. mercial production of these fungi,
acute phase followed by recovery ingredients, concentrations, quantity
occurs 1°. Larger doses cause patho- Fungi and quality of the fermentation
logical changes resulting in larval There are several entomopatho- methods used are virtually identical
death 11'12. The commercial produc- genic and saprophytic fungi which to those required for the synthesis of
tion, targeted hosts and manufac- have been used for the control of either fungalbiomass or metabolites 15

Table 2
Commercial Bacillus products a
Species Serotype/variety C o m m e r c i a l name Target Manufacturer/Country
Bacillus moritai -- Lavillus M Diptera Sumitomo Chemical Co./
Japan
Bacillus popilliae -- Doom, Japidemic Coleopteran Fairfax Biological Lab./
larvae USA
Milky spore disease Reuter Laboratory, Inc./
USA
Bacillus thuringiensis H3(HD-1)/Kurtsaki Dipel Numerous Abbott Laboratories/USA
(B. thuringiensis H3(HD-1)/Kurtsaki Thuricide Lepidoptera Sandoz-Wander, Inc./
5-endotoxin + USA
spores) Certan Zoecan (Sandoz) Corp./
USA
-- Bactur Thompson-Hayward
Chemical Co.
(distributor)/USA
H-1 thuringiensis Bactospeine Roger Bellon/France
H3(HD-1 )/Kurtsaki Plantibac Procida France
H-3(HD-1 )Kurtsaki BugTime Biochem. Products/
France
H-3(HD-1 )Kurtsaki Sporeine LIBEC/France
H-3(HD-1 )/Kurtsaki Biospor Farbwerbe Hoechst/
West Germany
H-1/thuringiensis Bathurin ChemapoI-Biokrma/
Czechoslovakia
H-I(HD-1 )/ Baktukal Serum zavod
Kalinovica/Yugoslavia
H4/dendrolimus Dendrobacillin Glavmikrobioprom/USSR
H-5/galleriae Entobacterin-3 Glavmikrobioprom/USSR
H-1/insectus Insektin Glavmikrobioprom/USSR
Bacillus thuringiensis Hl/thuringiensis Biotoksybacillin Agricultural
(B. thuringiensis Microbiology/USSR
exotoxin) -- Eksotokin Glavmikrobioprom/USSR
-- Toxobaktedn Glavmikrobioprom/USSR
Bacillus thuringiensis israelenis H-14 Bactmos, Vectobac Culex spp. Biochem Products/France
Teknar Aedes spp. Zoecan (Sandoz) Corp./
Anopheles USA
aAdapted from Refs 2, 3 and 4.

by fermentation technology.
T h e r e are several entomopatho-
genic fungi which do not growin vitro
or require very complex media. For
example, Entomophthora gry]li is
cultivated in vivo, using either field-
collected diseased insects or healthy
insects infected in the laboratory 16.
Fungal formulations currently being
evaluated contain a mixture of fungal
mycelial fragments and conidia or
blastospores. Active ingredients are
formulated in a car~ier base which
often is made of bentonite, kaolin
clay, or a mixture of various agri-
cultural by-products. Commercial
products are formulated either as
wettable powders or as dust formu-
lations and to these, antioxidant and
UV-screening materials are added.
Nematodes and Protozoa
Very few nematodes and protozoa
have been mass produced industri-
ally or used as biological pest control
agents in large scale field trials (Table
4). Nosema ]ocustae is an obligate
parasite and is cultured in the host
insect, e.g. grasshoppers 2, necessitat-
ing extraction from ground whole
insects by filtration and centri-
fugation, to arrive at a pure prepar-
ation. The major obstacle for com-
mercialization has been production of
these organisms.
-- Table 3
Entomopathogenic fungi in commercial and experimental production a
Fungus
Beauveria bassiana
Culicinomyces clavisporus
Hirsutella thompsonii
Metarhizium anisopliae
Nomuraea rileyi
Verticillium lecanii
Colletotrichum gloeosporioides
Cercospora rodrnanfi
Peniophora gigantea
Trichoderma virdae
aAdapted from Refs I and 29.
bEAO, experimental application only.
c
Marketed under tradenames Biocontrol ® , Biomax ® , Combio ® , Metabiol ® and Metapol ®.
Biotechnology and biological pest
control agent production
Biotechnology can affect industrial
production and cost effectiveness of
biological pest control agents. The
cost of gross input for in vitro
production of bacteria and fungi
includes the use of media, energy
(both for culture growth and steriliz-
ation of the equipment), time and
labour. Biotechnological innovations
will lead to improvements in the
process technology 2. Cost of produc-
tion could be reduced by using
cheaper fermentation media and
shorter fermentation cycles. A great
many biotechnological advances for
the production of such insecticides
are aimed at genetic engineering of the
insecticides or their toxins to develop
more potent and broader host range
toxigenic bacteria, exclude other
toxins and metabolites from the
preparation and maximize the par-
ameters that govern fungal sporu-
lation15.17.
Where insecticidal crystal protein
(ICP) production is desired, research
has been directed to the production
of B. thuringiensis asporogenous
mutants ~8"~9, and hence an improved
diversion of cell biomass to the
production of the ICP. It can be
estimated that such improvements
would increase the productivity 3-5-
Target Product
Colorado potato beetle Boverin
codling moth
European corn borer
pine caterpillar
mosquito larvae EAO b
citrus rust mite Mycar
spittle bug Metaquinoc
sugarcane frog hopper
lepidopteran larvae EAO
aphids Vertalec
coffee green bug
greenhouse whitefly Mycotal
thrips Thriptal
Northern Joint-vetch --
water hyacinths EAO
H. annosum of conifers tree --
roots
apple canker disease --
TIBTECH - MAY 1986
fold. There are important differences
between ICPs and the plasmids which
encode them vary depending on
the strain of B. thuringiensis from
which they are derived. Toxicity and
host range of the ICPs vary for these
toxins 2°. These are two character-
istics which might be altered effect-
ively by genetic manipulations. The
serological and molecular character-
istics of the ICPs from various isolates
of B. thuringiensis have been ana-
lysed 17. The respective DNAs have
been sequenced 21 and cloned into
E. coil and B. subti]is zz'23. In spite of
major accomplishments in molecular
studies the widespread use of this
insecticide is still very limited.
The main method for the com-
mercial production of B. thuringien-
sis has been submerged batch
fermentation. We are examining
continuous phase-production or con-
tinuous culturing and harvesting of
the ICP-containing culture material.
The end product from the batch
fermentation process contains cells,
spores, extracellular enzymes and
proteins, other low molecular weight
material, and ICPs. These are har-
vested by high speed continuous flow
centrifugation and acetone precipi-
tation into a thick paste containing all
of the sedimentable material. During
formulation the paste is mixed with
Manufacturer/Country
USSR
People's Republic of China
USA
Abbott Laboratories/USA
Brazil
USA
Tate and Lylein Ltd/UK
Tate and Lylein Ltd/UK
Tate and Lylein Ltd/UK
USA
Abbott Laboratories/USA
Bio-Basic Ltd./UK
--
TIBTECH - MAY 1986
_ Table 4
Entomopathogenic protozoa in experimental production a
Protozoa
Nosema Iocustae
Nosema pyrausta
Nosema fumiferanae
Vairimorpha necatrix
aAdapted from various authors in Ref. 2.
various adjuvants, wetting agents,
and sticky material (stickers) and is
spray-dried for final commercial
packaging 24. The quality of the mater-
ial is determined using standardized
bioassays and/or monoclonal anti-
body assays.
Production of fungi or their meta-
bolites offers the greatest prospects in
this area, especially when the tech-
niques for genetic manipulation of
antibiotic producers become readily
available. The major obstacles in the
area of fungal bioinsecticides are the
genetic instability of the fungi,
identifying the mechanism of action
of toxic material or secondary meta-
bolites, and understanding the mole-
cular genetics of pathogenicity. Pro-
duction of metabolites toxic to non-
targeted insects or the environment
could be reduced or prevented by
genetic manipulation.
For Micromonosporidiae (e.g.
Nosema) and viruses, productivity
could be increased substantially if in
vitro tissue culture systems were
available. Some media for in vitro
cultivation of insect viruses need
animal sera, which are expensive and
may contain animal viruses. Use of
fermentation technology for culturing
insect cells and biotechnologically
derived animal sera substitutes
should be of help here. Equally
helpful would be the cloning of insect
virus material into soil bacteria such
as B. thuringiensis, or into plants.
The economics o f bioinsecticide
production and use
It has been suggested that the cost of
research development and obtaining
US Environmental Protection Agency
approval for a new bioinsecticide will
limit introduction of products with
potential markets of less than $10
million. In fact, most companies
Target
grasshoppers
European corn borer
spruce budworm
cabbage looper
corn earworm
tobacco budworm
involved in biotechnology would
want to aim at a market of $50 million
or more 25. It is suggested that as a
result of this, bioinsecticides are not
likely to be developed for many low
volume applications. However, bio-
insecticides may still sell signifi-
cantly. The cost of research, develop-
ment and product safety analysis for a
new chemical pesticide (i.e. $15
million or more) means that equi-
valent work on bioinsecticides be-
comes an attractive alternative (i.e.
under $1 million). Additionally, the
quantity of bacterial or viral insecti-
cides needed for treating certain
agricultural acreages or forests may
be significantly lower (0.025-0.25 kg
ha -1) compared with that of chemical
insecticides. This must be related to
the costs and economics of produc-
tion. The cost of production of 10
litres of B. thuringiensis israelensis
(BTi) culture using complex and
defined liquid salts media is $7-12
compared with $0.02-0.03 if pro-
duced from by-products of industrial
factories 26, Another example of cost-
effective production data is from the
study of Herflin eta]. 27. They showed
that mosquito larvicidal strains of
B. sphaericus 1593 and BTi H14 grow
on commercial powders of soy
products, fish meal, dried milk pro-
ducts, raw sewage, blood and serum
from animal, bone meal, chicken
parts, animal dung and agricultural
waste material, yielding sufficient
culturebiomass with toxic properties.
In the case of viral insecticides the
successful adoption of any biological
pest control agent technology would
be judged by the reduction of the
production costs. Work at US Depart-
ment of Agriculture by Bell and
Shapiro 28 has resulted in a doubling
of the active life of Gypcheck (see
Table 1) and a drop in the production
cost from $75 to $5 per hectare.
Remarks
Peaceful Valiey Farm Supply/USA
Conclusions
The present advantages of bio-
logical pest control agents are: (1)
their high degree of specificity for pest
control; (2) little or no effect on non-
targeted and beneficial insects and
man; (3) absence of insect resistance;
(4) absence of residue build-up in the
environment; and (5) potential im-
pact from biotechnologicat research
and development. However, bio-
logical pest control agents have a
number of limitations: (1) of under
100 known agents only 20 are EPA
registered; (2) killing time may be
slow; (3) some are environmentally
unstable although some can recycle;
and (4) their production is expensive.
It is hoped that biotechnology and a
responsible environmental outlook
will help us change the limitations
and make biological pest control
agents products for the agriculture of
today. For this, future research and
development in the areas of identi-
fication of new etiological agents of
insect disease, and mechanism and
genetic basis of bioinsecticide-in-
duced insect disease are needed.
It can be concluded that biotech-
nology has a significant role in the
production and manufacturing of
biological pest control agents and
ultimately lowering the input costs of
agriculture. The challenge in this area
has not been fully met. However, it is
anticipated that with many firms
producing or developing bioinsecti-
cides there will be greater activity in
the area. Ultimately the production of
effective bioinsecticides in sufficient
numbers will displace the synthetic
chemical insecticides and the associ-
ated ecological disadvantages.
References
1 Papavizas, G.C. (ed) (1981). Bio-
logical Control in Crop Production,
Granada Publishing
 TIBTECH - MAY 1986

2 Burgess, H. D. (ed) (1981). Microbial
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1970-1980, Academic Press
3 Kurtsak, E. (ed) (1982). Microbial and
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4 Miller, L. K., Lingg, A. J. and Bulla,
L. A. Jr (1983) Science 219, 715-721
5 Kirschbaum, J. B. (1985) Annu. Rev.
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6 Payne, C. C. (1982) Parasitology 85,
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7 Burgess, H. D. (1982) Parasitology 84,
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packaged and used either as a seed

Benefits of Rhizobium to dressing or incorporated into drills at
sowing.
agriculture The contribution of Rhizobium
Properly managed, legumes can fix
all the nitrogen for their o w n needs
Janet I. Sprent and m a y enrich soil nitrogen for
associated or subsequent crops. Be-
Crops fixing nitrogen by means of endosymbiotic rhizobia are a major fore the mineral boom, the e c o n o m i e s
world source of protein and soil nitrogen. Interactions between the of N e w Zealand and Australia relied
bacteria and host plant are currently being unravelled. This will almost entirely on legume-based
enable current rhizobial biotechnology and developing plant agriculture, using introduced
biotechnology to be targetted towards more efficient nitrogen fixation legumes such as w h i t e clover (Tri-
and new nodulated, nitrogen fixing crops. f o l i u m repens), and subterranean
clover ( T r i f o l i u m subterraneum), re-
spectively, together with suitable
Nitrogen fixation research celebrates p r o d u c t i o n of these root nodules. rhizobia. Even at a fairly conservative
its centenary this year. In 1886 it was Inside the nodule, R h i z o b i u m re- estimate of 50 kg nitrogen fixed ha -1
first s h o w n c o n c l u s i v e l y that pea duces atmospheric nitrogen to am- year -1, the 108 ha of l e g u m e pasture in
plants grow better in nitrogen de- m o n i a w h i c h the host l e g u m e in- Australia w o u l d save $2.7 x 109 at
ficient soil if they have n o d u l e s on corporates into organic c o m p o u n d s 2. 1986 prices for nitrogen as urea. This
their plant roots I (Fig. 1). A bacter- Searches were soon u n d e r w a y for assumes that all of the fertilizer
ium, t h e n called B a c i l l u s radicicola strains of R h i z o b i u m to give i m p r o v e d nitrogen w o u l d be incorporated into
but n o w k n o w n as R h i z o b i u m nitrogen fixation and in 1895 a patent the plant, w h i c h it is not (see also
t e g u m & o s a r u m , was subsequently leading to the biotechnology of Table 1). The true value is more likely
isolated from and found to stimulate R h i z o b i u m i n o c u l a n t p r o d u c t i o n was to be at least $3.7 x 109. The central
taken out 3. Strains of rhizobia for use highlands of Brazil, covering about
w i t h particular legumes (e.g. clover, 2 x 108 ha, are nutrient poor but
]. I. Sprent is at the Department o f peas and lentils) were grown in bulk, because there is reasonable rainfall,
Biological Sciences, University o f added to a suitable carrier (sterile plant yields of grain legumes such as
Dundee, Dundee DD1 4HN, UK. ground peat being the most common), dry bean (Phaseolus vulgaris) a n d
~) 1986, Elsevier Science Publishers B.V., Amsterdarn 0166-9430/86/$02.00