lecture-biopesticides [compatibility mode].pdf

8/10/2019 Lecture-Biopesticides [Compatibility Mode].pdf

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Biopesticides with special reference to Bt toxin

A P = (bacteria, fungi, plant,animal)

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Losses caused by different Pests

O 18% (A : R .60,000 C )

: P P , P C , I

37%

12%

29%

22%

Insects

Rodents & Others

Weeds

Diseases

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Pest management methods

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85% A .


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Development of pesticide resistance - insects andweeds.(insect resistance -nearly 200 species).Disrupt the natural ecosystem and natural biodiversity.Hazardous effect on the non-target, useful arthropods. Theykill beneficial insects and plants (non selective).Pollution runoff of herbicides and insecticides into

irrigation water and then into rivers - damages wildlifehabitat, kills fish.Reappearance of residues in things of human use, Residueson crops and soilCause cancer organophosphatesBioaccumulation

Concerns regarding use of chemical pesticides


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Bio-Pesticides

Bio-pesticides are certain types of pesticidesderived from natural material such as animals,plants, bacteria, and certain minerals.

Many bio-pesticides are less toxic and pose alower risk than conventional pesticides which

can be quite toxic.

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(Copping, 2004)

Insecticide, 50Herbicide, 12

Fungicide, 35

Acaricide, 1 Bactericide, 2Fungicide, 35 Fungicide and bactericide, 2Fungicide and nematicide, 1 Fungicide and plant growth regulator, 1Herbicide, 12 Insecticide, 50Insecticide and acaricide, 1 MolluscicideNematicide, 5 Plant growth promoter, 1

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Bio-pesticidesLimit : Contact products so adequate coverage is essential to

have a good efficacy. Often time consuming

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Use of Biopesticides in Weed Management

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Microbial management of weed

Commercialname

Pathogen Target weed

ABG5003 Cercospora rodmani Water hyacinth

BioChon Chondrostereum purpureum Prunus serotina

DeVine Phytophthora palmivora Milkweed vine

BioMal Colletotrichum gloeosporioides Malva pusilla

Camperico Xanthomonas campestris Zoysia tenuifolia

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Effect of Alternaria alternata on waterhyacinth


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Phytotoxins from microbes

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Mycotoxin Pathogen Target weed

AAL-Toxin Alternaria alternata Jimson weed

Colletotrichin Colletotrichum sp. Solanum sp.

Curvulin Drechslera indica Spiny amaranth

MoniliforminFusarium

moniliformeSpiny amaranth

Anisomysin Streptomyces sp.Barnyard grass

and crab grass

Use of Biopesticides in Insect Pest Management

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Microbial control of insects

Microbial insecticides are comprised ofmicroscopic living organisms (viruses, bacteria,fungi, protozoa, or nematodes) or the toxinsproduced by these organisms.

They are formulated to be applied as conventional

insecticidal sprays, dusts, liquid drenches, liquidconcentrates, wettable powders, or granules.

Each product's specific properties determine theways in which it can be used most effectively.

Some entomogenous nematodes have characteristics that allow them to be

considered with the pathogens.

The most important insect pathogenic nematodes for biological control are verysmall and use mutualistic bacteria to kill the host.

Eutectona machaeralis, pest of teak


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Although nematode species in at least 20 families are primary or facultativeparasites of insects, those in the order Rhabditida have been most exploited asbiological control agents.

Species in the genera Steinernema and Heterorhabditi s (Steinernematidae andHeterorhabditae, respectively), are particularly amenable to mass production andapplication in a variety of pest systems.

Entomopathogenic nematodes enter the host via natural body openings orthrough the cuticle.

Some species utilize an anterior stylet or a tooth to rasp the cuticle and gainentrance into the hemocoel.

Others ingress by ovipositing on the host food source and the eggs hatch in thehost midgut.

Effects of nematode parasitism on the hosts can be sterility, reduced fecundity,reduced mobility and life span, behavioural and morphological changes, and death.

The non-feeding, developmentally arrested infective juvenile seeks outinsect hosts and initiates infections.

When a host has been located, the nematodes penetrate into the insectbody cavity, usually via natural body openings (mouth, anus, spiracles) orareas of thin cuticle.

Once in the body cavity, a symbiotic bacterium ( Xenorhabdus for

steinernematids, Photorhabdus for heterorhabditids) is released from thenematode gut, which multiplies rapidly and causes rapid insect death.

The nematodes feed upon the bacteria and liquefying host, and matureinto adults. Steinernematid infective juveniles may become males orfemales, where as heterorhabditids develop into self-fertilizinghermaphrodites although subsequent generations within a host producemales and females as well.


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The life cycle is completed in a few days, and hundreds ofthousands of new infective juveniles emerge in search of freshhosts.Thus, entomopathogenic nematodes are a nematode-bacteriumcomplex.The nematode may appear as little more than a biological syringefor its bacterial partner, yet the relationship between theseorganisms is one of classic mutualism.

Nematode growth and reproduction depend upon conditionsestablished in the host cadaver by the bacterium.The bacterium further contributes anti-immune proteins to assistthe nematode in overcoming host defenses, and anti-microbialsthat suppress colonization of the cadaver by competing secondaryinvaders.Conversely, the bacterium lacks invasive powers and isdependent upon the nematode to locate and penetrate suitablehosts.


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Insects infected with Steinernema nematodes are usually light tan incolor.

Note the adults (larger nematodes)and the infective juveniles (the tinynematodes forming a cloud around thegrub.

Insects infected withHeterorhabditis nematodes are usuallya reddish color.

Of some 14,000 described species of Protozoa, about 500 are pathogens of

insects. Many are chronic pathogens that may debilitate a host without producingobvious disease symptoms but some species are extremely virulent, causingstunted growth, slow development, and early death.

Entry into the host is typically by ingestion, but some can invade through thecuticle.

Some species may be transovarially transmitted from infected females to theiroffspring.

Species that invade the cells of the host are usually found in the cell cytoplasmand are typically more pathogenic than extracellular species.

Some protozoans exhibit tissue tropism , infecting only certain tissues or organs,others are systemic.

No toxins have been found to be associated with protozoa in insects.

Death or debilitation of infected hosts may be, for example, the result ofcompetition for metabolites, disruption of normal cell and tissue function, orblockage of the gut or other organs by extracellular species.

The insect-pathogenic Protozoa are currently recorded from four major groups:Amoebas, Gregarines, Flagellates and Ciliates.


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Viral diseases have been found in 13 insect orders and most likely occur in allorders. Viruses that are primarily or exclusively found in insects are currentlyplaced in 12 families (Miller, 1998):

DNA Viruses: Baculoviruses (Nuclear polyhedrosis viruses- NPV andGranuloviruses-GV), Ascoviruses, Iridoviruses, Parvoviruses, Polydnavirusesand Poxviruses.

RNA Viruses: Reoviruses (Cytoplasmic polyhedrosis viruses), Nodaviruses,Picorna-like viruses and Tetraviruses.

they do not infect vertebrates, non-arthropod invertebrates, microorganisms orplants.

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Table 16.7

Entomopathogenic fungi are able to invade their insect hosts by

penetrating directly through the cuticle.

The fungal spore first adheres to the cuticle.

Under appropriate conditions the spore germinates, penetrates thecuticle of the host and enters the hemocoel.

Fungal reproduction occurs in the hemocoel of the insect host.

As the hemocoel becomes filled with hyphal bodies, the insectusually dies and the fungus continues to develop saprophytically.

After the body of the dead insect is filled with mycelia, fruitingstructures emerge from the cadaver and produce infectious spores.

Dead insect has the consistency of a moist loaf of bread and,depending on the colour of the spores or conidia, may appear whiteor some darker colour.


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Tanada and Kaya (1993) listed 8 classes, 13 orders and 57 genera that containentomopathogenic species of fungi.

There are five major groups of fungi: the Flagellate fungi or Chytridiomycetes, theOomycetes (also flagellate but also not true fungi), the Zygomycetes, theAscomycetes, and the Basidiomycota.

The Zygomycota and the Ascomycota contain common insect pathogens that arealso useful in biological control programs.

Examples of Entomopathogenic fungi are:1. Metarrhizium anisopliae 2. Beauveria bassiana 3. Entomophthora spp.


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Entomopathogenic Fungi Beauveria spp. "White" Fungus

Metarhizium spp. "Green" Fungus

Beauveria bassiana is called the white fungus of insectsbecause most of the strains produce external spores thatmake the infected insect appear coated with a white powderor cottony material.

Metarhizium is usually called the green fungus of insects.The fungus has white mycelia within the body, but when it isready to form spores, the spores coat the killed host with avelvety covering of olive-green.

A bluegrass billbug adult (above) andJapanese beetle larva (right) infected withBeauveria .


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Green June beetlegrubs killed byMetarhizium .

Japanese beetle grubinfected withMetarhizium .

They can be divided into two broad categories, non-spore-forming bacteria and spore-forming bacteria.

Although most of the species isolated from diseasedinsects are non-spore-forming, spore-forming bacteria inthe genus Bacillus are the most important from thestandpoint of biological control since they are resistant to

environmental changes.Among the spore formers crystalliferous ones are better

than non-crystalliferous ones because of the toxic natureof crystals they produce.


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Serratia

Obligate

Entomogenous Bacteria

Facultative

Sporeformers Non-sporeformers

B. thringensis

Non-crystaliferous

Paenibacillus popilliae

Clostridium sp. Aerobacter sp.

Crystalliferous

FacultativePotential

ExExEx Ex

B. cereus Pseudomonas sp.

Ex

Ex. 1: Milky disease bacterium, Paenibacillus popilliae , onwhite grubs of Japanese beetles There are numerous bacteria that can infect and kill insects.

The most common ones encountered in landscapes are theones that kill white grubs milky disease bacteria.

Spores of Paenibacillus popilliae (formerly Bacillus popilliae )infect larvae of Japanese beetles ( Popillia japonica ).

The milky disease bacterium, Paenibacillus popilliae , causesthe blood of white grubs to turn a milky white color. Eventually,the bacteria overwhelm the grub.

Though milky disease spores are commercially available,there is little evidence that use of such products actuallyincrease infection over the long run.


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The adult Japanese beetles

Milky disease bacterium, Paenibacillus popilliae

It is a soil-dwelling, gram-positive, spore forming, rod-shapedbacterium. It is responsible for a disease (commonly called milkyspore ) of the white grubs of Japanese beetles.

Milky Spore in the soil is not harmful to beneficial insects, birds,bees, pets or man

Milky Spore, like other bacteria, is highly survivable in cold anddrought conditions.


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Milky disease bacterium, Paenibacillus popilliae ,on white grubs of Japanese beetles


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Normal grub (left) and a milky disease infected grub (right). Note colorof blood droplet where the tip of the leg was pinched off.

Resident spores in the soil are swallowed by grubs during their normalpattern of feeding on roots. This ingestion of the spore by the hostactivates reproduction of the bacteria inside the grub. Within 721 daysthe grub will eventually die and as the grub decomposes, billions of new spores are released into the soil.

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Ex. 2: Bacillus thuringiensis

A Short History of Bacillus Thuringiensis (BT)

Bt is a naturally occurring soil bacterium.

It was first detected in 1902 in the dying larvaeof Bombyx mori by Ishiwata,

he reported his finding in the book: "Pathologyof the Silkworm".

It was first isolated from the larvae of Ephestia kuehniella by Berliner in 1911 afterhe noted that it had the capacity to kill certaininsects in their larva stage.

Ephestia kuehniella


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Ex. 2: Bacillus thuringiensis

A Short History of Bacillus Thuringiensis (BT)

It was regarded as potential biocontrol agent bySteinhaus in 1956.

1st commercial product containing this bacilluswas available in USA in 1958.

Natural Bt is highly specific, with toxicitylimited to only some species of one of themajor groups of insectstypically Lepidoptera(butterflies/moths), Coleoptera (beetles),hymenoptera (wasps, bees, ants) or Diptera(flies/ mosquitoes).

Scientific classification of Bacillus thuringiensis


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Characteristics of BT

Bt subspecies can synthesize more than one parasporal inclusion.The parasporal inclusions are formed by different insecticidalcrystal proteins (ICP).

The crystals have various shapes (bipyramidal, cuboidal, flat,rhomboid, spherical or composite with crystal types), depending ontheir ICP composition.

During sporulation many Bt strains produce crystal proteins(proteinaceous inclusions), called Cry proteins which are encodedby cry genes, and have insecticidal action. This has lead to their useas insecticides and more recently to genetically modified cropsusing Bt genes.

In most strains of B. thuringiensis the cry genes are located on theplasmid.

Different domains of the ICP are responsible for host susceptibility(receptor recognition) and toxicity (pore formation).

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Normal gut bacteria

BT SPORES


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1. The Bacillus thuringiensis is only effective when eaten byspecific family of insects with a specific (usually alkaline) gutpH and the specific gut membrane structures required to bindthe toxin. (typically butterflies, moths, beetles, flies andmosquitoes).

2. Not only must the insect have the correct and be at asusceptible stage of development , but the bacterium must beeaten in sufficient quantity.

3. When ingested by a susceptible insect, the spores feed on naturalintestinal flora then it burst releasing the protein toxin(Crystalline protein) damaging the gut lining (the intestinalwalls), leading to a kind of leaky gut condition.

4. Affected insects stop feeding and die from the combinedeffects of starvation, tissue damage and gastrointestinalinfections by other pathogens like bacteria and funguses.

5. The natural Bt spores do not usually spread to other insectsor cause disease outbreaks on their own as occurs with manypathogens.


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Figure 16.1

B. thuringienis parasporal crystal composed of Cry1protoxin protein. Conversion of the 130-kDa protoxininto an active 68-kKa toxin requires an alkalineenvironment (pH 7.5 to 8) and the action of a specificprotease, both of which are found in the insect gut.The activated toxin binds to protein receptors on theinsect gut epithelial cells.

Figure 16.3The toxin is inserted in gut epithelial cell membranes of theinsect and forms an ion channel between the cell cytoplasmand the external environment, leading to loss of cellular ATPand insect death.


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NOTES:Technically, the bacterium, Bacillus thuringiensis (Bt), is not atrue biological control. This is because the bacterium doesnot cause an infection within the insect. The bacteria actuallygrows in soils. However, many strains of this bacteriumcontain a protein toxin crystal that is released if the bacteriumis digested. The protein toxin destroys the insect gut liningwhich causes a secondary infection or starving of the insect.

Many strains of Bt are known and only a few have been foundto have insect killing properties.


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Bacillus thuringiensis Bt -endotoxins

Variety Target Examples

Bt. kurstaki Bt. aizawai Bt. israelensis Bt. tenebrionis

(=san diego )Bt. japonensis

(strain 'Buibui')

caterpillarscaterpillarsmosquitoesleaf beetles

scarab grubs

Dipel, MVP Mattch Vectobac M-one

M-press

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Strain/subsp. Protein size Target Insects Cry #berliner 130-140 kDa Lepidoptera CryIkurstaki KTP, HD1 130-140 kDa Lepidoptera CryIentomocidus 6.01 130-140 kDa Lepidoptera CryI

aizawai 7.29 130-140 kDa Lepidoptera CryIaizawai IC 1 135 kDa Lepidoptera, Diptera CryIIkurstaki HD-1 71 kDa Lepidoptera, Diptera CryIItenebrionis (sd) 66-73 kDa Coleoptera CryIIImorrisoni PG14 125-145 kDa Diptera CryIVisraelensis 68 kDa Diptera CryIV


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Change in pest Scenario

a. Excessive and indiscriminate use of pesticidesb. Improper choice, quantity and application of pesticides.c. Use of pesticide mixtures

(Has lead to)

a. Resurgence of minor pests

b. Resistance to pesticides

c. Increase in cost of protection

Constraints of Cotton Production

Cotton also carries environmental controversy, particularly in the developingworld, where dangerous pesticides are heavily employed.

Pesticides compromise as much as 50% of the costs of cultivation for manyfarmers and the increasing use of pesticides has led to high levels of debt andbankruptcy among poorer farmers

An alternative to increased pesticide use is the encouragement of naturalpredators of the bollworm, including ladybirds, lacewings, damsel bugs, nightstalking spiders, common brown earwigs, fire ants, muc wasps, and thetrichogramma wasp.

Pest traps, spot treatment and organic pesticides are also more sustainablealternatives.

A more common alternative today is to turn to genetically-modified cropsproduced by companies such as Monsanto. The most common species, Bt orBacillus thuringiensis, is advertised as fully resistant to the common bollworm.Early use shows that this is true, as such large portions of cotton production inthe US and China (20 and 30% respectively) now use Bt strains.

Bt Cotton seeds were introduced by Bollgard Cotton, a trademark of theMonsanto group. Bt Cotton was first introduced to the U.S. in 1996


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