2. ELEC 230 (2+1):BIOPESTICIDES AND
BIO FERTILIZERS
Course In-Charge
Mr.S.Srinivasnaik
Assistant Professor
Department of Entomology
Lecture No.4
MICROBIAL BIO PESTICIDES: BACTERIA, VIRUSES,
FUNGI, NEMATODES, PROTOZOA & RICKETTSIAE
4. MICROBIAL CONTROL
Microbial control
“Microbial control refers to the exploitation of diseases causing
organisms to reduce the population of insect pests below the
economic injury level
Entomopathogens
Word derived from two Greek words
“Entomon” - Insects
“Genes” - Arising In
Therefore, the etymological meaning of
entomogenous microorganism is “microorganisms which arise
in insects.”
5.
6. 1.ENTOMOPATHOGENIC BACTERIA
Bacteria are unicellular organisms, small in size and lack
defined nucleus. Two categories of bacteria have been noted.
Those with rigid cell wall are spherical (coocal), rod (bacilli) or
spiral (spirilla) shaped.
The other category lacks rigid cell wall and is called
pleomorphic (mollicutes). Bacteria occur in regular and irregular
aggregations, may develop chains or packets of individual cells
and may be motile.
Bacteria reproduce by binary fission (asexual mode) and
conjugation (sexual mode). They develop aerobically in the
presence of oxygen or in its absence anaerobically.
7.
8.
9. HISTORY-ENTOMOPATHOGENIC BACTERIA
The Bacillus thuringiensis Berliner story began in the first
decade of the 20th Century when the Japanese bacteriologist S.
Ishiwata isolated the bacillus from diseased Bombyx mori (L.)
larvae.
He named it Sottokin, which means "sudden death bacillus."
He described the pathology it causes in silkworm larvae and its
cultural characteristics
10.
11. HISTORY-ENTOMOPATHOGENIC BACTERIA
He also noted that many of the larvae that did not die when
exposed to the bacillus were very weak and stunted.
In a subsequent report (Ishiwata 1905b) he stated that "From
these experiments the intoxication seems to be caused by some
toxine, not only because of the alimentation of bacillus, the death
occurs before the multiplication of the bacillus..."
This showed that from the very beginning it was realized that a
toxin was involved in the pathogenicity of B. thuringiensis
12. Ernst Berliner isolated a similar organism from diseased
granary populations of Ephestia kuehniella (Zeller) larvae from
Thuringia, Germany, which he named Bacillus thuringiensis,
and because Ishiwata did not formally describe the organism he
found, Berliner is credited with naming it.
HISTORY-ENTOMOPATHOGENIC BACTERIA
13.
14.
15. Aoki & Chigasaki (1916) reported on their studies of
Ishiwata's isolate, noting that its activity was due to a toxin present
in sporulated cultures, but not in young cultures of vegetative
cells.
The toxin was not an exotoxin because it was not found in
culture filtrates. It is obvious from their data on inactivation of the
toxin by acids, phenol, mercuric chloride, and heat that they had a
protein
HISTORY-ENTOMOPATHOGENIC BACTERIA
16. Berliner's isolate was lost, but in 1927 Mattes reisolated the
same organism from the same host as did Berliner (Heimpel &
Angus 1960a).
Mattes' isolate was widely distributed to laboratories in various
parts of the world, and most of the early commercial B.
thuringiensis-based products and most of the early microbial
control attempts used this isolate (Norris 1970).
Both Berliner and Mattes observed in addition to the spore, a
second body, which they called a Restkörper in the developing
sporangia.
HISTORY-ENTOMOPATHOGENIC BACTERIA
17. CLASSIFICATION OF BIOPESTICIDES
Cutter Laboratories then produced B. thuringiensis
preparations for Steinhaus which he used successfully against
Colias eurytheme larvae (Briggs 1986).
In 1956 Steinhaus and R. A. Fisher met with the president of
Pacific Yeast Products, J.M. Sudarsky, to explore the
practicality of producing a B. thuringiensis-based product. Pacific
Yeast Products was a yeast and vitamin B-12 producer in Wasco,
CA. The decision was made to produce B. thuringiensis and by
1957 a product called Thuricide was available for testing.
Thuricide was formulated as liquid concentrates, dusts, and
wettable powders.
18. Several other U.S. Companies (Merck, Agritrol; Rohm &
Haas, Bakthane; and Grain Producers, Parasporine) produced
B. thuringiensis for short periods (van der Geest & van der Laan
1971).
Besides the production of Sporeine in France in the late
1930s, there was the development of B. thuringiensis production
and usage in European socialist countries in the 1950s.
Angus, Hannay and Fitz James alkalinity of soluble proteins
determined the toxicity of the crystals in 1955
Goldberg and margalit found B. thuringiensis israelensis in
mosquito breeding pond and negev desert which highly toxic to
the mosquitoes and black flies during 1970s
HISTORY-ENTOMOPATHOGENIC BACTERIA
19.
20. Schenepf and whitely first time identified the insecticidal
activity of crystal proteins and first cloning of the Bt sub sp,
kurstaki in E.cloi in 1981
Kreig found Bacillus thuringiensis tenebrionis effective against
meal worm: Tenebrio molitor in 1983
Crickmore given the classification of the crystal proteins
based on the amino acid homology
HISTORY-ENTOMOPATHOGENIC BACTERIA
21. Bacteremia : Occurs when the bacteria multiplies in the insect
haemocoel without the production of toxins.
Observed frequently with symbionts and rarely
with insect pathogenic forms
Septicemia : It occurs frequently in insect pathogenic bacteria
that invade the haemocoel, multiply and produce
the toxins. These bacteria generally kill the
insects
Toxaemia : Occurs when the bacteria produce the toxins and
the bacteria is usually confined to the gut lumen
Bacterial infections in insects are broadly classified into:
SYMPTOMS AND PATHOLOGIES
23. TYPES OF E.BACTERIA
Family Species
Bacillaceae Bacillus cereus
Bacillus thuringiensis subspecies
Israeliensis, thuringiensis, alesti, sotto,
kurstaki, galleria
Bacillus sphaericus
Bacillus popilliae
Bacillus lentimorbus
Pseudomonadaceae Serratia marcescens
Pseudomonas sp
Vibrionaceae Aeromonas
Streptococcaceae Sterptococcus apis European foul brood)
Streptococcus faecalis
Types of entomopathogenic bacteria
The insect pathogenic bacteria occur in the following families
24. CLASSIFICATION OF E.BACTERIA
A. Spore producers: Two types:
i) Obligate: Bacillus popillae
ii) Facultative: Two types:
a) Crystalliferous: Bacillus thuringiensis
b) Non-Crystalliferous: Bacillus cereus
B. Non spore producers: Pseudomonas spp.
25. TARGET INSECT PESTS
1. Bacillus thuringiensis sub sp. kurstaki, sotto, aizwai,
entomocidus and berliner : Lepidoptera
2. Bacillus thuringiensis sub sp.israelensis, galleriae:
Mosquito larvae
3. Bacillus thuringiensis sub sp.tenebrionis: Coleoptera
4. Bacillus popilliae: Japanese beetle larvae, Popillia japonica
(Milky disease)
26.
27.
28. INSECITICIDE PROTEIN TYPES-ENDOTOXINS
S.No. Toxin Shape and molecular weight Target pest
1. Cry I Bipyrimidal, 130-140 kDa Lepidoptera
2. Cry II Cuboidal, 65-71 kDa Lepidoptera and Diptera
3. Cry III Rhomboidal, 73 kDa Coleoptera
4. Cry IV Polypeptides, 135, 128, 74 and
72 kDa
Diptera
5. Cry V 80 kDa Coleoptera & Lepidoptera
6. Cry VI - Nematodes
29.
30.
31.
32.
33.
34.
35. ENTOMOPATHOGENIC VIRUS
The etymology of the term virus is from Latin meaning slimy
liquid, poison or stench.
Matthews (1991) defined virus “A virus is a set of one or
more nucleic acid template molecules, normally encased in a
protective coat or coats of protein or lipoprotein, that is able
to organize its own replication only within suitable host
cells”.
Viruses are sub microscopic, obligate, intracellular
pathogenic entities. These are pathogenic arthropods belongs at
least 11 families
36. ENTOMOPATHOGENIC VIRUS
Viruses in the family Baculoviridae are the best known of all the
insect viruses because the disease symptoms are easily
recognised and they have the potential for development as
microbial insecticides. Baculoviruses are the double stranded
DNA viruses having bacilliform or rod shaped virions
37. HISTORY OF ENTOMOPATHOGENIC VIRUS
Historically the first symptom of virus was observed in silk
worm in 16th century. Bergold in 1947 given the definitive nature
the viral disease in insects. ELCAR is the first commercial product
from Heliothis zea/ Spodoptera litura NPV.
38. CLASSIFICATION OF ENTOMOPATHOGENIC VIRUS
S.No. Family Genetic material Shape Example
1 Ascoviridae dsDNA Allantoid -
2 Baculoviridae dsDNA Bacilliform NPV & GV
3 Calciviridae ssRNA Isometric -
4 Iridoviridae dsDNA Isometric -
5 Nodaviridae ssRNA Isometric -
6 Parvoviridae ssDNA Isometric -
7 Picornaviridae ssRNA Isometric -
8 PloyDNAviridae dsDNA Ovoid -
9 Poxviridae dsDNA Ovoid/spheroid -
10 Reoviridae dsRNA Isometric CPV
11 Rhabdaviridae ssRNA Helical -
According to the ICNV (International Commission on
Nomenclature of Viruses) there are 11 families comes under
entomopathogenic virus category
39.
40.
41.
42.
43. ENTOMOPATHOGENIC VIRUS
Important sub groups within families are NPV Nuclear
Polyhedrosis Virus (NPV) and Granulosis Virus( GV).
The grasserie of silkworm was a good French descriptor of
nuclear polyhedrosis virus (NPV) (Baculoviridae) infection which
resulted in liquefaction and disintegration of the affected insects.
The NPV of nun moth (Lymantria monacha) causes changes in
infected larvae that gives rise to aberrant behaviour involving
larvae climbing upwards to die in the topmost branches of trees.
This was described in German as wipfelkrankheit or tree top
disease or caterpillar wilt.
45. ENTOMOPATHOGENIC VIRUS
Nuclear Polyhedrosis Virus (NPV)
•Occluded (rod shaped) singly/in groups in polyhedral (many
sides) inclusion bodies
•Site of multiplication is cell nucleus of epidermis, fat bodies, blood
cells and trachea
Granulosis Virus (GV)
•Virions (Oval or egg shaped) are occluded singly in small
inclusion bodies called capsules
•Site of multiplication is either cytoplasm or nucleus of epidermis,
tracheae and fat body
Cytoplasmic Polyhedrosis Virus (CPV)
•Spherical virions occluded singly in polyhedral inclusion bodies
Site of multiplication is cytoplasm of midgut epithelium
47. SYMPTOMS
Dosage
•250-500 LE (1 LE=6x109Poly Occlusion Bodies). 1 LE POBs can
be harvested from 3 matured caterpillars.
•250LE=1.2X 1012 POBs
•500 LE=1.5X1012 POBs
49. ENTOMOPATHOGENIC FUNGI
Entomopathogenic fungi are important regulators that are present
naturally to control the pest population. Myco-biocontrol offers an
attractive alternative to the use of chemical pesticides. It is
naturally occurring organisms which cause less damaging to the
environment. Entomopathogenic fungi are first organisms to be
used for the biological control of pests. More than 700 species of
these fungi from around 90 genera are pathogenic to insects.
Disease caused by fungus is called ‘Mycosis’
50. ENTOMOPATHOGENIC FUNGI
•2700bc: Chinese people recognise diseases of honey bee and
silkworm
•Ancient Time : Indian literature refers the diseases of same
insects at the same time in Europe Aristotle was the first person
mention about the diseases of honey bees
•1835: Agostino bessi experiment on silk worm disease
•1879 : E. metschinikoff (1879) experiment control of wheat
cockchafer (Anisoplia austriacea), sugarbeet weevil (Cleomus
punctiventris)
51. ENTOMOPATHOGENIC FUNGI
•2700bc: Chinese people recognise diseases of honey bee and
silkworm
•Ancient Time : Indian literature refers the diseases of same
insects at the same time in Europe Aristotle was the first person
mention about the diseases of honey bees
•1835: Agostino bessi experiment on silk worm disease
•1879 : E. metschinikoff (1879) experiment control of wheat
cockchafer (Anisoplia austriacea), sugarbeet weevil (Cleomus
punctiventris)
52. ENTOMOPATHOGENIC FUNGI
1. Beauveria bassiana / White Muscardine Fungus
2. Metarhizium anisopliea / Green Muscardine Fungus
3. Verticillium lecanii / White Halo Fungus (Recent name is
Lecanicillium lecanii)
4. Nomuraea rileyi
5. Paecilomyces fumoroseus
6. Hirsutella thomsonii
53. MODE OF ACTION-ENTOMOPATHOGENIC FUNGI
•Adhesion of fungal infective units or conidium to the insect
epicuticle
•Germination of infective units on cuticle
•Penetration of the cuticle
•Multiplication in the haemolymph
•Death of the host (Nutritional deficiency , destruction of tissues
and releasing toxins) Mycelial growth with invasion of all host
organs
•Penetration of hyphae from the interior through the cuticle to
exterior of the insect
•Production of infective conidia on the exterior of the insect. Most
of the entomopathogenic fungi infect their hosts by penetration of
the cuticle by producing cuticle digesting enzymes (Proteases ,
lipases chitinases).The typical symptoms of fungal infection are,
mummified body of insects and it does not disintegrate in water
and body covered with filamentous mycelium
54.
55.
56. TOXINS
Entomopathogenic fungi Toxin produced
Beauveria bassiana Beauvericin
Beauverolides
Bassinolide
Metarhizium anisopliae Destruxins A,B,C,D,E,F
Paecilomyces fumoroseus Beauvericin
Verticillium lecanii Similar to Bassinolide
57. SYMPTOMS
1. Beauveria bassiana
•Soft and breakable
•Dried and giving milky liquid
2. Nomouraea rileyi
•Yellow to brown spots on the integument
•Swelling of posterior abdominal segments
•Covered with pale green spores
3. Metarhizium anisopliae
Mummified
Hard
Covered olive green powdery mass of spores
4. Verticillium lecanii
Mummified
Hard
Covered filamentous white hyphae
58. SUCCESSFUL MULTIPLICATION
1. Fungal Isolate
•Rapid growth
•High pathogenesis
•To target pests
•Sporulate profuse
2. Medium should be cheap and easily available
3.The production procedure should be easy and production
cost low
4. Formulation should have long shelf life and no loss of
infectivity up to 12-18 months
59. ENTOMOPATHOGENIC NEMATODES
Nematodes, commonly referred to as
roundworms, eelworms, or
threadworms, are translucent, usually
elongate, and more or less cylindrical
throughout their body length.
The body is covered by a non cellular
elastic cuticle that differs chemically from
the chitinous cuticle of arthropods.
Nematodes have excretory, nervous,
digestive, reproductive, and muscular
systems but lack circulatory and
respiratory systems.
61. PORTALS OF ENTRY
•The majority of protozoa enter the insects by way of the mouth
and digestive tract. Penetration through the integument occurs in
the ciliates.
•Those protozoa that remain in the lumen of the digestive tract are
attached to the epithelium, or enter appendages associated with
the digestive tract and generally cause no obvious pathology.
These forms are mainly ciliates, flagellates, and gregarines.
Others penetrate into the haemocoel and exist extracellular in the
hemolymph or intracellularly within the cells of various tissues and
organs, and cause pathologies.
•They are mainly the apicomplexans and microsporidia.
62. ENTOMOPATHOGENIC NEMATODES
Representatives of this family offer much promist as biological
control agents because of their high virulence and broad host
range.
Steinernema carpocapsae, for example, kills its hosts within 48
h and will infect many insect species in the laboratory and field.
A number of Steinernema species have been described from
natural infections of insects, and all have a mutualistic association
with bacteria. The bacteria, in the genus Xenorhabdus have been
studied in great detail.
63. ENTOMOPATHOGENIC NEMATODES
Heterorhabditids have a similar life cycle to the steinernematids,
but major differences also exist. Infective juveniles, which invade
the haemocoel, release the bacterium Photorhabdus
luminescens, killing the host within 48 h, and reach adulthood
rapidly.
64. ENTOMOPATHOGENIC PROTOZOA
The protozoa ("first animals") are a heterogeneous group of
microorganisms of very diverse characters, behaviour and life
cycles. The protozoa, because of their minute size, remained
unobserved until the development of the microscope. Anton van
Leeuwenhoek (1632-1723), who produced lenses and built
microscopes, discovered free-living, fresh-water protozoa (Dobell,
1932). From the descriptions of the animalcules provided by van
Leeuwenhoek, Dobell (1932) believes that he also observed
coccidians in cats and flagellates in the digestive tract of the
horse fly (tabanid). Van Leeuwenhoek is generally recognized
as the father of protozoology for this observations on numerous
protozoa
65. TAXONOMIC POSITION
Taxa Representative genera
Phylum Apicomplexa
Class Gregarinia
Order Eugregarinida Gregarina, Ascogregarina
Order Microsporida Mattesia, Farinocystis,
Ophryocystis
66. TRANSMISSION
•Vertical transmission from parent to offspring occurs in many
protozoa, especially the microporidia.
•The transmission is transovum by way of the ovary
(transovarial) or by surface-contaminated eggs.
•The egg surfaces are contaminated from spore-containing faces
of females with protozoan – infected digestive tracts.
•These types of transovum transmissions are in addition to the
common per os route, and their significance, as a means of
vertical transmission, varies greatly with the protozoa and their
hosts.
•Transmission through surface-contaminated eggs is probably not
important in regulating insect populations, but transovarial
transmission is often highly significant in the transmission of
microsporidia
67. PATHOGENESIS, SIGNS , SYMPTOMS
•Most entomopathogenic protozoa have low virulence and cause a chronic
infection that often does not kill an insect. Such a chronically infected insect
frequently does not exhibit marked external signs and symptoms (e.g., color
changes and abnormal movement or behaviour).
•The enormous numbers of protozoan spores in the fat, midgut, or hemolymph
may cause these structures to turn milky white. The integument of dead
insects (mainly larvae) generally remains firm and does not readily disintegrate.
•The intercellular forms usually occur in the cytoplasm. No toxins have been
detected in protozoan infections in insects, but Weiser (1961) has suggested
that toxins may be produced by microsporidia that cause tumorlike growths
and inflammatory responses in insects.
•Some protozoa exhibit tissue tropism and infect only certain tissues or organs
(e.g., certain microsporidia and neogregarines infect only midgut epithelium or
fat tissues). Others invade nearly all major tissues and organs to cause a
systemic infection.
•The members of class Microsporea are commonly called microsporidia. The
disease they cause is called microsporidiosis.
68. HOSTS
About 700 species have been recorded from these hosts. Insects
in nearly all taxonomic orders are susceptible to microsporidia and
over half of the hosts occur in two orders, Lepidoptera and
Diptera.
•Nosema locustae (trade name: Nolo bait): Grasshoppers and
desert locusts
•Varimorpha necatrix-Noctuid pests
•Nosema bombycis:Bombyx mori
•Malpighamoeba locusate :Grasshoppers
•Farinocystis triboli:Tribolium casataneum
Virulence: debilitative pathogen: it kills the insect in 30 days
indirectly
69. ENTOMOPATHOGENIC RICKETTSIAE
Intermediate between bacteria and virus. Vago and Martuja
identified the specificity of Rickettsiae grylli against crickets
and Rickettsiae gregaria against Locusta migratoria.
Diseases
1. Lorsch disease: Rickettsiae melolanthe on lamellicorn beetle
2. Blue disease: Rickettsiae popilliae on Japanese beetle,
Popillia japonica
Virulence: debilitative pathogen: it kills the insect in 30-90
days indirectly
