MICROBIAL CONTROL

MICROBIAL CONTROL

Defination: Microbial control refers to the exploitation of disease causing organisms(viruses, bacteria, fungi, protozoa, rickettsia, mycoplasma and nematodes) to reduce the population of insects pests below the damaging levels. Steinhaus (1949) Coined the term ‘Microbial Control’ when microbial organisms or other products (toxins) are employed by man for the control of pests on plants, animals or man.

            Insect diseases and their symptoms have been recognized as far back as 2700 BC in China with the honeybee, Apis mellifera and silkworm, Bombyx mori. In Europe, Aristotle was the first to mention that the bees suffered from diseases. In 1835, Agostino Bassi published his great work on muscardine disease of silkworm.  A. Bassi is called the Father of Insect Pathology. A Russian Entomologist, Metschnikoff, conducted the first systematic experiments on the control on the control of injurious insects with microorganisms by infecting grubs of the grain beetle with the green muscardine fungus, Metarrhizium anisopliae.

Characteristic of an ideal microbial insecticide:

Ø  It should be economical for  mass production

Ø  It should be available in formulations which should have long shelf life, remain stable in the target insect and disseminate quickly and uniformly.

Ø  It should be safe to non-target organisms and man

Ø  It should ensure consistent suppression of pest populations to acceptable low densities

Terminology:

Potential pathogens are those organisms that are incapable of invading the host, either through the body wall or through the digestive tract, without assistance of external factors that lower the insect’s resistance or enhance the ability of the microorganism to invade the insect.

Facultative pathogens are those microorganisms that do not require an insect weakened by external factors to cause infection

Obligate pathogens require living insect hosts for survival and replication.

Infectivity is the ability of a microorganism to produce infection.

Virulence is the disease producing power of a microorganism.

Pathogenecity refers to the disease producing power of a group of species of microorganism.

Viruses

Virus are submicroscopic, obligate, intracellular, pathogenic entities, normally encased in a protective coat or coats of protein or lipoprotein, that is able to organize its own replication only within the suitable host cells.

Insect pathogenic viruses belong at least 11 families among with baculoviruses is most important.

Baculoviruses are double-standard DNA viruses having baciliform or rod shaped virions. The two most important subgroups within the family are the nuclear polyhedrosis virsus (NPV) and granulosis virsus (GV).

Mode of infection of NPV: Upon ingestion by a larva, the protective polyhedron coat of the virus is dissolved in the midgut (Alkaline gut juice). The virions cause primary infection in midgut cells, which in turn produce the budded virus responsible for spreading the infection to other tissues like fat bodies, epidermis, tracheal cells, muscles, gonads etc. The virus particles infect the blood cells, tracheal matrix or epidermal cells. Inside these cells, they enter the nuclei, attach themselves to the chromatin and multiply. Eventually, the nuclear and cell memberane rupture, releasing the polyhydra into body cavity to invade other cells. After 5-7 days of infection the symptoms are

Symptoms:

Ø  The insect stop feeding, become sluggish and pale in colour,  swells slightly and then become limp and flaccid

Ø  The integument becomes fragile, ruptures easily to emit blood with disintegrate tissues and polyhydra

Ø  Prior to death, insect climb to higher positions and the dead larvae usually hang by their prolegs. This is called tree top diseases or ‘wipfel krankeit’

Ø  Dry up to a dark brown cadaver

Cytoplasmic polyhedrosis viruses (CPV):

The CPV viruses attack the columnar cells of the mid gut epithelium. The infection is restricted to the midgut but at best spread to the foregut and midgut. The polyhedra develop in the cytoplasm where they enlarge to lose their polyhedral shape and become spherical.

Symptoms:

Ø  Small size of the larvae

Ø  Disproportionately large heads

Ø  Loss of appetite

Granulosis Viruses:

It inhibit the fat body, epidermis, frequently blood cells and the tracheal matrix. Yellowing of skin is generally observed on the ventral side. The infected tissues eventually disintegrate and the body fluid becomes filled with inclusions. If the skin is ruptured, a whitish liquid oozes out.

Host range: Lepidioptera, Hymenoptera, Diptera and Coleoptera. A granulosis virus isolated from codling moth (Cydia pomonella) and Indian meal moth (Plodia interpunctella) is effective against codling moth and Indian meal moth.

Examples of the exploitation of NPVs for pest contrl:

Ø  Field trials conducted in Maharastra on chickpea have revealed that two sprays of NPV of Helicoverpa armigera at the rate of 500LE/ha were as effective as two sprays of endosulfan (0.05%) in reducing the number of insects/plant and pod damage and increasing the yield.

Ø  In Tamil Nadu, upto 81.3 to 71.6% reduction in larval populations of H. armigera was obtained on chickpea, when NPV was used @ 250 and 125LE/ha respectively.

Ø  In India a number of field trials have also been conducted for the management of H. armigera on tomato, sunflower, cotton, g.nut etc.

Ø  NPV combined with sub lethal doses of quinalphos or dichlorvos inflicted 60-80 % mortality of 5^th and 6^th instar larvae of Amsacta sp.

Ø  Other insects which have been controlled with NPVs with varying degrees of success include Spilosoma oblique, Achaea janata and Oryctes rhinoceros

What is larval equivalent (LE): It is the average number of PIB found in a larva. One LE for H. armigera is 6 x 10⁹ PIB

Commercially available viral pesticides:

Virin-HS(H. armigera), SPOD-X(S. exigua), Spodopterin (S.littoralis)

Limitation of the use of baculoviruses in pest management:

Ø  Lack of quick knockdown effect and take about one week to cause mortality

Ø  They are easily inactivated by sunlight and ultraviolet rays

Ø  They are highly host specific and each species of insect will require specific virus of its own

Ø  In general, the early instar of the insects are more susceptible and hence require the correct timing of application.

Advantage:

Ø  They are safe to non-target organisms, humans and environment

Ø  They can be applied easily using methods similar to those employed for application of chemical pesticides

Tolerance of baculoviruses to ultraviolet radiations be increased:

Ø  Various additives/adjuvants like charcoal, Indian ink, egg albumin, molasses etc. may be used along with the formulations

Ø  The viral preparations should preferably be applied in the field in the evening

           

The sporeforming or sporulating bacteria form endospores and producing bacterial cell on being ingested. Amongst the sporeformers again the crystalliferous due to its toxic (delta-endotoxin) producing ability are more promising in insect pest control. One of the best studied species amongst the crystalliferous bacteria, Bacillus thuringensis which was also the first to be regarded as a potential microbial control.

            The protein crystal or the parasporal body is formed only when sporulation is to take place and not before in the vegetative cell. The crystal are highly toxic to a majority of the lepidopterans. The crystal toxic are referred to as endotoxin.

B. thuringiensis is a gram positive, rod-shaped aerobic, endospore forming soil bacterium and produces a protenaceous parasporal crystal in the sporangium at the time of sporulation. B. thuringiensis was first isolated in 1902 from diseased larvae of silkworm, Bombyx mori in Japan by Ishiwata. However, Berliner who reisolated it from the Mediterranean flour moth in Thuringia, Germany first gave the name B. thuringiensis in 1905.

Mode of Action: On being ingested by the susceptible host, the endospore germinates in the gut (alkaline pH<9), producing the bacterial cell. The cell migrates into the haemocoel where they multiply rapidly and destroy certain tissues and soon fill much of the haemocoel cause septisemia and finally death of insect. Ex. Ostrinia nubilalis

            In insects (Caterpillars) having a highly alkaline gut (pH>9), the endospores fails to germinate and only the protein crystal is effective. The proteolytic enzymes working at that pH dissolve the crystal releasing their endotoxin, which cause paralysis of gut and mouth parts, leading to cessation of feeding, regurgitation and diarrhea followed by general body paralysis. Blockade of the nerve conduction, precipitation of blood proteins, inhibition of enzyme and finally insect die. Ex. Bombyx mori

Symptoms:

• Stop feeding, regurgitation and diarrhea to gut paralysis followed by general body paralysis
• Body becomes soft and dried to a scale
• The tissues get disintegrated giving a foul smell
• The body usually becomes darkened in colour due to dark body fluid.

Bt based product marketed in India are Halt, Biolep, Dipel, Delfin, Bioasp, Biobit, Spicturin etc.

Host Range of Bt : Lepidoptera, Coleoptera, Diptera, Hymenoptera, Isoptera, Orthoptera, Hemiptera and Neuroptera

Classification of Insecticidal Crystal Proteins:

Five major classes are

1.                                                            Cry I: Lepidoptera

2.                                                            Cry II: Lepidoptera and Diptera

3.                                                            Cry III : Coleoptera

4.                                                            Cry IV : Diptera

5.                                                            Cry V : Coleoptera and Lepidoptera

Problems with the use of Bacterial pathogens:

• Have slow knockdown effect and may take long time to kill insect
• They are generally specific and have narrow host range
• Rapidly inactivated by sun light and washed by rain as such repeated application may be necessary
• Effective against foliage feeder only

Milky Disease: The bacteria which attck beetles of family, Scarabidae, B. popillae causes milky disease in Japanese beetle, Popillae japonica. It produces endospores which upon ingestion by a susceptible host germinate in the gut and the vegetative cells invade into the hemocoel where they multiply. When vegetative forms becomes numerous sporulation begins. The blood becomes milky white, hence the name milky disease. Doon is promising against white grub infesting ground nut.

Field Efficacy:

Bacillus thuringienis var kurstaki effective against Lepidoptera

Bacillus thuringienis var galleriaeeffective against Lepidoptera

Bacillus thuringienis var israelensis effective against Diptera

Bacillus thuringienis var tenebrionis effective against Coleoptera

Bacillus popilliae effective against Japanese beetle

Bacillus sphaericus effective against mosquitoes and black flies

Bt has been recommended for the suppression of Plutella xylostella and pieris brassicae (0.75 kg a.i/ha) on cabbage. Crocidolmia binotalis on cauliflower, H. armigera on tomato, tobacco, sunflower and pulses.

Spodoptera litura on tobacco, Papilio demoleuson citrus, Amsacta moorei on pulses and oil seed. Moderate to high level of control was also achived in case of a number of pests including cotton bollworm, rice stem borere, brinjal fruit and shoot borere and cabbage semilooper.  Thr DBM, Plutella xylostella is the most notable because it evolved high levels of resistance in the field as a result of repeated use of Bt.

FUNGI

Major classes to which entomogenous fungi belongs: phycomycetes, Ascomycetes, Basidiomycetes and deuteromycetes (Ex: Beauveria, Metarrhizium, Aspergillus etc.). Fungi provide only satisfactory microbial means of biocontrol of sucking insects such as aphid, whitefly, which are not susceptible to bacteria and virus.

Mode of Infection:

• The most common root of host invasion is through the external integuments.
• The infective stage in fungi is a spore, usually a conidium
• Under favourable conditions the conidium attach to the cuticle, germinate to a short germ tube and penetrate the cuticle.
• The penetration is both mechanical (pressure exerted by germ tube) and enzymatic through the action of proteinase, lipase and chitinase on the cuticle.
• In the haemocoel, the mycelium remifies throughout the host, forming yeast like hyphal bodies and death of the host.
• Host death is often due to a combination of the action of a fungal toxin, chocking of tissues, physical obstruction of blood circulation and nutrition depletion.
• Death generally occurs about 1-2 days later depending on dose, temp and larval age.
• Under suitable environmental condition, death is followed by external sporulation, which helps to spread the fungus and establish an epizootic which may result in very high levels of kill.

Symptoms:

• Losses of appetite and an attempt to climb higher are the early symptoms
• General or partial body paralysis
• Discoloured patch on integuments
• The body gets hardened and the insect is upright on its legs at the time of death.

Toxin produced by fungi:

• B. bassinae: Beauvericin, Beauverolides, Bassianolide
• Metarrhizium anisopliae: Destruxins A, B, C, D, E and F
• Aspergillus ssp: Aflatoxin
• Aspergillus ochraceus: Ochratoxin
• Streptomyces moberaensis: Piericidin A and Piericidin A
• Paecilomyces: Beauvericin
• Codyceps militaris: Cordycepin

Commercial formulation of diff. fungi  and Field Efficacy:

Micro-organism	Trade Name	Effective against
B. bassinae	Naturalis-L, Mycotrol	sucking peest
	Conidia, Ostrinol	Borers
Metarrhizium anisopliae	Bio-1020, Bio-path	Black vine weevil
	Bio- Blast	Termites
Verticillium lecanii	Verticillin, Mycotal	whiteflies and thrips
Paecelomyces fumosorosus	PFRE-97, Pre Feral	whiteflies and thrips

Limitation:

• Fungi need a high degree of atmospheric humidity to germinate. So fungi are not effective in dry and or cold conditions. So need to apply in fairly warm and humid conditions
• Fungal pathogens are not always spp. specific and may also attack predators and parasitoids of the pest.
• The maintainance of virulency is often a limiting factors.

Potential:

• Have wide scope for utilization of insect pathogenic fungi particularly on pests of rice, cole crops and other irrigated crops as the high humidity is most congenial for their multiplication.
• The pathogens makes the pest sick and make more sucsceptible to pesticides.

Host range and field efficacy: Hemiptera, Diptera, Coleoptera, Lepidoptera, Orthoptera and Hymenoptera

• Beauveria bassiana(white muscardine fungus): Having largest host range. Effective against colarado potato beetle, Helicoverpa armigera, mango mealu bug, cotton white fly, rice BPH, GLHand stem borer etc.
• Metarhizium anisopliae (Green muscardine fungus): Effective against chrysomelid, curculionid and scarabaeid beetles. It is effective against sugarcane spittle bug, rice BPH, rhinocerous beetle, sugarcane pyrilla, DBM etc.
• Verticillium lecanii: Common pathogens for scale insects. It also infests aphids, thrips, white fly, mites and nematodes.
• Nomuraea rileyi: Pathogenic to a majority of noctuids moth. Effective against Spodoptera, Helicoverpa etc.
• Neozygites fresenii: Efficient natural pathogen of cotton aphids.

NEMATODES

            Nematodes have a symbiotic relationship with the bacteria which are released into the haemolymph of the host causing septicemia. The best known complex is known as DD-136 in which the nematode involved is Steinernema carpocapsae and the bacterium is Xenorhabdus nematophilus. The nematodes serves as a vector for the bacterium which ccauses septicemia in the insect body. When the nematode enters the insect body, the bacteria are rreleased and they multiply. The nematode ingests on both the dead host tissue and the bacteria. The bacteria are retained in nematode intestine, as the later does not feed during the free living existence. When such bacteria carrying nematodes invade fresh insect hosts they are also killed.

            In this association, the nematode is dependent on the bacterium for (1) quickly killing its insect host (ii) creating suitable environment for its development by producing antibiotics that suppress competing secondary microorganisms (iii) and transforming the host tissues into a food source. The bacterium requires the nematode for (i) protection from the external environment (ii) penetration into host hemocoel and (ii) inhibition of the hosts antibacterial proteins.

Symptoms :

Ø  Dead insect’s body discolours only slightly and remain intact and unputrified for several weeks due to antibiotics produced by bacteria.

Ø  Cuticle becomes fragile and dried to scale

Ø  The emerging nematodes are usually visible and the minute exit holes of nematodes turn black.

Importance in pest management:

            DD-136 has been applied as aqueous spray for the control of codling moth, Cydia pomonella in USA. In India foliar application of S. carpocapsae has been found to be effective against the leafminer on groundnut, Spodoptera litura on sunflower, paddy cutworm and yellow stem borer in rice.

Commercial formulation of diff. Nematodes  and Field Efficacy:

Micro-organism	Trade Name	Effective against
Steinernema carpocapsae	Bio Vector, Ecomask, Exhibit, green cooandoss	Lepidopterous larvae
Steinernema feltiae	Entonem	Diptrous insect
Steinernema scapterisci	proactant	Adult mole cricket
Heterrhabditis bacteriophora	Heteromask	Oil dwelling insect

Limitation:

• Intolerance or mortality of the free living stages of nematodes to low humidity and extreme temperatures.
• Nematodes need to be protected from U.V light which soon reduces infectivity and may kill the infective stages.

Potential:

The attributes which make nematodes as ideal biological control agents include their broad host range, high virulence, safety to non-target organisms, ability to search hosts, ease of production and application and compatibility with other control tactics.

Protozoa:

            Portal entry of the protozoan is mainly through the gut by ingestion of spores along with contaminated food but less frequently it could be congenitally through transovum transmission or even through infected parasites (by ovipositors) and predators (by mandibles). Crowdling of insects encourages the spread of pathogens.

Symptoms:

• The body becomes soft, breakable and may dry to scale that may yield a milky mass in water
• The white masses irregularly distributed in the fat body becomes visible in aquatic larvae.

Importance in pest management:

• Nosema fumiferae is being exploited for the management of spruce budworm.
• Nosema locustae, the only registered material and is marketed for the control of grass hopper.
• Vairimorpha necatrix infects a number of important lepidopterous peasts.

Limitation:

• The protozoa breed only in vivo (i.e, in host) and are difficult to culture on artificial media.
• Protozoa are generally non-specific in nature
• They are slow acting and hence not suitable for short term control programmes which demand quick results.

Advantage:

• They reduce pest populations to a level that permits control by other means
• They reduce fecundity of the host by their devitalizing effect and thus help the spread of the disease by not killing the hosts outrightly.

Micrbial insecticides aree not popular with farmers:

Ø  Because a single microbial insecticide is toxic to only a specific or group of inseeects, each application may control only a portion of the pests present in a field.

Ø  Heat, desiccation or exposure to UV radiations reduces the effectiveness of several types of microbial insecticides.

Ø  Because several microbial insecticides are pest specific, the potential market for these products may be limited.

Ø  The fficacy of microbial pathogens is dependent on a wide range of environmental factors.

Ø  The relatively slow sspeed with which microorganism kill their hosts has hampered their effectiveness.

Ø  It is necessary to maintain the virulence and viability of the pathogens till use.

Ø  Special formulation and storage procedures ar necessary for some microbial pesticides.

Ø  Proper timing and application procedures are especially important for some products.