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2.1 Define the Objectives

Standardization is the most important process and is based on theoretical background of industrial microbiology. The selection of a fungal strain, or a species in the case of a target pest susceptible to several pathogens, is a critical step since the aggressiveness of a fungus is highly dependent on it. The assessment of pathogenicity is usually based on the results of pest, or progeny (in the case of fast reproducing insects), mortality obtained from laboratory tests. It should be stressed, however, that bioassays conducted under laboratory conditions invariably optimize the potentialities of the fungus and thus the data should be interpreted carefully. For example, bioassays are run in the absence of any microbial competitors, in ambient conditions ideally chosen for the pathogen and with in vitro reared insects, the physiology of which may be different from that of the wild types. These bioassays, which are useful to compare strains or species, should represent, therefore, only the preliminary step before field experimentation. The latter is essential in order to determine if the microclimatic requirements of the pathogen and the host coincide and thereby to assess the true potential of an entomopathogenic fungus as a biocontrol agent. The pathogenic stability of a strain during repetitive transfers should also be checked. The media used for these transfers and for subsequent production should be chosen with care since it is known that nutrients can markedly influence conidial viability. Preferably, the strain selected should not have too narrow and specific, it being commercially advantageous if a product has a relatively wide host range within an insect group containing several pest genera. However, the host range within an insect group of several pest genera cannot be too wide and obviously must exclude beneficial insects as well as other invertebrates and vertebrates. Experiments have been carried out with several entomopathogenic fungi to test their effects on vertebrates, specifically to evaluate any allergic, irritation or toxic properties. Only minor allergic responses have been detected amongst a few of the entomopathogenic fungi screened so far.

Strains with the highest sporulation capacities should be selected since variations both in the amount of spores produced and in their mode of production have been reported. Industry will also screen for strains with the simplest nutritional requirements.

Most strain selections have been made from wild isolates of entomopathogenic fungi from naturally infected hosts. However, recent developments in fungal genetics suggest that the natural properties of a strain can be improved through genetic manipulation. In the past, mutagenesis has been used to enhance the virulence or sporulation of Metarhisium anisopliae. Conceivably, this process could also be exploited to produce mutants resistant to the pesticides normally encountered in the crop habitat of the target pest. Recombination of selected strains, markers, has been attempted amongst the Deuteromycetes. Parasexual recombinants from heterozygous diploids, produced by hyphal anastomosis, have also been identified in M. anisopliae. Recombination by protoplast fusion has been investigated in Beauveria brongniartii, M. anisopliae and Verticillium lecanii. One of the problems typically encountered in recombination is the poor stability of the highly sporulating strain and this is why the spectrum of insecticide activity to the target must be continuously monitored. The aggressiveness or the sporulation potential of a fungal strain should be controlled by numerous genes. Thus, genetic improvement, either by classical techniques or the new methods of genetic engineering, is fraught with difficulties. Nevertheless, it is feasible that the genes responsible for toxin excretion could be cloned and that their reinsertion into genome of another strain or species would be a method of increasing the efficiency of a mycoinsecticide.

All entomopathogenic fungi are characterized by a biphasic biological cycle: a mycelium vegetative phase and reproductive phase. Two spore types are usually found: asexual spores "anamorpha", for promoting rapid dissemination of the fungus and resting spores (sexual spores "telomorpha" or vegetative chlamydospores), responsible for survival of the pathogen during adverse conditions or in absence of suitable hosts. Theoretically, any of these fungal propagules could be considered for the production of mycoinsecticides.

Because of their primary role in the infection process, spores have been considered since the beginning of biological control history as the most adapted fungal propagule to produce. Conidia of the Deuteromycetes are readily mass-produced on solid media under aerated conditions. Conidia can also be obtained in liquid media, being produced on typical conidiophores arising from hyphal filaments or directly from the spore through a sporulation microcycle. This microcycle, typically induced by nutrient and/or temperature manipulation, has been developed as a model to study the biochemical events occurring during sporulation of the conidial fungi. This potential for microcyclic sporogenesis has been of particular interest in the case of entomopathogenic fungi since it shortens the culture time and increases spore yields. The other type of mass producible spore is the resting spore of the Mastigomycotina and Zygomycotina which have a role in disease carryover. These spores offer the advantage of being highly resistant and can survive for several months both in vitro and in nature. They can also be produced in liquid culture as well as on solid media. However, these spores, like the mycelium, are not directly infectious; their pathogenicity is dependent upon their potential to produce infective spores by germination.

The production of mycelium has been also contemplated especially in the case of Oomycetes and Zygomycetes for the reasons explained above. Mycelium propagules of entomopathogenic fungi are non-infective and thus the successful use of a mycelium formulation in biological control is dependent upon the ability of the mycelium to sporulate under natural conditions. On solid media, a continuous segmented mycelium is usually produced, whilst in shake-liquid cultures, as in the insect body, fungal development is most often characterized by the formation of yeast-like cells able to reproduce by fission. The terminology applied to these propagules depends on the fungal group under consideration, being termed: blastospores (Deuteromycetes); hyphal bodies (Entomophthorales, Coleomomyces); hyphal segments or subthalli (Lageniales). The yeast-like cells are often produced by hyphal constriction and thus their wall structure is mycelial. The fungal dimorphism exhibited by entomopathogenic fungi needs to be investigated more thoroughly in vitro since the multiplication of yeast-like forms would facilitate not only mass production but would also be an indication of the virulence of a strain as these cells are responsible for the rapid colonization of the insect haemolymph.

A further disadvantage of using mycelial propagules from in vitro cultivation, or even those formed in vivo, is their short viability in comparison with spores. However, formulation can significantly improve the longevity of the in vitro mycelium.

The growth requirements of most entomopathogenic fungi have been poorly defined despite the fact that this information is essential for mass production. In particular, such knowledge may permit simplification of the medium, cutting cost without affecting yield. The choice of industrial nutrients will obviously be directly related to the nutritional requirements of the selected fungus. For example, Entomophthorales do not metabolize sucrose; as a result all industrial media for the production of these fungi should exclude carbon sources extracted from sugar beet or sugar cane, which have high sucrose contents, and should be based on corn residues rich in dextrose.

Entomopathogenic fungi require oxygen, water, an organic source of carbon and energy, a source of inorganic or organic nitrogen and additional elements amongst which are minerals and growth factors. The carbon source is usually dextrose but can be replaced by polysacharides (such as starch) or lipids. Nitrogen can be supplied in the form of nitrate, ammonia or organic compounds such as amino acids or proteins. Other essential macronutrients are phosphorous (as phosphates), potassium, magnesium and sulphur, the latter supplied either in an inorganic form (as sulphate) or organic (cystein or methionine). Essential microelements usually include calcium, copper, iron, manganese, molybdenum, zinc and water soluble B-complex vitamins, especially biotin and thiamine. All these micronutrients usually occur in the raw materials included in industrial media but can be supplied as protein hydrolysate or yeast extract.

The nutritional requirements of entomopathogenic fungi vary with the fungal species or even the fungal strain under consideration. Deuteromycetes typically have low requirements and substantial growth of B. bassiana and M. anisopliae can be obtained in media containing only dextrose, a nitrate and a macromineral solution. However, semi-defined media, including protein hydrolysates, or natural undefined media rich in starch (rice, oatmeal, potato) have proved to consistently give the highest yields. Nevertheless, certain fungi, particularly those belonging to the genera Hirsutella, may have more specific requirements than the majority of this group since some species of these genera have failed to establish a culture.

The entomopathogenic Mastigomycotina and Zygomycotina have the most complex growth demands. These fungi require an organic form of nitrogen to attain substantial growth and cannot metabolize nitrates. Often vitamins and oligominerals have to be supplied to the medium. In these subdivisions of the fungi, usually considered phytogenetically less advanced than the Deuteromycotina, interconnections between the anabolic pathways are limited and the precursors of all the essential metabolic pathways, or the host, have to be added to the medium. Their absence in the culture medium is probably responsible for the lack of success in establishing in vitro cultures of any of the Coelomomyces species and many of the Entomophthorales and Trichomycetes. Most of the Mastigomycotina which have been cultured have been grown on complex media such a coagulated egg-yolk or mixtures of protein hydrolysates with a lipid or sugar source. Nevertheless, some species can be grown in relatively simply defined media.

The more wide spread acceptance and use of mycoinsecticides will depend on improvement in a number of key areas.

1) Production methods need to be cheaper. This will require greater yield over a reduced time scale. The preferred method of liquid fermentation, is at present not available for production of the most infective and/or stable spore types. This is particularly a problem for Entomophthoralean fungi. McCabe and Soper (1985, patent USA) have described a system for the production of dry viable preparations viable mycelium. The draw back with this method is that mycelial fragments are not infective; contact with water in the field stimulates required conidiogenesis. The extra step prolongs the response time.Semi-solid fermentation of Deuteromycotina using cereal grains holds much promise.

2) Develop new formulations that will extend the shelf life improve efficiency of application and field persistence. A water retaining formulation would be particularly helpful.

3) Produce stress tolerant strains which are less affected by low RH, UV, high temperatures and fungicides.

4) Produce more virulent strains which kill faster such that they could be used against larger pest population and obviate the need for prophylactic use.

5) Investigate the possibility of integrating mycoinsecticides with existing control measures e.g. Applications with low doses of insecticide have received little attention. Chitin synthesis inhibitors by weakening insect cuticle can facilitate entry of enthomopathogenic fungi.

Laboratory-reared insects are used in screening pesticides, and production of microbial pesticides. Their many uses, which are becoming more and more sophisticated, require that these insects be of a definite quality.

It is now known that changes in genetic variability occur during domestication of insect populations. Greater attention needs to be given to colony establishment and the changes that occur over time because the variability within a genetic pool has a significant impact on the quality of the insect.

The basic steps in standardization are: (1) define the objective, (2) establish standards, (3) design and test the production methods, and (4) implement quality insect.

a) Fecundity
b) Percent hatch
c) Yield of pupae or adults
d) Yield of adults/unit (amount of diet)
e) Size of larvae, pupae, or adults (weight)
f) Life history-development time for each stage

The quality of diet components can be controlled.

Microbial control during colonization of the wild insect, in dietetics, in facility maintenance, and personnel management is recognized as a part of good rearing techniques that contribute to increased efficiency of rearing (higher yields) and improved biological vigour of the insect.

Environmental control involves the regulation of the external conditions that affect the growth, development, and behaviour of the insect being produced. Usually the conditions that must be controlled are temperature, humidity, light, and the movement and cleanliness of air. Noise and vibration must also be considered.

Ultimately, it is the degree of performance of the product in meeting its intended use that determines overall quality.

a) Control
b) Release and recapture
c) Ratio of fertile/sterile

a) Mating competitiveness
b) Mating compatibility
c) Sterility
d) Flight ability
e) Flight capacity
f) Flight propensivity
g) Pheromone response
h) Circadian activities
i) Longevity
j) Irritability

a) Genetic diversity
b) Electrophysiological
c) Pheromones
d) Biological chemistry

Insects rearing see Annex.

2.2 Microbial Insecticide Based on Bacillus Thuringiensis

Cultures of this species nowadays serve as the basis for large-scale production of microbial insecticides. World-wide production now constitutes a few thousand tons annually. The cultures of B. thuringiensis are closely related to those of B. cereus, which are widely distributed in nature. They differ from the former only by the formation of crystalline inclusions.

For the purpose of systematics and identification of B. thuringiensis cultures, an efficient method is serotyping by H-antigen along with some biochemical properties of the cultures. Up to now 23 serotypes have also been differentiated within some of the species. From a practical point of view it is essential that there exist certain correlations in the spectrum and activity of entomocide effect between the various subspecies as well as subserotypes (Krieg, 1986).

There are three basic questions that can be asked in any study of production of toxin by B. thuringiensis.

What toxins are produced?
In what quality?
How reproducible is the fermentation?

In this discussion of our program which follows we will direct most of our attention to ask what toxins are produced. However, our interpretation must not be confused by the quantities of toxin present in product powders.

This is important to remember. We will use several concepts, ratios, distributions of toxicities, crystal-types, H-types, etc. It is impossible to eliminate quantity as a variable. The quantity of toxin produced in a fermentation can be influenced by the isolate of B. thuringiensis used and by medium on which it is grown. Thus two powders may differ many-fold in toxicity yet still contain the same toxin. All analyses will be made in the light of this factor and will attempt to pinpoint type as differentiated from quantity of toxin.

The basic active agent, called d-endotoxin, is produced in the form of crystalline parasporal inclusions during sporulation and liberated in the medium after its completion.

Principally important especially for production purposes is that the level of endotoxin insecticide activity is not correlated with its size and quantity but is defined by subspecies and the strain. For example: the strain HD-1 belonging to the subserotype kurstaki of the subspecies alesti and having the same mass of crystalline inclusions reveals 100 times more insecticide activity than these of other strains. Industrial production of more than 30 preparations has been developed in different countries and the products have been introduced in the market under different trade names since the sixties.

The main sources for the production of B.t. preparations are strains of the subspecies kurstaki, galeriae and dendrolimus. The cultures of serotype-I berliner, thuringiensis and some other subspecies also produce soluble termostabile á-exotoxin of a nucleotide nature, beside crystalline inclusions. A number of preparations manufactured from exotoxin producing strains are characterized by a broad spectrum of entomopathogenous action. Some producers exclude exotoxin in ready-made preparative forms because of possible side effects.

Bacillus thuringiensis (B.t.) is a gramm-positive bacterium forming elliptical spores, contained in unswollen sporangia, and a parasporal body (or crystal) which appears mainly as a bipyramidal shape. B. thuringiensis is a complex species divisible into subspecies and H-serotypes by serological and biochemical tests. These produce several insecticidal toxins, two of which are used in agriculture.

The relative activity of each isolate against different insect species "spectrum activity" arrives partly from the combined effects of the potencies of the varying concentrations of the different insecticides that it produces. The d-endotoxin of different isolates of B.t. can kill different insect species or differ in the degree of their activity toward them.

This variation of activity spectrum according to the B.t. isolate is very important. Failure to control a pest insect with a particular B.t. preparation does not mean that all preparations will fail; it mean just that the wrong isolate was selected for use against the target insect. Similarly, even though a pest species may be satisfactorily controlled by the toxins in the present commercial preparations, it is possible that a different isolate may be more effective and thus cheaper to use.

The picture is further complicated because different isolates can produce more or less of the same d-endotoxin than other (Dulmage 1970). Also maximum toxin production can be achieved only by careful attention to the interaction of fermentation conditions, media and the isolates involved - there is, for example, no one medium best suited to all isolates (Dulmage 1981).

The b-exotoxin, "heat-stable exotoxin", is a water soluble toxin highly toxic to larvae of several species of flies, while alfa-exotoxin "heat-unstable" is toxic per os to mice and to the diamond moth, Plutella xylostella (maculipennis). á-exotoxin has been defined chemically as an adenine nucleotide and ATP analogue and given the name "thuringiensin". Many regulatory authorities opted to prevent its use in agriculture, because it has a terratogenic effect in insects and has mutagenic activity.

The d-endotoxin in crystals of B.t. has a limited infectivity spectrum limited, so far as we know, to certain Lepidoptera, mosquitoes, chiromonids and blackflies. The crystalline glycoprotein is formed during sporulation, it is variously called the crystal, parasporal body or d-endotoxin. There is evidence that plasmids are related to crystal formation. Different numbers of plasmids are found in most serotypes of B.t. The plasmids DNA (CCC - covalently closed circular DNA) has different values from 2 to 32 Mda and depend on subspecies and serotype. The strains which loss the large plasmid > 32 Mda, loss insecticide activity. There are two categories of plasmids:

36. < 15 Mda.
37. > 15 Mda.

The role of phages in B.t. genetics is most important. They provide a mechanism for a specialized and generalized transduction which constitute functional genetic transmission systems. The discovery of lysogeny in B.t. has also opened the possibility that one of the toxins is coded by a prophage.

Three types of phages lyse B.t., and these are virulent, pseudolysogenic and temperate. They respectively lyse cells immediately, or form temporary or permanent associations with mitomycin C or UV light, but none have formed a lysogenic relationship with the host.

The crystals from various subspecies are composed of up to four proteins with molecular weights ranging from 26 to 140 kDa. In the case of the lepidopteran-specific subspecies, the major components of this crystal is a 130 - 140 kDa protein referred to as the protoxin. Upon dissolution at alkaline pH in the mitgut of targeted larvae, the protoxin is further "activated" by specific proteolysis and the toxic moiety released. Upon its attachment to specific receptors of the columnar cells in the midgut epithelium, the cells swell and are released from the basement membrane and finally burst. The insect (larvae) stops feeding, becomes rapidly dehydrated and generally dies within the next 48 hours.

Many strains of B.t. have now been isolated and classified upon biochemical, enzymatic and serological criteria. The generally accepted key for the taxonomic division of the species of B. thuringiensis is based on the antigenic properties of the flagella as developed by de Barjac and Bonnefoi (1962). However, B.t. strains can also be allocated to different subspecies or varieties based on their pathotypes or insecticidal activity for different insects.

For the purpose of the systematics and identification of B.t. cultures, an efficient method is serotyping by H-antigen along with determining some biochemical properties of the cultures. Up to now 23 serotypes have been described. Subspecies and a number of serotypes have also been differentiated within some of the species. From a practical point of view it is essential that there exist certain correlations in the spectrum and activity of insecticide effect between the various subspecies as well as subserotypes.

The cultures of a majority of serotypes of these species B.t. var. kurstaki are characterized by strong entomocide activity to Lepidoptera, products DIPEL (Abbott), Bactospeine (Philips Duphar), THURICIDE, JAVELIN (Sandoz). Some differences in the spectrum have also been observed. Thus, the cultures of serotype 5 (galleriae) are very active to Galleria mellonella, while representatives of serotype 10 (darmstadiensis-caucasicus) do not reveal any insecticide activity towards this insect. The cultures of serotype 4 are very active to Siberian silkworm and comparatively less virulent to silkworm Bombyx mori.

Of great interest was serotype 14 that was describes in 1978 as B.t. subsp. israelensis. Cultures of this serotype thus produce crystalline parasporal toxin with strong larvicide activity to mosquitoes, black fly and other insect of the Simallidae. It is worth mentioning that they are characterized by specific larvicide pathogenicity and are practically harmless to mammals, plants and useful hydrobionts.

A number of countries have organized industrial production of larvicide preparations on the basis of B.t. subsp. israelensis cultures like TECNAR, VECTOBAC, BACTIMOS and others.

Of special practical interest was the description of cultures named as B.t. var. tenebrionis with activity to Coleoptera. Other pathotype of B.t. var. san diego with insecticide activity to Coleoptera products like TRIDENT (Sandoz) and M-ONE (Mycogen) and pathotype of B.t. var. aizawai with insecticide activity to Lepidoptera and Diptera products like CERTAN (Sandoz).

In the case of B.t. , the subspecies (varieties) and H-serotypes as well as biotypes with different enzymatic features are defined on a biological and serological basis. This is the case with the serotypes H4a4b and H6, these biotypes have a different spectrum of pathogenic properties, the discovery of antigenic subfactors (fractions) in five of the serotypes of H3, H4, H5, and H11 allows for further differentiation of various subtypes or biotypes.

The first three letters of the subspecies name can be used as an abbreviation of the strain name. For instance, KUR H-3a3b (serotype) kurstaki isolated from Ephestia kuhniella (Kurstak 1962); KUR H-3a3b HD-1 kurstaki isolated from Pectinophora gossipiella (Dulmage 1970); ISR H14- israelensis.

Lepidoptera B.t. var. aizawai Certan (Sandoz) Coleoptera thuringiensis var. kurstaki (mainly HD1 and HD12 isolates) has been used for some years on a limited scale in both agriculture and forestry. It is well known that B.t. is very selective in its biological action, and kills only a limited range of insects; birds, mammals and fish are not affected. This selectivity is a key to marketable product; very favorable toxicology and environmental profile, zero pre-harvest interval on vegetable crops, "bio-rational" registration like in North America.

Table 2.1. Pathotypes of Bacillus: thuringiensis

However B. thuringiensis var. kurstaki has drawbacks including speed and mode of action, and solar radiation sensitivity which have limited its usage. Combining these problems, B.t. application on foliage remains a challenge in most agricultural situations. Anti-feeding effects observed in various B.t. formulations combined with non-optimised application rates and spray technology especially in agriculture have decreased significantly its attractiveness to farmers.

Exotoxin formulations of sufficient purity can be obtained by rechromatography on DEAE-cellulose or silicagel. The purification process which does not use any adsorption on carbon makes use of the supernatant of the B.t. cultivation medium which is thickened to 1/10 of its original volume by boiling. The inactive material is then precipitated out by stepwise ethanol additions. At 90 vol. % of ethanol an active precipitate is obtained which can be further purified on cellulose or ion exchange columns.

The b-exotoxin, called thuringiensin, is a nucleotide composed of adenine, ribose, glucose, and phosphorylated allaric acid.

It inhibits the synthesis of ribonucleic acid by stopping off the polymeration catalyzed by DNA-dependent RNA-polymerase. The toxicity of b-exotoxin to caterpillars.

Galleria mellonella is LD₅₀ = 0.5 ug/g, that to mice is LD₅₀ = 18 ug/g. The spectrum of effectiveness of b-exotoxin is much broader than that of delta-endotoxin and it is lethal to insects of Lepidoptera, Coleoptera, Isoptera, and Orthoptera groups. Effectiveness varies depending on dose, time, and mode of application.

Autoclaved preparation was effective against Two-spotted mite (Tetranychus urticae), citrus mite (Panonychus citri), and Nematode Meloidogyne). Formulations containing b-exotoxin are prohibited in Europe and the U.S. A formulation called Bitoxibacillin containing 0.5-0.8 % exotoxin is in use in the USSR. In production where the bacterial mass is separated from the liquid of the cultivation medium by centrifugation, the exotoxin is removed.

Living cycle of B. thuringiensis has two phases: vegetative and sporogenic.

Using the lactose-acetone technique (after Dulmage et al., 1970)

In all assays, the powder is administrated to the insects by mixing it into their diet, with larvae being allowed to feed ad libitum on the powder-diet mixtures. 2) The effect of this exposure is judged by a single criterion: death. A severely retarded or moribund larva is considered alive if it could move. The assays measured only per cent dead. 3) Preliminary assays determined kill at two levels, usually 500 ug and 50 ug powder/unit of diet. If activity is sufficient, repeat assays with an appropriate series of dilutions to determine the LC₅₀.

The first generally accepted standard was prepared in France from a fermentation of H-type thuringiensis and called "E-61". E-61 was assigned a potency of 1000 IU/mg and recommended as an international standard in 1966 (Burges, 1967). When the HD-1 strain of H-type kurstaki was selected for commercial production of B.t. in the USA, a formulation of HD-1, labelled HD-l-S-1971 was assigned a potency of 18,000 IU/mg on the basis of assays against E-61, using Trichoplusia ni as test insect (see ANNEX 5).

Basic formula:

LC₅₀ Standard
--------------------------------- x Potency of Standard IU/mg = potency of LC₅₀ Test Sample,
IU/mg test sample

IU/mg = Potency of Test Sample

When HD-1-S-1971 is used as the standard, the equation

becomes:

LC₅₀ HD-1-S-1971
------------------------------ x 18 000 IU/mg = Potency of Test Sample,
LC₅₀ Test Sample IU/mg

The numerical value of the ratio gives a quantitative evaluation of the difference between a sample and a standard, and this can to further comparisons e.g. toxin with a any two insect species can form ratio, even if the assays are performed in different laboratories, as long as the assays are performed on the same powder.

BARJAC DE, H., BONNEFOI, A, 1962: Essai de classification biochimique et sérologique de 24 souches de Bacillus thuringiensis. Entomophaga 7:5-31.

BURGES H.D., 1967: in Insect Patholotgy and Microbial Control" (P.A. van der Laan, edd.) pp. 306-338. North-Holland Publ. Co., Amsterdam.

BULLA, L.A., BECHTEL, D.B., KRAMER, K.J., SHETHNA, Y.I., ARONSON, A.I., FITZ-JAMES, P.C. 1980: Ultrastructure, physiology, and biochemistry of Bacillus thuringiensis subsp. kurstaki. Biochem.. Biophys. Res. Commun. 9:1123:1130.

DULMAGE, H.T., 1970: Insecticidal activity of HD-1, a new isolate of Bacillus thuringiensis var. alesti. J. Invetebr. Pathol. 15: 232-239.

DULMAGE, H.T., et cooperators, 1981: Insecticidal activity of isolates of Bacillus thuringiensis and their potential for pest control. In: Microbial control of pests and plant diseases 1970-1980. Edit. Burges, H.D.,Acad. Press London-New-York 1981, pp. 193-222.

KRIEG A.,1986: Bacillus thuringiensis, ein mikrobielles Insecticid. Grundlagen und Anwendung. Acta Phytomed. Suppl. J. Phytopathol. 10, Paul Parey Berlin-Hamburg 1986, 191 pp.

KURSTAK, E.S. 1962: Données sur l`épizootie bacterienne naturelle

provoquée par un Bacillus du type Bacillus thuringiensis sur Ephestia kühniella Zeller. Entomophaga, Mem. Hors. Ser. 2:245-247.

VAOKOVA, J., 1990: Bacillus thuringiensis bakteri ln¡ insekticid. Academia Praha, 116 pp

2.3 Microbial Insecticide Based on Metarhizium Anisopliae

The first attempt at using Metarhizium anisopliae = M.a. for biological control was by Krassilstchik (1888). He was successful in destroying 55-80 % of Clones punctiventris insects in small areas. Varied results have also been obtained when M.a. was used against other insects (Picles 1945, James 1946). Presently M.a. were applied successfully against insect pests of several crops (Ferron 1981), including pests of rice in the tropics (Rombach et al. 1987). In this region annually treated about several hundred thousand hectares, and using around 2.5 x 10¹² viable spores/ha.

M.a. "Metaquino" is produced in Brazil by some 5 commercial companies as well as grower cooperatives and individual plantation owners. It is used for control of the sugarcane spittle bug, Mahanarva postica, in the NE of the country. M. anisopliae var. majus is also used for control in parts of the Pacific and SE-Asia to augment baculovirus at high larval densities (Gillespie, 1988).

According to Zimmermann and Simmons (1986) it is possible to recommend M.a. against Otiorrhynchus sulcatus in greenhouse and to lesser degree in the field. Bayer AG produce Bio 1020 granule, Chabchoul and Táborsky (1990) have also been used M.a. against Colorado beetle Leptinotarsa decemlineata.

Adhesion appears to be a prerequisite for successful invasion as noted for hypovirulent strains of M. anisopliae. Dillon and Charnley (1989) showed that germination of M.a. is initiated by water but progress to the first overt stage of germination (swelling) is depended on an exogenous nutrient. Prior exposure to water "soaking" synchronized and accelerated swelling, germ tube and appressorial formation when a nutrient was finally provided. Soak spores were significantly more pathogenic than the controls (Hassan et al. 1989). Vegetative spores "conidia" are strong hydrophobic and keep their viability for more than two weeks after spraying on target insects. For example; larvae of L. decemlineata are compleat bay destroyed in the soil by green muscardine (Táborsky unpubl. data). For germination of conidia M.a. by the test of viability is possible put into drop on microscopy slides special nutrients like orange juice (0.05 %). Current evidence for M.a. suggests that differentiation of appressoria is strictly governed by the concentration of low molecular weight nitrogen compounds on a conductive surface.

Penetration of host exosceleton appears to involve both mechanical and enzymic components.

Figure 2.2. Penetration of host cuticle by a Deuteromycetes entomopathogens 1 = appressorial complex, 2 = penetration peg, 3 = penetration plate

In soft cuticles e.g. caterpillars growth across the cuticle is more or less direct, in hard cuticles e.g. wireworms the fungus proceeds in a step-wise fashion. The production of cuticle-degrading endoproteases with similar modes of action by all Deuteromycetes studied suggests that it is unlikely that they contribute to host specific or virulence, though the common occurrence implies an indispensable fungi differ only in charge. However, this does have practical significance as binding to cuticle with different charge may be favorable or unfavourable to binding by individual enzymes, with consequences for the parts of the body which can be invaded by enzymic action. This can influence the speed of penetration and thus virulence.

Deposition of oxidized phenols (melanin) in cuticle by host phenoloxidase is the first overt response to infection. Antimicrobial effects of phenols are well established, but in insect cuticles melanization appears to be primarily an effective defense against more virulent pathogens. Protease inhibitors within the cuticle may serve to restrict pathogen enzyme activity. Within the haemocoel the main cellular response of the insect is a multihaemocytic encapsulation of the fungal element following initial recognition of the fungus by the haemocytes. The yeast like blastospores, produced by Deuteromycetes in the insect haemolymph, reduce the effectiveness of the cellular defences by sheer weigh of numbers and not being as antigenic as the mycelium (Charnley, 1989). Finally the cyclodepsipeptide toxins, destruxin produced by M.a. appear to interfere with haemocyte function, specially by suppressing prophenoloxidase activation (Huxham et al., 1989). However, recent evidence is consistent with destruxins being a determinant of virulence for M.a.. Destruxins are active in causing symptoms, principally by paralyzing muscles of caterpillars, while in other insect hosts of M.a. such as Orthoptera, whose muscles are not susceptible to destruxins, and in susceptible insects infected with low destruxins producing strains, the toxin may act indirectly to assist the pathogen to overcome host defences perhaps as stated earlier by interfering with haemocyte activity Samuels et al., 1988).

Deuteromycetes do not have a sexual cycle and thus recombination can only be achieved either by use of the parasexual cycle or by direct genetic manipulation (Heale et al., 1989). Recombinants from heterozygous diploids produced by hyphal anastomosis or protoplast fusion have been identified from M.a. and V. lecanii. (Heale, 1988). Frequently parasexual recombinants exhibit reduced pathogenicity in comparison with the wild-type parents due to the possibility to disrupt of clusters of pathogenicity genes.

The recent demonstrations of transformation in a number of filamentous fungi have indicated that molecular cloning techniques could be used to investigate pathogenicity determinants of entomopathogenic fungi, isolate genes coding for specific pathogenicity determinants and produce organisms with enhanced virulence. At the present time M.a. seems to be the most appropriate fungus for this approach as there are two putative pathogenicity/virulence determinants like endoprotease, chymoelastase and destruxins. The development of a transformation system and cloning vectors for M.a. is an essential prerequisite for such approach. For the future once recombinant plasmids containing genes coding for virulence factors have been identified it may be possible to use them in a programme of strain improvement, particularly as transformation in filamentous fungi is frequently accompanied by gene amplification.

M.a. is possible to produce like local product in plastic fermenters (see Verticillium lecanii). The isolate M.a. must be passed through target host and then be cultivated on Sabouraud`s liquid medium. As far as the concentration of conidia was very high and reached after 8 days 3.19 x 10¹⁰ conidia/1 g. Light and darkness have not any decisive effect on mass production of conidia. The temperature is most important for the yield of conidia. At first 6 days is the best temperature 24-25^oC and then it is possible to decrease temperature to 22-20^oC.

Submersed culture produce through 3-4 days blastospores, which are suitable like seed culture with medium for plastic fermenters.

Process harvesting must be done with Tween 80 or Triton 100, and then is used Siloxyd 125 - 150 g per 1 liter mud and dry during 24 hours. Formulation WP is suitable for conidia M.a. and shelf life during storage is good at 4 - 10^oC.

100 g of conidia powder ( 2.5 x 10¹⁰ conidia/1 g) must be mix with Siloxyd and diluent so, that the total weight will be 500 g. The recommended dose per ha is in this case 500 g of formulated product (e.g. 2.5 x 10¹² of conidia) .

CHABCHOUL H., and TABORSKY, V., 1990: Use of Metarhizium anisopliae (Metsch.) Sorokin against Colorado beetles Leptinotarsa decemlineata (Say). Agricultural Tropica et Subtropica, Universitas Agriculturae Praga, 23 (in press).

CHARNLEY, A.K., 1989: Mechanisms of fungal pathogenicity in insects. In The Biotechnology of Fungi for Improving Plant Growth. J.M.Wipps and R.D.Lumsden (Eds), Cambridge: University Press.

DILLON, R.J., CHARNLEY, A.K., 1989: Initiation on germination in conidia of the entomopathogenic fungus, Metarhizium anisopliae. in A.K. Charnley: Mycoinsecticides: Present use and Future prospects. pp.165-181. Progress and Prospects in Insect control. Monograph No.43, British Crop Protect. Council

FERRON, P., 1981: Pests control by the fungi Beauveria and Metarhisium. In: Microbial control of pests and plant diseases 1970-1980 (Ed. M.D. Burges). Acad. Press, pp.465-482.

GILLESPIE, A.T.,1988: Use of fungi to control pests of agricultural importance. In: Fungi in Biological Control Systems M.N. Burge (Ed), Manchester: University Press, pp. 37-60.

HASSAN, A.E.M., DILLON, R.M. and CHARNLEY, A.K., 1989: Influence of accelerated germination of conidia on the pathogenicity of Metarhizium anisopliae for Manduca sexta Journal of Invertebrate Pathology.

HEALE, J.B., ISAAC, J.E., CHANDLER, D., 1989: Prospect for strain improvement in entomopathogenic fungi. Pesticide Science 26: 79-92.

HUXHAM, I.M., LACKIE, A.M., MCCORKINDALE, N.J. 1989: Inhibitory effects of cyclodepsipeptides, destruxins, from the fungus Metarhizium anisopliae, on cellular immunity in insects. Journal of Insects Physiology 35: 97-107.

JAMES, H.C., 1946: The bionomics and control of Tomaspis fluvilatera Ur., The Demerara sugar cane froghopper. Proc. Brit. W.Ind. Sug.Tech., 34-79.

KRASSILSTCHIK, I.M., 1888: La production industrial des parasites végétaux pour la destruction des insects nuisibles. Bull. Scient. Fr. Belg., 19: 461-472.

PICKLES, A., 1945: Entomology. Adm. Rep. Dir. Agric. Trin. Tob., pp. 17-18.

ROMBACH, M.C., SHEPARD, B.M., and NELSON, F.R.S. 1987: Compatibility of M. anisopliae var. anisopliae with chemical pesticides. Mycopathologia, 99: 99-105.

SAMUELS, R.I., REYNOLDS, S.E., CHARNLEY, A.K. 1988: Calcium channel activation of insect muscle by destruxins, insecticidal compounds produced by the entomopathogenic fungus Metarhizium anisopliae. Comparative Biochemistry and Physiology, 90C, 403-412.

ZIMMERMANN, G. and SIMONS, W.R. 1986: Experiences with biological control of the black vine weevil Otiorhynchus sulcatus (F.). In Fundamental and applied aspects of invertebrate pathology (Ed. R.A. Samson, J.M. Vlak and D. Peters). Found. Forth Int. Colloquium Invert. Path., Wageningen, Netherlands: pp. 529-553.

2.4 Microbial Insecticide Based on Beauveria Bassiana

Beauveria bassiana = B.b. is best known as the causal agent of the disastrous muscardine in silkworms. It is the most widely distributed species of the genus and is generally found forming white dusty raised tufts on Coleoptera, Lepidoptera, Diptera and other insects in both temperate and tropical areas. B.b. is sensitive to soil-mycostatic factors. Survival in soil is favored by darkness and reductions in both temperature and soil moisture; at 8^oC and under dry conditions, 90 % of the conidia survive for over 635 days (Clark, G.C. and M.F. Mandelin, 1965, Walstad, J.D., R.F. Anderson and W.J. Stambaugh, 1970); on silica gel conidia survived up to 36 months of storage at -20^oC (Bell, J.V. and R.J. Hamalle, 1974). After spraying on the crops conidia survival only 3 days on the same level of germination before application.

Biological control experiments with it are numerous and have been directed particularly to the Colorado beetle and other Coleoptera and Lepidoptera. Various species of mosquito larvae can be killed by dusting conidia on the water surface (Clark, T.B. et al. 1968); conversely, bees larvae were not susceptible while adult bees succumbed (Toumanoff, C. 1931). It has also been isolated from the lungs of giant tortoises and box turtles affected wit a pulmonary disease (Georg, L.K. et al. 1962); an increase in the air spore has also led to allergic responses in man (Roberts, D.W., 1973). B.b. is employed on a large scale in the People`s Republic of China to control pine caterpillars (Dendrolimus punctatus), green leaf hoppers (Nephotettix spp.) and corn borer (Ostrinia nubilalis). In the USSR B.b. like "Boverin" is produced for the control of the Colorado beetle (Leptinotarsa decemlineata) and the codling moth (Laspeyresia pomonella), in CSRF "Boverol" against Colorado beetles, Whiteflies (Trialeurodes vaporariorum) and black vine beetle Otiorhynchus sulcatus.

On agar media conidiogenesis starts after six days, while in liquid culture this takes only 3-4 days (Sam1iòáková, 1966). In stirred liquid cultures employed in mass production of this fungus, so-called "blastospores" developed which are thin-walled, larger (3-5 x 2-3 mm) and less resistant than conidia (Mueller-Koegler, and Sam1iòáková , 1970); blastospores germinated in 6-10 hours at 18-24 0C, while conidia require 15-20 hours. The germination of conidia requires a saturated atmosphere and the optimal temperature for growth is in the range 25-30^oC, minimum 10^oC, and maximum 32^oC apparently depending on the geographic origin of the isolate; no germination occurs either below 10^oC or above 35^oC; the thermal death point of conidia has been determined as 50^oC for 10 min in water. The optimal pH for growth is 5.7-5.9, and for conidia formation 7-8 (Goral, Lappa, 1972).

Good growth occurs on maltose and sucrose and, among others, on the N sources glutamic and aspartic acids, and ammonium oxalate, citrate or tartrate. A medium recommended for optimal growth contains 2% corn steep liquor, 2.5% glucose, 2.5 starch, 0.5% NaCl, and 0.2% CaCO3. The fungus produces lipase, protease, urease, amylase, chitinase, cellulase and 1,2-á- glucanase. Chitinase can be realized into the medium during autolysis, but was also found to act jointly with other enzymes in decomposing insect integuments. The production of a toxic substance is proteins complex consisting of two fractions with different molecular weights (Kuèera and Sam1iòáková, 1968). This toxic metabolite is produced most abundantly on complex media (e.g. cornmeal, yeast or beef extract); inorganic N sources are ineffective (Kuèera, 1971). A red bibenzoquinone pigment, oosporein, with antifungal properties (Whine et al. 1962) and the yellow pigments tenellin and bassianin have been found. Small scale production, harvesting an formulation see in Verticillium lecanii.

BELL, M.R., and HAMALE, R.J. 1974: Viability and pathogenicity of entomogenous fungi after prolonged storage on silica gel at -20^oC. Can. J. Microbial, 20, 639-642.

CLARK, T.B., KELLEN, W.R., FUKUDA, R. and LINDEGREN, J.E. 1968: Field and Laboratory studies on the pathogenicity of the fungus Beauveria basiana to three genera of mosquitoes. J. Invertebrate Path. 11, 1-8.

CLARK, T.B., MANDELLIN, M.F. 1965: The longevity of conidia of three insect-parazitisms hyphomycetes. Trans.Br. Mycol. Soc. 48, 193-209

GEORG, L.K., WILLIAMSON, W.M., TILDEN, E.B., and GETTY, R.E. 1962: Mycotic pulmonary disease of captive giant tortoises to Beauvaria bassiana and Paecilomyces Fumoso-RoseusSabouraudia 2, 80-86.

GORAL, W.M., and LAPPA, N.V. 1972: The effect of medium pH on growth and virulence of Beauveria bassiana (Bals.) Vuill. Mikrobiol. Zh. 34 )4), 454-457.

KUEERA, M. and SAMSINAKOVA, A. 1968: Toxins of the entomophagous fungus Beauveria bassiana. J. Invertebr. Path. 12: 316-320.

KUCERA, M., 1971: Toxins of entomophagous fungus Beauveria bassiana. 2. effect on nitrogen sources on formation of the toxic protease in submerged culture. J. Invertbr. Path. 17: 211-320.

MULLER-KOGLER, E., SAMSINAKOVA, A. 1970: Zur Massenkultur des Insektenpathogenen Pilzes Beauveria bassiana. Experientia 26, 1400.

ROBERTS, D.V., 1973: Means for insect regulation - Fungi. Ann. N.Y. Acad. Sci. 217: 76-84.

SAMSINAKOVA, A., 1966: Growth and sporulation of submerged cultures of the fungus Beauveria bassiana in various media. J.Invertebr.Path. 8: 395-400.

TOUMANOFF, C., 1931: Action des champignons entomophytes sur les abeilles. Annls Parasit. Hum.Comp. 9: 464-482.

VINING, L.C., KELLEHER, W.J., and SCHWARTING, A.E. 1962: Oosporein production by a strain of Beauveria bassiana originally Identified as Amanita muscaria. Can.J.Microbiol. 8: 931-933.

WALSTAD, J.D., ANDERSON, R.F., STAMBAUGH, W.J. 1970: Effect of environmental conditions on two species of muscardine Fungi (Beauveria bassiana and Metarhizium anisopliae). J.Invertebr. Path., 16: 221-226.

2.5 Microbial Insecticides Based on Verticillium Lecanii

Verticillium lecanii is a well documented entomopathogen of insect order Homoptera, most commonly aphids, scale insects and whiteflies in tropical and subtropical regions. Also, V. lecanii sometimes hyperparasitizes phytopathogenic fungi, mostly rusts and powdery mildews.

Laboratory culture is possible on all conventional mycological media, but the best growth is on Sabouraud`s agar (Khalil at al.1983). On solid media, conidia are produced and in submerged culture are produced blastospores. Colonial growth rate was optimal at 23^oC - 24^oC. Both germination and growth declined steeply above 25^oC and ceased above 30^oC. Sporulation responded over slightly narrower temperature range than growth or germination, ceasing at 30^oC.

Virtually all fungi require humidity for spore germination, growth and sporulation. Thus, to ensure maximum germination of spores and hence highest possible levels of infection of insects, spore sprays should be synchronized with optimal humidity, which for most crops should occur in the evening as ambient temperature falls.

Virulence of V. lecanii spores can be measured by bioassay LD₅₀ for a 100 % viability conidia suspension in which adult, apterous Macrosiphoniella sanborni are immersed briefly in to the concentrations ca. 10⁵ spores/ml and that blastospores is slightly lower. However, when untreated aphids are placed on still wet spore-treated chrysanthemum leaves, the LD₅₀ is increased by a factor of 100. Furthermore, after drying, spores are preasumatly not readily dislodged from the leaves by the aphids. This suggests that a prophylactic spore-spray applied to an aphid-free crop may be wasted and ineffective when aphids invade.

Virulence of single and multi-spore isolates of the strain remained remarkably stable on most artificial media. Passaging of V. lecanii through an aphid host according Hall (1981) did not increase virulence, but there are many data, according Passaging through an aphid host or white fly increased virulents.

The half-life of conidia in distilled water varies, both at 2^oC (110 - 160 days) and -17^oC (60-120 days), blastospores on the whole are even shorter-lived and more variable (100-150 days at 2^oC). When conidia were equilibria at a range of humidities at 20^oC, only high humidity permitted good survival. In contrast, dried conidia (whether in slime-heads separated from their parent mycelium or washed) at 58 % R.H. died in less than 24 hours. However, conidia in slime heads still attached to the parent mycelium on aphids or on culture mycelium (without agar) survived for up to 13 days at 58 R.H. A favorable microclimate humidity was probably responsible for good survival of spores on aphid bodies killed by V. lecanii in glasshouses; 80/90 of conidia survived for at least 30 days after death of aphids despite daytime air temperatures of well over the upper temperature limit for growth.

Fungal spores in slime/heads adhere firmly to the mycelium when dry V. lecanii conidia did not become airborne from dried cultures or from V. lecanii killed aphids. Presumably, infection of a new aphid population on a new crop originates from soil.

Control of Aphids and Scale Insect 0.02 % Triton X-100 or Tween 20 in the same concentration are used as wetting agent with concentration (5 x 10⁷ blastospores/ml). Concentrations (10⁷-10⁸ blastospores/ml) died 2-3 times faster (LD₅₀ 1.9-2.3 days) than those treated with a lower concentration, 10⁵ spores/ml (LD₅₀ 4.8 - 6.2 days). Aphis gossypii is controlled within 14 days.

Once the spore has attached to the insect, it must germinated to produce a germ tube which will then penetrated the host cuticle. However, highly pathogenic strains germinate quicker and penetrate the epicuticle directly whilst strains of low pathogenicity took longer to germinate and grew extensively over the cuticle surface with only limited penetration. Virulence of V. lecanii has been associated with high extracelluar chitinase activity.

Most entomopathogenics are also able to excrete toxins; mycelium of V. lecanii can contain a cyclodepsipeptide.

The choice of infectious material is between conidia and blastospores. Production of conidia on agar is too expensive and it is also difficult to ensure culture purity. Alternatively, conidia can be produced on a cheap granular solid media such as grain, jack seeds (Arthocarpus) pearl millet or potato extract for 5-6 days in aerated vessels.

Kybal and Vlèek (1976) used polyethylene cushions made of large thin walled, polyethylene tubing sealed into sections which were partially filled (1 cm high layer absolutely horizontal) with submerged culture of V. lecanii after 2 + 1 days cultivation and inflated with sterile air. Product was harvested (ca. after 14-16 days) by discarding the medium and retaining the mad. Yield from a 0.8 % peptone, 1 % sorbitol medium was 1 x 10^12-13 conidia/1 m².

Harvesting of mud is done by mixing it with Siloxyd (ca. 100-125 g/ 1 liter of mad and wetting agent (Tween 80) and, after predrying at 30^oC one day, the wet cake is crushed using a meat-mincer, and the product if dry to perfection at 30^oC can be stored or milled (Condux Universal Mühle Typ 150/S-D) and packaged like water-dispersable powders which is prepared for dilution with water into a final spray. The application dose per hectare or meter square is determined depending of the conidia content.

Despite theoretical strain stability, a commercial product must be shown to have constant potency. This should be measured by viable spore count and bioassay. The viability of V. lecannii conidia and blastospores can easily be assessed by an agar-slide technique (Hall, 1976).

Pathogenicity of production batches of spores should be measured by bioassay in comparison with a standard since variation between consecutive assays is often significant.

V. lecanii is a clearly promising biological control agent against aphids, scales, thrips under glass, humid tropic and as mycofungicide against rust diseases.

The absence of records of V. lecanii in man and other vertebrates is an impressive evidence for its innocuity. All V. lecanii strains so far examined by Hall (1981) cannot grow at 37^oC and so the likelihood of infecting warm-blooded vertebrates internally is exceedingly remote. As reaction to the dose of 10⁶ conidia injected intravenously, no adverse symptoms were observed and, 28 days later, no gross pathological changes were apparent in the internal organs and no signs of the fungus could be found either in sectioned organs or in agar cultures from these. Safety tests are being carried out preparatory to commercial exploitation.

In the field, critical parameters like temperature and humidity cannot be readily manipulated. Temperate weather is often unpredictable and often unfavourable to fungi, except perhaps where the pest occupies a moist micro-climate. However, great potential may exist in tropical and subtropical zones where high humidity is normal for prolonged periods.

Verticillium lecanii strains were first introduced commercially in the U.K. for the control of aphids "Vertalex" and

whitefly "Mycotal" on protected ornamental and vegetable crops. The products were discontinued in 1986.

HALL, R.A.,1976: A bioassay of the pathogenicity of Verticillium lecanii conidiospores on the aphid Macrosiphoniella sanborni. Journal of Inv.Pathology 27: 41-48.

HALL, R.A., 1981: The fungus Verticillium lecanii as a Microbial Insecticide against Aphids and Scales. In: Microbial Control of Pests and Plant Deseases 1970-1980. Acad. Press 1981, Ed. Burges H.D., p.482-498.

KHALIL, S.K., BARTOS, J. and TABORSKY, V. 1983: Effect of temperature, pH of the medium and sugar on the germination of spores, development of mycelium and sporulation of the entomopathogenic fungus, Verticilium lecanii (Zimm.) Viìgas. Agric. Trop. et Subtr., Universitas Agriculture Praga, 16: 255 - 274.

KYBAL, J. and VLEEK, V. 1976: A simple Device for Stationary Cultivation of Microorganisms. Biotechnol. Bioeng. 18: 1713 - 1718.

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