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Table 17.3 Scheme for screening and evaluating the efficacy, safety, and environmental impact of biological agents for control of disease

vectors designed by the WHO

Stage I Stage II Stage III Stage IV Stage V

Preliminary

Laboratory Laboratory field trials Laboratory Large-scale field trials

A. Identification A. Mammalian Results of Strictly A. More detailed Review of stages To be conducted

and charateri- infectivity Review of regulated tests on I, II, III and IV by under WHO auspices.

zation tests to stages I and II ponds tests under mammalian by informal Not presently defined,

ensure safet WHO supervision infectivity using consultation group and will vary according

to laboratory to determine appropriate to target vector,

and field efficacy against techniques habitat(s), mode of

personnel disease vectors Laboratory and application, etc.

under natural field trials

conditions

B. Assessment B. Preliminary B. Detailed studies

against assessment on non-target

selected against range especially

target certain other fauna in

vectors non-target habitats where

species stage V trials may

be conducted

C. Preliminary

evaluation Formulation

of ease of C. Studies on

rearing in stability of suitable

formulations and

delivery system

Production of Microbial Insecticides ! 

found particularly active against mites which attack citrus. It is applied as the conidial

powder and maximum effectiveness occurs at 27°C and under moist conditions or at

relative humidities of 79-100%. Coelomomyces sp. is very effective against mosquitoes but

its production is difficult because of the need of a secondary host. Most effective and

specific against mosquitoes are Culicinomyces sp. which was isolated in Australia and

produced a mortality rate on mosquitoes of 90-100%. Tolypocladium cylindrosporum is

essentially like Culicinomyces in being highly specific for mosquitoes. Lagenidium

giganteum and Leptolegnia sp have been shown to have high mortality for mosquitoes. All

the above (except Coelomomyces) can be mass-produced by fermentation. Beauvaria and

Metarrhizium already discussed have broad activity against mosquitoes.

(iv) Protozoa: In constrast to the rapid action of viruses and spore-forming bacteria, killing

by protozoa is slow and may take weeks. Furthermore they are difficult to produce, being

accomplished only in vivo. Nevertheless they have been produced and successfully used

experimentally for stored-product pests (Matosia trogoderina) mosquitoes (Nosema algerae)

and grasshoppers (Nosema pyrasta).

Vavra vilivis is also effective against mosquitoes and has properties similar to those of

Nogema algerae. Studies sponsored by the WHO have shown that N. algerae does not seem

to constitute a safety hazard for man. Factors favoring the use of N. algerae are spore-

longevity, ease of spore-production under laboratory and especially cottage industry

conditions and the probable impact on disease transmission by reducing the longevity of

infected female mosquitoes.

So far however protozoa have not been produced on an industrial scale for biological

control.

17.2.3 Bacillus thuringiensis Insecticidal Toxin

B. thuringiensis strains produce two types of toxin. The main types are the Cry (crystal)

toxins, encoded by different cry genes, and this is how different types of Bt are classified.

The second types are the Cyt (cytolytic) toxins, which can augment the Cry toxins,

enhancing the effectiveness of insect control. Over 50 of the genes that encode the Cry

toxins have now been sequenced and enable the toxins to be assigned to more than 15

groups on the basis of sequence similarities. The table below shows the state of such a

classification in 1995. An alternative classification has recently been proposed based on

the degree of evolutionary diversity of the amino acid sequences of the toxins, but this has

not yet been widely adopted.

Cry toxins are encoded by genes on plasmids of B. thuringiensis. There can be five or six

different plasmids in a single Bt strain, and these plasmids can encode different toxin

genes. The plasmids can be exchanged between Bt strains by a conjugation-like process,

so there is a potentially wide variety of strains with different combinations of Cry toxins.

In addition to this, Bt contains transposons (transposable genetic elements that flank

genes and that can be excised from one part of the genome and inserted elsewhere). All

these properties increase the variety of toxins produced naturally by Bt strains, and

provide the basis for commercial companies to create genetically engineered strains with

novel toxin combinations.

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Table 17.4 Bt toxins and their classification

Gene Crystal shape Protein size (kDa) Insect activity

cry I [several subgroups: bipyramidal 130-138 lepidoptera larvae

A(a), A(b), A(c), B, C, D, E, F, G]

cry II [subgroups A, B, C] cuboidal 69-71 lepidoptera and diptera

cry III [subgroups A, B, C] flat/irregular 73-74 coleoptera

cry IV [subgroups A, B, C, D] bipyramidal 73-134 diptera

cry V-IX various 35-129 various

Mode of Action of Bt Toxin

The toxin of Bt is lodges in a large structure, the parasporal structure, which is produced

during sporulation. The parasporal crystal is not the toxin. However, once it is

solubulized a protoxin is released.

The crystals are aggregates of a large protein (about 130-140 kDa) that is actually a

protoxin, which must be activated before it has any effect. The crystal protein is highly

insoluble in normal conditions, so it is entirely safe to humans, higher animals and most

insects. However, it is solubilised in reducing conditions of high pH (above about pH

9.5), the conditions commonly found in the mid-gut of lepidopteran larvae. For this

reason, Bt is a highly specific insecticidal agent.

Once it has been solubilized in the insect gut, the protoxin is cleaved by a gut protease

to produce an active toxin of about 60 kDa. This toxin is termed delta-endotoxin. It binds

to the mid-gut epithelial cells, creating pores in the cell membranes and leading to

equilibration of ions. As a result, the gut is rapidly immobilized, the epithelial cells lyse,

the larva stops feeding, and the gut pH is lowered by equilibration with the blood pH.

This lower pH enables the bacterial spores to germinate, and the bacterium can then

invade the host, causing lethal septicaemia.

17.3 PRODUCTION OF BIOLOGICAL INSECTICIDES

Microbiological insecticides are produced in one of three ways: submerged fermentation;

surface or semi-solid fermentation; and in vivo production. The first two are for

facultative pathogens and the third is for obligate pathogens.

17.3.1 Submerged Fermentations

These have been used for the production of Bacillus spp. (excluding production of B.

poppillae which is produced in vivo) and to a lesser extent, fungi.

Medium: In fermentation for Bacillus thuringiensis the active principle sought is the delta

toxin found in the crystals. Media for submerged fermentation have been compounded by

various workers in a number of patents. In one such preparation, the initial growth in a

shake flask occurred in nutrient broth; in the second shake flask, and in the seed

fermentor best molasses (1%), corn steep liquour (0.85%) and CaCO

(0.1%) were used. A

typical medium for production would be beet molasses (1.86%), pharmamedia (1.4%)

and CaCO

(0.1%). Other production media contain corn starch (6.8%), sucrose (0.64%),

casein (9.94%), corn steep liquor (4.7%), yeast extract (0.6%) and phosphate buffer (0.6%).

Production of Microbial Insecticides ! !

A third medium contained soya bean meal (15%), dextrose (5%), corn starch (5%), MgSO

(0.3%), FeSO

(0.02%), ZnSO

(0.02%) and CaCO

(1.0%).

The above media were used for agricultural strains of B. thuringiensis but could no

doubt be used also for B. thuringiensis var israelensis.

Bacillus thuringiensis var insraelensis and Bacillus sphericus do not require carbohydrates

for growth and can grow well and produce materials which will kill the larvae of

mosquitoes in a variety of proteinaceous materials such as commercial powders of soy

products, dried milk products, blood and even materials from primary sewage tanks.

Effective powders of B. sphericus 1593 and B. thuringiensis var israelensis have been

produced using discarded cow blood from abattoirs and various legumes.

Extraction: At the end of the fermentation, the active components of the broth are recovered

by centrifugation, vacuum filtration with filter aid or by precipitation. Precipitation has

been done with CaCl

and the acetone method yields products of very high potency. The

fermentation beer may readily be diluted and used directly.

17.3.2 Surface Culture

Surface culture techniques are used for fungi and for spore formers. The organisms after

shake-flask growth are cultured in a seed tank from where the broth is transferred to flat

bins with perforated bottoms. The semi-solid medium is a mixture of an agricultural by-

product such as bran, an inert product such as kisselghur, soy bean meal, dextrose, and

mineral salts. The use of this medium increases the surface area and hence aeration

because of the thinness of its spread in the bins. Hot air is passed through the

perforations to dry the material. It is ground, assayed and compounded to any required

strength with inert material. Submerged, culture in which the hyphae are used have been

carried out with good results in the United States using Hirsutella thompsonii.

17.3.3 In vivo Culture

In vivo culture methods are used for producing caterpillar viruses, mosquito protozoa

and Bacillus popillae. The method is labor-intensive and could be easily applied for

suitable candidates in developing countries where expertise for submerged culture

production is usually lacking.

Once the organism has been obtained in a sufficient quantity to last for several years it

is lyophilized and stored at low temperature. The viruses are introduced into the food of

the larvae and the dead larvae are crushed, centrifuged to remove large particles and the

rest are dried. The amount of viruses in each larva is variable but the virus content of

between one and one hundred caterpillars should be sufficient to treat one acre in the

case of cotton moths. Usually separate facilities are used for rearing the caterpillars, for

infecting them and for the extraction of the virus particles. The preparation is then bio-

assayed and mixed with a suitable carrier.

17.4 BIOASSAY OF BIOLOGICAL INSECTICIDES

It is obvious that a reference standard must be set up against which various preparations

can be compared. The standard will differ with each particular bioinsecticide. Thus,

! " Modern Industrial Microbiology and Biotechnology

standards do exist for Bacillus thuringiensis serotypes H3 and H3A used against

caterpillars and a standard for B. thuringiensis var israelensis against mosquito ‘IPS82’

exists. Both standards are prepared and deposited at the Institute Pasteur in Paris. In the

simplest terms a standard is based on the LD

, the dose of the insecticide which will kill

50% of the population must be clearly defined; the age and type of insect to be used; the

food of the insect; the temperature conditions and a host of other parameters.

17.5 FORMULATION AND USE OF BIOINSECTICIDES

The formulation of the bioinsecticides is extremely important. An insecticide shown to be

highly potent under laboratory experimental conditions may prove valueless in the field

unless the formulation has been correctly done. Since microorganisms cannot by

themselves be patented, industrial firms producing bioinsecticides depend for their

profits on the efficiency of their formulation (i.e., the inert material which ensures

adequate presentation of the larvicide to the target insect). The inert material is referred to

as a carrier or an extender. Carriers or extenders are the solids or liquids in which the active

principle is diluted. When the carrier is a liquid and the active principle is suitable in it

the application is a spray. There are thus two types of formulation: (a) powders and dusts

(b) flowable liquid; which of the two is manufactured depends to a large extent on the

method of production and intended use of the insecticide.

17.5.1 Dusts

Semi-solid preparations based on waste plant products usually are compounded as

dusts or powders because making them into liquid causes the bran to absorb water and

prevent free flow thus leading to the clogging of conventional liquid applicators. The

advantage of dusts is greater stability of the preparation. They are also useful when the

insecticide is intended to reach the underside of low lying crops such as cabbages. Heavy

rains unfortunately wash off dusts. They may also lead to inhalation of the

bioinsecticides by the persons applying them. Diluents which have been used in

commercial dust of Bacillus thuringiensis are celite, chalk, kaolin, bentonite, starch, and

lactose. Lactose has also been used for diluting virus insecticide dusts. When the active

principle is absorbed on to the extender (or filler), the extender is referred to as a carrier.

If the extender or carrier is attractive to the insect as a food, oviposition site etc., then the

extender or filler is known as a bait. Baits for Bacillus thuringiensis include ground corn

meal, and for protozoa, cotton seed oil, honey, hydroxyethyl cellulose.

17.5.2 Liquid Formulation

Liquid formulations are usually made from water in which both the crystal and spores

are stable. Sometimes oils and water/oil emulsions may be used. When liquids other

than water are used it must be ascertained that they do not inactivate the active agent.

Emulsifiers may be added to stabilize emulsions when these are used. Some emulsifiers

which have been used for B. thuringiensis and viruses are Tween 80, Triton B 1956, and

Span 20.

The nature of the surface on which the insecticide is applied and which may be oily,

smooth or waxy may prevent the liquid from wetting the sprayed surface. Spreaders or

Production of Microbial Insecticides ! #

wetting agents which are surface-tension reducers may be added. Wetting agents may be

added to dusts to produce wettable-powders which are more easily suspended in water.

Some wetting agents and spreaders which have been used for agricultural Bacillus

thuringienses include alkyl phenols Tween 20, Triton X114 and for viruses Triton X100

and Arlacel ‘C’ which are all commercial surface-tension reducing agents.

To prevent run-off of liquids or wettable powders, stickers or adhesives are added to

hold the insecticide to the surface. Stickers which have been used for bacteria and viruses

include skim milk, dried blood, corn syrup, casein, molasses, and polyvinyl chloride

latexes.

Protectants are often added to insecticides which protect the active agent from the effect

of ultra violet light, oxidation, desiccation, heat and other environmental factors which

reduce the effectiveness of the active agent. These are usually trade secrets and their

composition is not disclosed. Dyes combined with proteins such as brewers yeast plus

charcoal, skin milk plus charcoal, and albumin plus charcoal have also proved effective

in protecting virus preparations from the effect of the ultraviolet light of the sun. Micro-

encapsulation of bioinsecticides with carbon also affords protection.

17.6 SAFETY TESTING OF BIOINSECTICIDES

Many individuals on first learning of the use of microorganisms to control insect pests

and vectors of disease express fear about the effect of these entomopathogens or their

effective components (e.g., crystals of B. thuringiensis). For this reason animal tests

including feeding by mouth, inhalation, intraperitoneal, intradermal and intravenous

inoculations, and teratogenicity and carcinogenicity tests are done. Test animals

include rats, mice, monkeys, rabbits, fish, and sometimes when appropriate, human

volunteers.

Tests conducted on the following agricultural entomopathogens in the United States,

Russia and Japan have shown them non-toxic for man, other animals or plants: Bacteria

(Bacillus popillae, B. thuringiensis, B. moritai), five viruses (Heliothus, Orgyia, Lymantria,

Autographa, Dendrolimus), three protozoa (Nosema locustae, N. algerae, N. troqodermae) and

two fungi (Beauveria bassiana, Hirsutella thompsonii).

Tests sponsored by the WHO and carried out in France and the United State have

shown the following useful or potentially useful entomopathogens to be safe. Bacillus

sphericus stain SS11-1, B. sphericus strain 1593-4; B. sphericus strain 1404-9, Bacillus

thuringiensis, var. israelensis (serotype H14) strain WHO/CCBC 1897; Metarrhuzium

anisopliae, Nosema algerae.

17.7 SEARCH AND DEVELOPMENT OF NEW

BIOINSECTICIDES

There are a number of stages in the development of a new bioinsecticide. The World

Health Organization has for some years followed the scheme given in Table 17.3 for the

screening, evaluation, safety, and environmental impact of entomopathogens to be used

for biological control.

Except where the material can be produced on a small scale, cottage industry, level,

production and sale of the final material will have to be done by industry, with its

! $ Modern Industrial Microbiology and Biotechnology

experience of formulation and sale distribution. It has been estimated that it will take five

to seven years to develop an entomopathogen into a biological insecticide; it will take less

than five years if some information on safety already exists on safety of a related

bioinsecticide. The cost of developing a biological insecticide is far less than that of

developing a chemical insecticide by between 20% and 50%.

SUGGESTED READINGS

Glare, R.E., Callaghan, M. 2000. Bacillus thuringiensis: Biology, Ecology and Safety. Wiley

Chichester UK.

Knowles, B.H. 1994. Mechanism of action of Bacillus thuringiensis insecticidal delta-endotoxins. In:

Advances in Insect Physiology, P.D. Evans, (ed.) Vol 24 Academic Press. London: UK. pp. 275-

308.

Obeta, J.A.N., Okafor, N. 1984. Medium for the production of the primary powder of Bacillus

thuringiensis sub-species israelensis. Appl. Environ. Microbiol. 47, 863-867.

Obeta, J.A.N., Okafor, N. 1983. Production of Bacillus sphaericus strain 1593 primary powder on

media made from locally obtainable Nigerian agricultural products. Canadian J. Microbiol. 29,

704-709.

World Health Organization. 1979a. Report of a meeting on standardization and industrial

development of Microbial Control Agents. TDR/BCV/79.01.

World Health Organization. 1979b. Biological Control Data Sheet: Bacillus thuringiensis de Barjac,

1978. VBC/BCDS/79.01.

World Health Organization. 1979c. Progress Report: Mammalian Safety Tests on the

Entomocidal Microbials Contract V2/181/113/(A): WHO/TDR/VBC.

World Health Organization. 1979d. Proposals for the adoption of a standardized bioassay

method for the evaluation of insecticidal formulations derived from serotype H14 of Bacillus

thuringiensis (Barjac de. H., and Larget, I.) WHO/VBC/79.744. Geneva.

World Health Organization. 1980. Annual Rept. Scientific Working Group on Biological Control

of Vectors, July 1979-June, 1980. WHO Geneva, Switzerland.

Yousten, A.Y., Federici, B., Roberts, D.W. 1991. Microbial Insecticides. In: Encyclopedia of

Microbiology Vol 2, Academic Press Sandiego, USA. pp. 521-531.

Nitrogen is a key element in the nutrition of living things because of its importance in

nucleic acids, which are concerned with heredity, and in proteins, which inter alia

provide the bases for enzymes. Gaseous nitrogen is present in abundance in the Earth’s

atmosphere forming about 80% of atmospheric gases. Indeed it has been estimated that

each acre of land has about 3,500 tons of N

above it. Unfortunately, most living

organisms cannot utilize gaseous nitrogen but require it in a fixed form; that is, when it

forms a compound with other elements. Nitrogen can be fixed both chemically and

biologically. Chemical fixation is employed in the production of nitrogenous chemical

fertilizers, which are used to replace nitrogen removed from the soil by plants. The ability

to carry out biological fixation is found only in the bacteria and blue-green algae. Some of

these organisms fix nitrogen in the free-living state and thereby contribute to the

improvement of the nitrogen status of the soil. Others do so closely associated (in

symbiosis) with higher plants. In some of these associations such as with some tropical

cereals, the organisms live on the surface of the plant roots and fix the nitrogen there. In

some others the microorganism penetrates the roots and forms outgrowths known as

nodules within which the nitrogen is fixed. Of the nodule-forming nitrogen fixing

associations between plant and micro-organisms, the most important are the legume-

bacteria associations. There are about 1,200 legumes species and their nodulation is

important because about 100 of them are used for food in various parts of the world.

Apart from serving as food for man and animals the legumes provide nitrogenous

fertilization to the soil for the better growth of crops in general; the importance of soil

fertilization can be seen from the fact that 100 kg of symbiotically-fixed nitrogen has been

estimated to be equivalent to an application of 50 kg of ammonium sulfate fertilizer.

The bacteria which form nitrogen-fixing nodules with legumes are members of the

genus Rhizobium. Inoculation of legumes with rhizobia started as long ago as 1896, eight

years after the beneficial association between the legumes and rhizobia was discovered.

Today there are thriving industries producing rhizobia inoculants in most parts of the

world. The inoculation of rhizobia into soils or on seeds is done where the specific

rhizobia which will form nodules with a given legume are absent in the soil because the

legume is new to the area or where because of a lapse of many years without the legume

the soil may become deficient in effective strains of rhizobia.

The Manufacture of

Rhizobium Inoculants

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The need for legume inoculation has become more urgent in recent years because of the

rise in the cost of chemical fertilizers, the inefficient use of chemical fertilizers by

agricultural crops and the short-term and long-term environmental consequences of

unused nitrate fertilizers which find their way into, and cause the pollution of, drinking

water.

Finally the problem of providing more protein for an ever-rising world population has

been compounded by the fact that areas of the world where protein shortage is most acute

are just those least able to afford the plants for the manufacture of chemical nitrogenous

fertilizers. By contrast investment in rhizobium inoculant production is relatively cheap

and the manufacture unsophisticated in comparison with chemical factories.

18.1 BIOLOGY OF RHIZOBIUM

18.1.1 General Properties

Members of the genus Rhizobium are aerobic, Gram-negative relatively large rods which

are motile and have peritrichuous flagellation. Older cells contain prominent granules of

poly->-hydroxybutyrate which give them a banded appearance. Within nodules they

form irregularly shaped cells known as ‘bacteroids”; no fixation occurs in the absence of

bacteroids. At the end of the growing season, the nodules occur in the absence of

bacteroids. At this time, the nodules disintegrate, releasing bacterioids which, once in the

soil, revert to rod-shaped cells and can survive there, sometimes for years. They invade

roots of appropriate legumes and form new modules when appropriate legumes are

planted.

18.1.2 Cross-inoculation Groups of Rhizobium

The most distinctive feature of Rhizobium spp. is their ability to form nodules with

legumes. Different rhizobia will form nodules only with some legumes. A group of

legumes with which a particular rhizobia bacterium will form nodules is known as a

cross-inoculation group. Over 20 cross-inoculation groups have been set-up out of which

seven (Table 18.1) are well-known. As will be seen from Table 18.1, rhizobia which lack a

more suitable group get lumped into the ‘cowpea rhizobia’ group. The group has also

been reclassified using numerical taxonomy, DNA base composition and homology,

serology phage susceptibility, patterns of isoenzymes, etc.

18.1.3 Properties Desirable in Strains to be Selected for

use as Rhizobium Inoculants

Before the production of a rhizobial inoculant is done the strain of the organism which

will yield the highest amount of fixed nitrogen in a given legume and a given

environment (or the most effective strain) is selected on the following basis.

(i) Effectiveness: ‘Effectiveness’ is a term used to describe the overall ability of a given

rhizobial strain to form nodules with a particular legume in a given environment

and fix useful quantities of nitrogen. Effectiveness itself is based on nitrogen-fixing

ability, invasiveness and competitiveness.

The Manufacture of Rhizobium Inoculants ! '

Table 18.1 Cross-inoculation groups of the genus Rhizobium

Rhizobium species Cross Legume host included Trigonella

Inoculation Group (Fenugreak)

1. Clover group R. Trifolii Trifolium (clover)

2. Alfalfa group Rhizobium meliloti Medicago (alfalfa)

Melilotus (sweet clover)

3. Bean group R. Phaseoli Phaseolus (Beans)

4. Pea group R. leguminosarum Pisum (pea); Vivia (Vetch)

Lathyrus (sweet pea)

5. Lupine group R. lupini Lens (Lentil)

Lupinus (lupines)

6. Soybean group R. japonium Ornithopus (Serradella)

7. Cowpea group Glycine (soy bean)

Vigna (cowpea); Grotolaria

(Crotolaria) Pueraria

(Kudzu) Arachis (Peanut)

Phaseolus (lima beans)

Nitrogen-fixing ability is an important factor as not all nodules fix nitrogen, or do

so to the same extent. Nitrogen-fixing nodules are usually comparatively few in

number, relatively large, and are pink in color. Invasiveness (also referred to as

infectiveness) is a measure of the ability of the legume to invade and nodulate the

roots of a high proportion of the plants to which it is applied. Competitiveness is

related to invasiness and is a measure of the ability of the rhizobium strain to

produce a large number of nodules in the presence of other infective or invasive

strains.

(ii) Ability to perform in the environment of a particular soil: When effectiveness is a

measure of properties inherent in the bacterium itself other factors relate to

performance in the soil. These are:

(a) ability to fix nitrogen in the presence of fertilizers used in the soil;

(b) tolerance of insecticide and seed disinfectants;

(d) adequate performance under the pH, aeration, and the mineral status of the

soil.

(iii) Growth and Survival in the carrier: The ability of the organism to survive and

multiply in the carrier (i.e., the inert support for distributing the bacteria) is

important; an otherwise adequate organism may be inhibited by the carrier.

18.1.4 Selection of Strains for Use as Rhizobial Inoculants

Methods available for assessing the performance of a rhizobium strain before choosing it

as an inoculant are discussed below:

(i) The agar method: Seeds sterilized with mercuric chloride, sulphuric acid and

alcohol are washed with sterile water are allowed to germinate in contact with the