lecture 1 : introduction and types and importance of
TRANSCRIPT
ELE PATH 243
LECTURE 1 : INTRODUCTION AND TYPES AND IMPORTANCE OF BIOFERTILIZERS,
BIOPESTICIDES AND BIOAGENTS IN AGRICULTURE AND ORGANIC FARMING SYSTEM
Introduction :
Although Green Revolution resulted in increased food production many fold but also
resulted in increased demand for fertilizers and agricultural chemicals. Modern agriculture
based on extensive cultivation, use of chemical fertilizers, high yielding varieties responsive
to nutrients and use of plant protection chemicals and its application increase day by day.
Due to discriminate use of fertilizers and pesticides have not only causes pollution of
natural resources but also created ecological imbalance between components of ecosystem.
Discriminate use of pesticides result in lowering down of efficiency and increase resistance
to pesticides in various crop pests.
Hence for sustainable agriculture, use of minimum inputs like fertilizers, pesticides
by adopting technologies which enhances natural reactions without disturbing the
ecological imbalance by maintaining soil-plant-animal-microbes balance.
Biofertilizers :
Biofertilizers is the product that containing living cells of effective strains of different
microorganisms which have an ability to mobilize nutritionally important elements from
non-available to available form through biological process.
Types of Biofertilizers :
A] Nitrogen Fixers : 1) Symbiotic Nitrogen Fixer – Rhizobium.
2) Non-Symbiotic Nitrogen Fixer – Azotobacter, Acetobacter.
3) Associatively Symbiotically Nitrogen Fixer – Azospirillum.
4) Azolla Symbiont Nitrogen Fixer with Anabaena azollae, Azolla
pinnata.
5) Blue Green Algae – It is also called as Cyanobacteria (free living
organism Nostoc and Anabaena.
B] Phosphate Solubilizers : Phosphate solubilising microorganisms,
1) Bacteria – Pseudomonas striata, Bacillus megatarium var. phosphaticum
Bacillus polymyzxa.
2) Fungi – Aspergillus awamari, Penicillium digitatum, Phosphate absorber,
Vesicular Arbuscular Mycorrhiza (VAM) fungi like Glomus and
Gigaspora species.
3) Actinomycetes – Streptomyces spp. , and Nocardia spp.
C] Sulphur Oxidizers : Thiobacillus thiooxidans.
D] Organic Matter Decomposer :
1) Bacteria – Cellulomonas folia, Bacillus stearothermophilus.
2) Fungi – Aspergillus niger, Rhizopus nigricans, Trichoderma viride.
3) Actinomycetes – Micromonospora vulgaris, Streptomyces thermofucus.
E] Potash Solubilizers : Fraturia aurantia, Bacillus mucilaginosus.
F] Silicate Solubilizers : A virulent silicate solubilizing bacterium (Si SOLB) of Bacillus spp.
Types of Bioagent :
1. Parasites –
a) Egg parasites : e.g. Trichogramma jopanicum – Rice Leaf Folder.
Trichogramma chilonis – Root Borer.
Trichogramma minutum – Sugarcane Borer.
b) Larval parasites : e.g. Bracon greeni – Cotton Bollworm.
c) Adult parasites : e.g. Epiricania melanoleuca – Sugarcane Pyrilla.
2. Predators –
1. Cryptolaemus : Mealy Bugs
e.g. Lady Bird Beetles had feed on Aphids, Mealy Bugs and White flies.
2. Chrysoperla : Jassids and White flies
Lacewing larva feeds on aphids, syrphid maggot feeds on aphids.
3. Zygogramma : Parthenium weeds
Some reptiles, insectivorous birds are regular feeder but they require suitable
conditions to grow and breed profusely.
Biopesticides :
Biopesticides is a population of pathogenic microorganisms that are antagonistic to
particular pest and provide natural control are called Biopesticides or Microbial Pesticides.
A] Viral Pesticides :-
1. Nuclear Polyhedrosis Virus (NPV) – They develop in insects cell nuclei and control
pest like cotton bollworm and tobacco budworm.
2. Cytoplasmic Polyhedrosis Virus (CPV) – They develop in the insect’s cell
cytoplasm and control pests like caterpillar.
3. Granulosis Virus (GV) – They develop either in nuclei or cytoplasm of insects cell
and control pests like sugarcane shoot borer.
B] Bacterial Pesticides :-
1. Bacillus thuringiensis var. kurstaki – Cotton Bollworms, Fruit Borer of Tomato and
Brinjal.
2. Bacillus papillae – Coleoptera – white grub.
3. Bacillus moritai – Diptera.
C] Fungal Pesticides :-
1. Beauveria bassiana – Sugarcane / Maize Stalk Borer.
2. Metarhizium anisopliae – Sugarcane pyrilla.
3. Verticillium lecani – Mealy bugs of Cotton, Papaya.
Importance in Agriculture and Organic Farming :
• Biofertilizers helps in the establishments and growth of crop plants and trees.
• They enhance biomass production and yield by 10 to 20 %.
• They are useful in sustainable agriculture.
• They are inputs containing microorganisms which are capable of mobilizing nutritive
element from non-available to available form through biological process.
• They are less expensive, eco-friendly and sustainable.
• Biofertilizers on application remain in soil, multiply and keep benefitting to the
growing crops. They do not get depleted as in case of chemical fertilizers and
therefore if the optimum soil conditions prevails, population of added microbes
build up continuously and no need of frequent application of biofertilizers.
• Biocontrol is exercised in a wide range of area and is safe for human and animal
health.
• Application of bioagents is easy and possible in inaccessible areas like dense crops
and tall trees.
• The bioagents survive in nature till the pest is prevalent and in absence of pest they
self precautions in nature.
• For bioagents farmers does not require any special treatment procedure expect
microbial formulations.
• No waiting period for harvesting a crop after release of bioagents or spraying of
biopesticides.
• Bioagents/Parasitoid/Predators may be multiplied at farmer level as these do not
require sophisticated instruments.
LECTURE 2 : HISTORY OF BIOFERTILIZER PRODUCTION.
Although the beneficial effects of legumes in improving soil fertility was known since
ancient times. But the field of the Biological Nitrogen Fixation opened with following
discoveries :
➢ Boussingault and Hellreigel (1886) – shown biological nitrogen fixation in legume
crops.
➢ Nobbe and Hiltner (1895) – Produced for the first time a labortary culture of
Rhizobia under the name “Nitragin”.
➢ Later vigorous research for other ‘N’ fixing microorganisms began and found that
therewere other non-symbiotic bacteria such as Azotobacter which could fix
atmospheric nitrogen.
➢ Then it was discovered that Blue Green Algae also fixed atmospheric nitrogen.
➢ After that in 1970 new group of bacteria Azospirillum was identified.
➢ Vesicular Arbuscular Mycorrhizae (VAM) are the latest introduction in the list of
biofertilizers which mobilize phosphorus. Some Important Research, Production and
Promotion of Biofertilizers in India.
➢ N. V. Joshi (1920) – first study on legume Rhizobium Symbiosis.
➢ M. R. Madhok (1934) – documented production of Rhizobium Inoculant.
➢ In 1956, first commercial production of biofertilizer.
➢ Sen and Pal (1957) – Study on solubilization of phosphorus by microorganisms.
➢ In 1964, Spurt in demand of Biofertilizer for Soyabean crops in Madhya Pradesh.
➢ V. Ishwaran (1970) – Shown use of charcoal, lignite and FYM as alternative carrier to
peat soil.
➢ In 1977, Use of ISI mark for Rhizobium Production.
➢ In 1983, Ministry of Agriculture, Government of India setting up of National Project
on Development and Use of Biofertilizers.
➢ In 1986, At IARI, New Delhi setting up of National Facility for Blue Green Algae by
Department of Biotechnology.
➢ In 1990, At IARI, New Delhi setting up of National Facility for Collection of Rhizobium
germplasm by Division of Microbiology, Department of Biotechnology.
LECTURE 3 : CLASSIFICATION OF BIOFERTILIZER
Classification of Biofertilizer :
A] Classification based on microorganisms used for management of Biological Fertility :
➢ Free Living :- (Non-Symbiotic Association)
This group includes many species of Blue Green Algae and Bacteria Azotobacter.
➢ Symbiotic Association :-
The bacteria Rhizobium live symbiotically on the roots of legumes by forming
nodules. Azolla fixes atmospheric nitrogen in symbiotic association with BGA (Blue
Green Algae).
➢ Associative Symbiotic Association :-
Azospirillum has associative symbiosis with higher plant root system.
B] Classification based on types of microorganisms :
Biofertilizers
Bacterial
Nitrogen Fixers
Symbiotic
e.g. Rhizobium
Non-Symbiotic
e.g. Azotobacter
Assciative
e.g. Azospirillum,
Acetobacter
Phosphate Solubilizers
Non-Symbiotic
e.g. Bacillus
Pseudomonas
Algal
Nitrogen Fixers
Non-Symbiotic
e.g. Blue Green Algae (BGA)
Fungal
Phosphate Solubilizer
Symbiotic
Mycorrhizae
e.g. Glomuse
Gigaspora
Non-Symbiotic
e.g. Aspergillus,
Penicillum
Actinomycetes
e.g. Frankia
LECTURE 5 : A STUDY OF GROWTH CHARACTERISTICS OF VARIOUS MICROBES USED IN
BIOFERTILIZER PRODUCTION.
A] Rhizobium :-
➢ Rhizobium is a genus of Gram Negative soil bacteria that fix nitrogen.
➢ Rhizobium species form an endosymbiotic nitrogen fixing association with roots of
legumes and parasponia.
➢ Rhizobium fixes atmospheric nitrogen symbiotically with legumes.
➢ Legumes can obtain their nitrogen from the atmosphere via the activity of nitrogen
fixing bacteria of the genus Rhizobium.
➢ Rhizobium colonizes the roots of specific legumes to form tumour like growth called
root nodules. Nodules act as a factories of Ammonia production.
➢ Rhizobium is abundant in soil and all of them not able to nodulate all types of
legumes.
➢ On the basis of ability of rhizobium to form effective nodules with specific legumes
are called Cross Inoculation.
➢ There are seven cross inoculation groups.
➢ With the formation of bacteroids a red pigment haemoglobin accumulate between
the bacteroids is called “Leghaemoglobin”.
➢ The amount of leghaemoglobin and bacteroid production had direct relations with
amount of N2 fixed by legumes.
➢ The process of N2 fixation is wholly dependent on the activity of enzyme
“Nitrogenase” which is present in bacteroids.
B] Azotobacter :-
➢ Based on morphological and physiological characters genus Azotobacter has been
classified into three types species –
Azotobacter chracoccum
Azotobacter beijrinekii
Azotobacter vinelandii
➢ Among these species first two are most commonly occurring species in India.
However, A.chrococcum found in acid soils while A. beijrinekii found in neutral to
alkali soils.
➢ Azotobacter not only provides the nitrogen but produce variety of growth promoting
substances like gibberllins, vitamin B and antifungal substances.
➢ Azotobacter has also ability to produce compounds against pathogen like fusarium
and alternaria.
➢ Due to these characteristics of azotobacter there was increased germination and
vigour of young plants.
➢ Another important characteristics of Azotobacter associated with crop improvement
is excretion of Ammonia in the rhizosphere in the presence of root exudates and
helps in nutrient uptake by plant.
➢ The results from larger number of experiments conducted in last four decades have
shown positive response to Azotobacter application on a crop like cereals, millets,
vegetables, cotton and sugarcane and increased the crop yield by 10 to 30%
C] Azospirillum :-
➢ Azospirillum an associative microphillic nitrogen fixer commonly found in association
with roots of cereals and grasses.
➢ High N2 fixation capacity, low energy requirement and abundant establishment in
the roots of cereals and tolerance to the high soil temperature (30 to 40° C).
➢ Due to these characteristic of Azospirillum, they are more suitable under tropical
conditions.
➢ Due to high ability of N2 fixing and beneficial response under saline condition
maintained high nitrogenase activity.
➢ They have ability to fix atmospheric nitrogen and produce phytoharmones on
Azospirillum application observed that there was increased in yield with wide
variation i.e. 10 to 64%.
➢ Most common species is Azospirillum brasilence is used and when applied with
Azotobacter chrococcum production synergistic effect on yield of maize, sorghum
and barley.
D] Blue Green Algae (BGA) :-
➢ It is also called Cyanobacteria.
➢ BGA are photosynthetic nitrogen fixing organisms passes nitrogen fixing species cells
called “Heterocysts”.
➢ This algae is suitable to rice ecosystem and its abundance in rice field and is often
referred as paddy organisms.
➢ It uses sunlight as a energy source and water as a source reducing agent for
photosynthesis and nitrogen fixation.
➢ Most of the N2 fixing BGA of rice fields are filamentous consisting of vegetable celles
including specialized called heterocysts.
➢ Heterocysts act as a micronodule for synthesis and N2 fixation BGA also synthesize
and liberate growth promoting substances such as auxin and amino acids.
➢ They supply additional humus to soil improve aeration to rice roots and reduce the
toxicity of sulphides.
E] Azolla :-
➢ Azolla is a primitive free floating water fern.
➢ There are six azolla species and out of these six species, Azolla pinnata is most
common fern in India.
➢ It is grown in a ditches and natural ponds along with other weeds.
➢ The Azolla usually forms green mate over the water which often becomes reddish
due to accumulation of anthocyanin pigment.
➢ It has an algal symbiont Anabena azolla with a central cavity.
➢ Anabena a BGA known for Nitrogen fixation is associated with aquatic weeds.
➢ Azolla growing in paddy fields upto 25 to 30 kg N/ha and recent studies indicate that
incorporations of azolla also reduces the requirement potash fertilizer to the tune of
25 to 35 %.
F] Phosphate Solubilizing Microorganisms :-
➢ Next to Nitrogen, Phosphorus is very important nutrient for plants and
microorganisms.
➢ Indian soils are poor to medium in available phosphorus status only 25 to 30 % of the
applied and phosphorus become available to crop and remaining 70 to 75%
converted into insoluble/unavailable forms.
➢ These insoluble soil phosphates are solubilized by a group of microorganisms are
called phosphate solubilizing microorganisms.
➢ These PSM producing organic and inorganic acids and phytase enzymes and
solubilize insoluble phosphate to soluble forms and make them available to crops.
➢ The PSM are bacteria, fungi and actinomycetes and they are as under :
Bacteria Fungi
Bacillus megatherium Aspergillus acva
Bacillus substilis Penicillium digitatum
Pseudomonas striata Trichoderma spp.
Pseudomonas rathonis
➢ It can also be used to enrich the compost.
➢ After the thermophillic phase of composting is over. i.e. after composting for two
months when temperature is stabilized around 30° C.
➢ The application of PSM like Aspergillus awamori improves the quality of compost.
G] Vesicular Arbuscular Mycorrhiza (VAM) :-
➢ These organisms are commonly found in association with agricultural crops, shrubs,
tropical trees.
➢ VAM is differ from PSM.
➢ VAM do not solubilize the insoluble phosphorus but assimilate phosphorus, zinc and
other nutrients for their own need i.e. for growth and reproduction and in addition
translocate them in different forms to the roots of host crops.
➢ There are 120 species of VAM and they are associated with wheat, maize, soyabean,
potato, grapes, banana, tea, sugarcane, coffee, etc. crops.
➢ These fungi formed by non-septate zygomycetous species belonging to genera
Glomus gigaspora and Acuolospora entrophospora.
➢ VAM increases root absorbing surface like phosphorus, zinc, copper, potassium,
aluminium, manganese and magnetite from the soil to the root cortex and increases
the growth of associated plants by producing auxins and antibiotics, etc.
LECTURE 6 : NITROGEN CYCLE IN NATURE AND ITS IMPORTANCE.
Atmosphere contains about 78 % nitrogen by volume. This nitrogen is in bound form
and not available to plants directly. Nitrogen is considered to be the “King Pin” of essential
nutrients and is an example of deficiency presence of 80000 tonnes in the atmosphere over
an 1 hectare land. The nitrogen present in the plants in the form of complex organic
nitrogenous materials like proteins, nucleic acids, etc. is further degraded by the microbial
activity and in the process nitrogen is again released in to the atmosphere.
As far as nitrogen fixation on earth surface through various sources, biological
nitrogen fixation contributes 67.30 % and 15.30 % contributes by Industrial fixation and
remaining contributing by lightening, combustion, etc. that means microorganisms play
very important role in nitrogen cycle.
A sequence of changes from free atmospheric nitrogen (N2) to fixed inorganic
nitrogen to simple organic compounds to complex organic compounds in the tissues of
plants, animals and microorganisms and the eventual release of this nitrogen back to the
atmosphere is called Nitrogen Cycle.
Biochemical Reactions in Nitrogen Cycle :
A] Proteolysis :-
The nitrogen present in proteins nucleic acid is in bound form and not available to
plants as nutrients. But some microbes convert proteins into amino acids with the help of
enzyme proteinases and peptidases.
Proteinases Peptidases
Proteins -------------------˃ Peptides --------------------˃ Amino Acids
Organisms involved – Pseudomonas, Bacillus, Clostridium.
B] Ammonification :-
Amino acids are further degraded by microbes by removing amino group (NH2) and end product is ammonia (NH3).
Alenine deaminase
CH3CHNH2COOH + ½ O2 -----------------------------------˃ CH3COCOOH + NH3
(Alanine Ammonia) (Pyruvic Acid) (Ammonia)
Production of ammonia from amino acids is called ammonification.
C] Nitrification :-
The oxidation of ammonia to nitrite (NO2) and further oxidation of nitrite to nitrate (NO3) is called nitrification.
2NH3 + ½ O2 --------------------- 2HNO2 + 2H2O
(Ammonia) (Nitrite)
Organisms involved – Nitrobacter, Nitrospina.
D] Nitrate Reduction :-
Nitrate is reduced to Ammonia in this process. Therefore in paddy field it is
recommended that to apply ammonical fertilizers like Ammonium sulphate or Ammonium
nitrate as Nitrogenous fertilizers instead of Urea. Because Urea is reducted to Ammonia
due to anaerobic condition existed in paddy fields due to waterlogging and to avoid leaching
losses of nitrogen present in urea, farmers suggest to directly apply ammonical nitrogenous
fertilizer for early and quick availability of nitrogen to paddy crops.
HNO3 + 4H2 ------------------------- NH3 + 3H2O
E] Denitrification :-
The transformation of nitrate to gaseous nitrogen by microorganisms in a series of
biochemical changes is called denitrification. It is an undesirable process for agriculture as
the available form of nitrogen (nitrate) is lost to the atmosphere as N2 gas which is not
directly available to plants.
2NO3 ------------ 2NO2 ------------ 2NO ------------- N2O --------------- N2
(Nitrate) (Nitrite) (Nitric Acid) (Nitrous Oxide) (Nitrogen)
LECTURE 7 : PROCESS OF NODULE FORMATION, ROLE OF NIF AND NOD GENE IN
BIOLOGICAL NITROGEN FIXATION.
➢ The role of legumes in enriching the fertility of soil by contributing nitrogen through
symbiotic nitrogen fixation by Rhizobium is well established since ancient times.
➢ Nodulation – The free living Rhizobia infects the host legume through infection
thread.
➢ Or at the point of emergence of lateral roots and are transferred into bacteroids
which are the site of Nitrogen fixation.
➢ The root cap cells secrete large amount of polysaccharides forming mucigel in which
Rhizobia are found enmeshed in large numbers.
➢ Curling and Branching of root hairs is the first visible plant response to Rhizobia.
➢ Rhizobium produced localised auxin at the root hair surface which softens the cell
wall for easy entry of infection thread for transfer of bacterial cell into root cortex.
➢ Thread grows with growth of root hair itself.
➢ Then Rhizobia get trapped with in the deformed root hair, inducing cell wall
synthesis and enclose the Rhizobia and finally forms nodule.
Mechanism Of Nitrogen Fixation :
➢ The nodule is simply a protective structure and that bacteroids are the seats of N
fixation.
➢ Nitrogen fixation is anaerobic process.
➢ In this process N2 is reduced to NH3 with the help of enzyme nitrogenase.
➢ Enzyme nitrogenase is made up of two components one with Fe + Mo and second
without Mo.
➢ The oxygen supply to bacteroids is excluded due to presence of leghaemoglobin
around it.
➢ The leghaemoglobin controlled the oxygen supply and helps in low oxygen
conditions near the bacteroids thus protect enzyme nitrogenase which is highly
sensitive to oxygen.
➢ However enough oxygen is made available at the site of ATP generation.
➢ The quantity of N fixed is closely related with amount of leghaemoglobin and
number of bacteroids present in nodules.
➢ Catalysis reaction of nitrogenous depends upon source of ATP and nitrogenous
enzyme.
➢ Products of photosynthesis mainly carbohydrates translocated from leaves to
nodules for ATP generation.
➢ The Fe component of haemoglobin is believed to be involved in binding of
nitrogenous while Mo component is responsible for decrease the strength of
Nitrogen bonds.
➢ By this strengthening and weakening process of nitrogen bonds facilitate optimum
reduction of N2 to NH3.
➢ The first intermediate compound of N fixation is ammonia.
LECTURE 9 : BIOCHEMISTRY IN NITROGEN FIXATION.
The overall biochemical reaction for nitrogen fixation can be expressed as :
Nitrogenase enzyme complex
N2 + 6H¯ + 12 ATP --------------------------------------˃ 2NH3 + 12ADP + 12P
(Atmospheric Nitrogen) (Ammonia) (Phosphate)
➢ Ammonia (NH3) is usually in the form of ammonium ions (NH+4) which is formed
when ammonia (NH3) dissolves in water.
NH3 + H2O -----------------------˃ NH+4 + OH
➢ The ammonia (NH3) is further oxidized by nitrifying autotrophic bacteria like
Nitrobacter and Nitrosomonas to Nitrate (NO3) in soil and then utilized by plants.
➢ Microorganisms which convert atmospheric molecular nitrogen (N2) into ammonia
(NH3) are known as “diazotrophs”. (Biological Nitrogen fixing organisms)
Nitrogen Enzyme Complex :
➢ The nitrogen fixation is accomplished by the presence of nitrogenase enzyme
complex present in microbes cells.
➢ It consist of two components and neither one is active without the other component
I is nitrogenase and component II is reductase.
➢ The component II is smaller and has low molecular weight and is known as Fe-
protein.
➢ Both components contain sulphur.
➢ The enzyme complex contains –
1) A strong reducing agents like Ferrodoxin or Flavodoxin.
2) Source of energy is ATP.
3) A reducing system for NH3 (ammonia).
4) A protection system for nitrogenase from molecular O2.
LECTURE 10 : CROSS INOCULATION GROUPS AMONGST RHIZOBIUM.
There are various cross inoculation groups of Rhizobium. Each group has specific
plant hosts which are colonized or nodulated by particular species of Rhizobium. Cross
inoculation grouping is based on ability of Rhizobium species to form nodules on a particular
legume species which are related to one another.
Cross Inoculation Groups
Rhizobium spp. Host it can nodulate N2 fixation kg/ha
Pea group Rhizobium leguminosarum
Pea, Lentil 62-132
Clover group R. trifolli Trifolium 130
Alfalfa group R. melioti Melilotus 100-150
Soyabean group R. jopanicum Soyabean 57-105
Cicer group Rhizobium spp. Bengal gram 75-117
Lupini group R. lupine Lupinus 70-90
Cowpea group Rhizobium spp. Mung, Redgram, Cowpea and Groundnut
57-105
Bean group R. phaseoli Bean
Summary of Biological Nitrogen Fixation System :
Sr. No.
Biological Nitrogen Fixation System
Organisms involved Nitrogen fixing capacity
1. Symbiotic Nitrogen Fixation : Organisms live in symbiotic association with the plants and fix atmospheric nitrogen. Rhizobium spp. From nodules on roots of legume crops. They derive CHO from plants and in turn make available nitrogen to plants. BGA lives in the ventral pores on dorsal lobe of leaf of Azolla BGA obtain CHO from leaves of Azolla and in turn fixes nitrogen for Azolla. The actinomycetes genus Frankia fix N2 symbiotically and like casurina, Alnus fix N2 by producing methods.
Bacteria – Rhizobium spp. BGA - Anabaena azollae Azolla pinnata Azolla indica Actinomycetes – F. alni on Alnus spp. F. elegnae on Eleghus spp. (Symbiotic nodulation in non-legume)
150 to 200 kg/ha/year
2. Non-Symbiotic Nitrogen Fixation : Organisms freely lives in soil and fix atmospheric nitrogen. They do not have symbiotic association with
Bacteria – Azotobacter chrococcuus Chroonatiuus spp.
25 to 50 kg/ha/year
plants. They derive CHO from soil and some autotrophic BGA by photosynthesis.
BGA – Anabaena spp. Nostoc spp.
3. Associatively Symbiotic Nitrogen Fixation : This stage of N2 fixation is noticed in graminaceous crops belonging to C3 to C4 type (high rate of photosynthesis and low rate of respiration). Bacteria lives in the cortex tissue of roots of crops like sorghum, pearl millet C3 and maize, sugarcane C4 and fix atmospherc nitrogen. No visible structure like nodules produced by them.
Azospirillum brasilence Azospirillum lipoferuus Acetobacter diazotrophicus
LECTURE 11 : METHODS USED FOR THE STUDYING SELECTIN OF EFFICIENT STRAIN OF
RHIZOBIUM.
Efficient strain of Rhizobium means their Nitrogen fixing ability i.e. nitrogenase
activity of Rhizobium. There are different methods of estimation of ‘N’ fixing efficiency of
Rhizobium culture.
1. By Acetylene Reduction Assay – Nitrogen Estimation.
2. By Kjeldahl Method – Nitrogen Estimation.
3. Number of effective nodule formation – By Physical Count.
4. Dry Matter Weight of Plants – On oven dry basis. Antogonistic effect again soil
borne plant pathogens.
5. Efficiency against soil borne plant pathogens – Imbition zone study in petri-dishes
under lab condition.
6. Efficiency against soil borne plant pathogens – Artificial inoculation of Rhizobium
strains and plant pathogen in sterilized soil as pot culture experiment.
LECTURE 12 : QUALITY STANDARDS FOR BIOFERLIZERS.
Indian Standard Institution (I.S.I.) now known as a Bureau of Indian Standards (B.I.S)
formulated an agriculturally useful microorganisms sectional committee specified the Indian
Standard for Rhizobium Inoculants (IS : 8268 – 1976) and Azotobacter Inoculants (IS : 9138 –
1979). These specifications are as below. ISI standards specified for Rhizobium and
Azotobacter Inoculants.
Sr. No. Parameters Rhizobium Azotobacter
1 Cell number at the time of manufacture
108/ gram of carrier within 15 days of manufacture.
107/ gram of carrier within 15 days of manufacture.
2 Cell number at the time of supply
107/ gram of carrier within 15 days before expiry date
106/ gram of carrier within 15 days before expiry date
3 Expiry Date 6 months from the date of manufacture
6 months from the date of manufacture
4 Permissible Contamination Level
No contamination at 108 dilution.
No contamination at 107
dilution.
5 pH 6.0 to 7.5 6.5 to 7.5
6 Strain Should be checked serologically.
Nothing specific.
7 Corner Should pass through 150-212 micron IS sieve.
Should pass through 160 micron IS sieve.
8 Nodulation Test Should be positive. -
9 Nitrogen fixation Above 20 mg/g of glucose. Not less than 10 mg/g of sucrose.
Quality control measures as per ISI specification. Biofertilizers should be assessed
for the following quality standards.
1) Inoculant should be carrier based or liquid based.
2) The inoculant should contain minimum of 108 viable cells of bioinoculant / gram of
carrier on dry weight basis when it is started at 25 to 30 ˚C.
3) The inoculant should have a maximum of 6 months of expiry period from
manufacture in case of carrier based and 9 months in case of liquid based.
4) The pH of inoculant should be in the range of 6.0 to 7.5.
5) Inoculant show effective nodulation/ nitrogen fixed on particular crop before expiry
date.
6) The carrier material should be in the form of powder i.e. peat, lignite, peat soil and
humus.
7) Inoculant should be packed in 50-75 micron low density polythene bags. (LDP bags)
LECTURE 13 : DIFFERENT METHODS OF APPLICATION OF BIOFERTILIZERS, BIOPESTICIDES
AND BIOAGENTS.
Biofertilizers :
There are two types of biofertilizer. a) Carrier based (powder form) biofertilizer b)
Liquid biofertilizer.
A] Application Methods of Carrier Based Biofertilizers :-
1) Seed Treatment – This method involves following steps.
➢ Preparation slurry 250 biofertilizer in 250 to 500 ml water.
➢ Pour this slurry with hands to get uniform coating of biofertilizer on seed.
➢ Dry the treated seeds in shade and then sow immediately.
2) Seedling Treatment – Generally this method is used in seedlings of transplanted crops
like chilli, vegetable seedlings, onion, etc. The seedling treatment involves following steps :
➢ Prepare solution of 1 to 2 kg biofertilizer in 10 to 15 litre of water.
➢ Dip the roots of seedlings in to the solution for 20 to 30 minutes.
➢ Transplant the seedling immediately.
3) When biofertilizer application to seed or seedlings is not possible, then soil application
method is followed. Soil application method involves following steps :
➢ Prepare the mixture of 2 to 4 kg biofertilizer in 40 to 60 kg sieved well decomposed
compost.
➢ Broadcast the mixture on acre area at the time of sowing or 24 hours before sowing.
➢ Soil application for fruit crops, 5 kg FYM + 25 gram PSB + 25 gram Azotobacter + 25
gram Trichoderma is applied per plant.
B] Application Methods of Liquid Biofertilizer :-
1) Seed Treatment –
Treatment 1 kg seed with 25 ml of liquid biofertilzers and seed are kept for 10
minutes. Then dry the seeds in shade and sow as early as possible preferably during
morning or evening hours for the seeds like cotton (hard coat) treatment should be carried
out overnight.
2) Seedling Treatment –
Root system of seedlings is to be dipped in liquid biofertilizer for 8-10 minutes so
that root system get high population of bioinoculant. The liquid biofertilizer 500 ml is
sufficient for seedling treatment of one acre.
3) Soil Application –
First 100 ml liquid biofertilizer diluted in 5 litre of water and then fix such solutions in
50 kg cowdung and 5 kg rock phosphate. Keep this mixture overnight and next day apply
the mixture over one acre.
4} Soil Pelleting –
Take 2 kg fine sieved soil sprinkle 25 ml liquid biofertilizer on it. Keep this mixture
overnight. Take about 8 to 10 kg seed and mix with mixture. Then allow the seeds to dry in
shade before sowing in the field.
5} Foliar Spray –
Dilute 3 litre liquid biofertilizer in 200 litres of water and spray the solution on one
acre crop preferably in the evening.
6} Drip Irrigation –
2 litre liquid biofertilizer is given through drip irrigation for one acre area.
Biopesticides :
A) Bacterial Pesticides :-
There are 100 species of pathogenic bacteria have been recorded from various
species of insects. Among 100 species of pathogenic bacteria following are
important in India.
1) Bacillus popilliae – soil treatment effective against Hototrichia (white grub)
infestation in Groundnut.
2) Bacillus thuringiensis var. aizurai – Effective against Diamond Black Moth in
cabbage.
3) Bacillus thuringiensis var. kurstaki – Effective against Lepidopteran insects
like Cotton bollworn, gram pod borer. Bacterial pesticides commonly used
as a foliar spray but it is important to maintain pH of spray solution at
neutral level 500 gm bacterial pesticide mix in 200 litres of water and spray
on one acre area.
B) Fungal Pesticides :-
The fungal pesticides can be applied as dusts, sprays and granular.
1) Beauveria bassiana – Mealy bugs, aphids and white flies.
2) Beauveria brongniatii – Army worm, cutworms and cabbage looper.
3) Metarhizium anisopliae – White grub, leaf hopper, pyrilla and beetles.
4) Verticillium leccani – Aphids, white flies, mealy bugs, onion thrips and leaf
hoppers.
C) Viral Pesticides :-
1) NPV used for control of cotton bollworms and tobacco cutworms.
2) CPV used for control of caterpillars on various crops.
3) GV used for control of Early Shoot Borer of Sugarcane.
Bioagents :
Augmentative biocontol in some selected crops are as under.
1) Rice :-
➢ Brown plant hopper of rice is control by release of mirid bug @ 100 bugs per
m2 or 50 to 75 egg/m2.
➢ Rice stem borer and leaf folder control by release of Trichogramma chilonis
and T. jopanicum @ 100000 eggs/ha and require 7 to 9 effective control as by
standard insecticides.
2) Sugarcane :-
➢ Early shoot borer and internode (stalk borer) control by T. chilonis release @
1,25,000 eggs/ha.
➢ Similarly for stalk borer release of comoron – Coteria flavipes, 6 release of
10000 eggs/ha at 10 days interval.
➢ Top shoot borer also control by release of larval parasitoid Isotima javensis.
➢ Early shoot borer also control by release of gravid female (Stumniopsis
inferens) @ 310 to 320 females/ha.
➢ Sugarcane pyrilla effectively controlled by release of 8000 to 10000 cocoons
or 8,00000 – 10,00000 eggs/ha of Epiricania melanuleuca.
3) Cotton :-
➢ Bollworms controlled by release of Trichogramma spp. @ 1,50,000 eggs/ha
at weekly intervals.
➢ Bollworms are also controlled by release of Green Lacewing Larvae @2
larvae/plant.
➢ Cotton sucking pest controlled by releasing of chrysopids @ 1,00000/ha at
fortnightly intervals.
4) Horticultural Crops :-
➢ Citrus mealy bugs controlled by release of exotic parasitoid Leptomastix
doctylopii.
➢ Cryptolaemus montrouzieri also found effective against mealy bugs of citrus,
grape and guava scale.
➢ Tomato and Brinjal Fruit Borer controlled by egg parasitoid @ 4000 adults/ha
LECTURE 15 : STRATEGIES OF MASS MULTIPLICATION OF BIOFERTILIZERS.
A] Carrier based Biofertilizer Production :
1) Preparation of Carrier –
Finely powdered peat, lignite or soil + compost or cellulose powder may be used as
carrier. The carrier should have following characteristics :
➢ Contained high organic matter, more than 60 %.
➢ Low soluble salt content, less than 60 %.
➢ High moisture holding capacity - 150 to 200 % by weight.
Carrier provides a nutritive medium for growth of bacteria and prolongs their
survival in culture as well as on inoculated seed. The carriers are powdered to 250 to
300 mesh about 75 micron pore size. If peat is used of 300 meshes is neutralized
with 1 % CaCO3 and sterilized at 15 PSI for 4 hours in autoclave.
2) Preparation of Inoculants in Powder Form –
In the preparation of inoculants moisture essential that the appropriate moisture
content of a specific carrier should be determined. The ideal moisture range must be set to
consider the bacterial growth, maximum population attainable after mixing, bacterial
survival and anticipated moisture loss from the package over the period of shelf life 5 – 6
months.
Usually about one part of broth (by weight) required two parts of dry carrier. Final
moisture content varies from 30 to 50 %, depending on quality of carriers. After adding
broth culture to carrier powder in 1 : 2 proportion by weight, it is kept for curing at room
temperature 28˚ C for 5 to 10 days inn 10 cm deep trays of convenient size.
After curing it is sieved to disperse the concentrated packets by breaking lumps. It is
then packed in polythene bags of 0.5 mm thickness leaving 2/3 space open for aeration of
the bacteria.
B] Liquid Biofertilizer Production :
1) Preparation of Starter Culture –
Pure culture of efficient strain of N2 fixing, phosphorus solubilizers and potash
solubilizers grown on respective agar medium in petri-plate or slants. A loopful inoculant
from petri-plates or slants is transferred in a 250 ml Liquid medium/ broth of conical flasks.
The flasks are kept on shaker at 260 rpm for 72 to 96 hrs (3 to 4 days). If shaker is not
available incubate it at 28˚ C for 5 to 6 days.
2) Preparation of Liquid Biofertilizer –
➢ Prepare liquid medium/broth for respective efficient strain of N2 fixing, phosphorus
and potash solubilizers.
➢ Cell protectants viz. trehalose dissolved separately and add in to broth before
sterilization.
➢ The sterilization of broth is to be carried out at 15 PSI for 15 minutes.
➢ Separately sterilized saturated stock solution of glucose and arabinose.
➢ This stock solution inoculated with pure starter culture at 10 ml/litre under aseptic
condition in conical flasks.
➢ Incubate the flask at 28˚ C for 2 to 5 days.
➢ Sterilize glycerol added to the inoculated broth.
➢ This broth fill up in previously sterilized polypropylene bottles and make it air tight
by screw cap.
➢ Labelled it properly with product of specific strain for which crop it is used
application instructions, expiry date and quantity required for seed treatment or
quantity required for soil application.
LECTURE 16 & 17 : REGISTRATION WITH CIB OF BIOAGENTS AND BIOPESTICIDES.
Guidelines detail for the Registration of Bioagents in India developed by Central
Insecticides Board and Registration Committee, Directorate of Plant Protection, Qaurantine
Storage. NH – IV, FARIDABAD, HARYANA.
I] Chemistry :
1 – 1 – Technical formulation.
1 – 1 – 1 – Physiochemical specification and composition.
1 – 1 – 2 – Systematic Name and Strain.
1 – 1 – 3 – Common Name.
1 – 1 – 4 – Natural occurrence of the organism (whether it is exotic or only different from
indigenously available strain.
1 – 1 – 5 – Manufacturing process.
1 – 1 – 6 – Test procedure and Criterion used for Identification.
6 – 1 – Morphology – Particle size, heat resistance, spore count, proteins per kg of
dry material.
6 – 2 – Proteins per kg of dry material – procedure required to be developed by
Lowry’s Method.
6 – 3 – ELISA ( Enzyme Linked Immuno Sorbent Assay) would have to be submitted
by the firm for Bacillus thuringiensis only.
6 – 4 – The methodology of determination of toxin content by Dot Blot ELISA assay.
1 – 1 – 7 – Method of Analysis.
7 – 1 – The protein content per Mg.
7 – 2 – Viable spore count.
7 – 3 – Determination of Toxin content by Dot Blot ELISA.
7 – 4 – Plasmid pattern if any.
7 – 5 – Technique for the separation and purification of crystals for raising antiserum
against pure crystals.
1 – 1 – 8 – Storage availability.
8 – 1 – Storage stability data has to be provided. Method of analysis will be bio-assay
method. Experimental details and calculations if any should be provided.
8 – 2 – The importer/ manufacturer has to provide the sample of technical material
for referral and storage purpose, which could be sent to Indian Institute of Microbial
Technology, Chandigarh under the Department of Biotechnology for storage and future
references.
Registration with CIB of Bioagents and Biopesticides :
II] Bioefficiency :-
2 – 1 – Technical.
2 – 1 – 1 – Labortary test for registration of microbial pesticides LC90 and LD95 various for
each test insect species under that lab conditions should be generated.
LC90 – Lethal concentration expressed in % of active ingredient and observations recorded
24 hours. After 1 hour exposure at 90 % concentration.
LD95 – Lethal dose is an indication of lethal toxicity of a given substances and killing % is 95
%. (LD50 – killing % is 50 %)
These LC90 and LD95 value’s data is required from two independent labs recognized
by ICAR/ SAO/ CSIR/ ICMR.
2 – 1 – 2 – Formulation.
2 – 1 – 2 – 1 – Lab test as above.
2 – 2 – Field Test – The data on bioefficiency based on two reasons under two
different agroclimatic conditions in the form of published authentic data along with
phytoxicity data has to be submitted.
2 – 3 – Data on persistence in soil water and plant should be submitted store count
method should be used for estimation.
2 – 4 – Data on residues by the method used for persistence to a period of 3 months
has to be submitted.
2 – 5 – Data on compatibility should not be required. However data on compatibility
will required in case it is recommended to be used in case it is recommended to be used in
mixture with some other pesticide.
2 – 6 – Time of application – Information on time of application, equipment and the
manner in which insecticides are used has to be submitted.
2 – 7 – Data on non-targeted organisms – Information on toxicity up to two species
of parasitoids and predators has to be submitted.
III] Toxicity :
3 – 1 – Single Exposure Studies Technical Formulation
3 – 1 – 1 – Oral Toxicity/ Pathogenicity Required Required
1 – 2 – Dermal Toxicity/ Pathogenicity Required Required
1 – 3 – Mucous Membrane Initiation Required Required
1 – 4 – Primary Skin Initiation Required Required
1 – 5 – Inhalation toxicity/ Pathogenicity Required Required
3 – 2 – Repeated Exposure Studies.
3 – 2 – 1 – Oral Toxicity/Pathogenicity (30 days) Required Required
2 – 2 – Dermal Toxicity/ Pathogenicity (20 days) Required Required
2 – 3 – Inhalation Toxicity/ Pathogenicity Required Required
3 – 3 – Supplementary Toxicity/ Pathogenicity
3 – 1 – Mutagenicity Required Required
3 – 2 – Teratogenicity Required Required
3 – 3 – Carrierogenicity Required Required
3 – 4 – Allergy/ Sensitization/ Immuno Suppression Required Required
3 – 4 – Eco-toxicity.
4 – 1 – Toxicity to birds Not Required Required
4 – 2 – Toxicity to fish Not Required Required
4 – 3 – Toxicity to silkworms Not Required Required
4 – 4 – Toxicity to honey bees Not Required Required
4 – Processing, Packaging and Labelling –
4 – 1 – 1 – Manufacturing/ formulation process.
4 – 1 – 1 – 1 – Raw material.
1 – 2 – Plant and machinery.
1 – 3 – Process unit operation/ unit process.
1 – 4 – Output (finished products and generation of water)
4 – 1 – 2 – Packaging.
2 – 1 – Classification of solid/ liquid/ other types.
2 – 2 – Size as per Indian Standard Specification.
2 – 3 – Compatibility – Glass bottles are not recommended.
2 – 4 – Packaging specifications/ system of packing.
I. Specification of primary pack.
II. Specification of secondary pack.
III. Specification of transport pack.
4 – 1 – 3 – Labels and Leaflets.
As per existing norms, indicating common name, composition, antidote, storage
statement, etc. The packing material should also be ensured free from contamination
during handling storage and transportation.
Note : 50 for Parasitoids and Predators ar excepted from registration.
LECTURE 18 : ROLE OF MICROORGANISMS IN DECOMPOSITION OF ORGANIC FORM
WASTE.
Decomposition (composting) is a self regulating thermophilic, aerobic process,
involving the microbial conversion of biodegradable organic waste to stable humus by
microorganisms including bacteria, fungi, actinomycetes and macrofauna like earthworms.
In decomposition process organic substances are reduced from large volumes of rapidly
decomposable materials to small volumes of materials which continue to decompose slowly
till the stable compound humus is formed.
Following groups of microorganisms involved in decomposition of organic wastes.
Bacteria :- Bacillus, Thiobacillus, Cellulomonas, Sporosarcina and Carophanon spp.
Fungi :- Aspergillus, Mucor, Humicola, Penicillium, Thermomyces, Torula and Chaetomium
spp.
Actinomycetes :- Streptomyces, Thermonospora, Saccharomonospora, Actinomudura,
Rhodococcus and Facnia spp.
Method of Decomposition of Organic Waste :
The organic matter consist of residues of plant and animal origin.
Organic matter contain –
Cellulose – 15 to 60 %.
Hemicellulose – 10 to 30 %.
Lignin – 5 to 30 %.
Proteins – 2 to 15 %.
Acids and Organic Acids – 10 %.
Above all mircoorganisms act on organic matter by production of enzymes and
degrades the complex compounds into simpler forms and later develop into humus which is
rich in nutrients.
A] Cellulose :
➢ It is decomposed by enzyme cellulose and converted into cellobiose.
➢ Cellobiose decomposed with the help of enzyme Beta-glucocidase and converted
into glucose.
➢ Glucose is further decomposed with the help of enzyme glucose and converted into
CO2, H2O and other simpler compounds.
➢ Organisms :-
Bacteria – Cellulomonas and Bacillus.
Fungi – Fusarium and Trichoderma.
Actinomycetes – Streptomyces and Nocardia.
B] Hemicellulose :
➢ It is pectin substances and pectin is degraded with the help of enzyme pectinase into
simpler compounds.
➢ Organisms :-
Bacteria – Bacillus and Pseudomonas.
Fungi – Aspergillus and Rhizopus.
Actinomycetes – Streptomycetes.
C] Lilgnin :
➢ It is more complex than cellulose and hemicellulose.
➢ Lignin is degraded by enzyme lignase into simpler compounds.
➢ Organisms :-
Bacteria – Pseudomonas and Flavobacterium.
Fungi – Clavaria and Humicola.
All these complex organic matter, finally degraded into stable compounds called
‘Humus’ by various enzymes secreted by microorganisms.
Decomposition of Soil Organic Matter :
The organic residue present in soil undergo decomposition by various
microorganisms. Organisms utilize the material as food. The process of decomposition
accompanied by evolution of CO2 and liberation of energy in the form of heat.
Soil organic matter consist of Cellulose, Hemicellulose, Sugar, Starch and Simple
Proteins. Cellulose, Hemicellulose and Lignin decomposed as material above. Starch/
sugars are converted into the humus by enzyme amylase or invertase.
Organisms :-
Bacteria – Bacillus and Cytophagus.
Fungi – Aspergillus and Rhizopus.
Actinomycetes – Streptomyces and Nocardia.
Properties of Humus :
➢ A tiny colloidal humus particles compound of ‘C’, ‘H’, and ‘O’ in the form of
polyphenol, polyglucones and polyuronides.
➢ The surface area of humus particle per unit mass very high.
➢ The colloidal surface of humus are negatively charged. The source of charge being –
OH phenolic and –COOH – carboxylic.
➢ A high pH value and C.E.C of humus on a mass basis is 150 – 300 centimetres/kg.
➢ The water holding capacity of humus is on mass basis 4 – 5 times more than silicate
clays.
➢ Humus has a favourable effect on aggregate formation and stability.
Functions of Organic Matter in Soil :
➢ It controls soil erosion and surface runoff.
➢ It helps in soil aggregation.
➢ It increases the water holding capacity of soil.
➢ It serve as a reservoir of chemical element.
➢ After decomposition it produces organic acids and CO2 which helps to dissolve
minerals.
➢ It helps to buffer soils against rapid chemical changes in pit.
Functions of Humus :
➢ It serve as a store house of exchangeable and available cations.
➢ It serves as a surface of energy for the growth of microorganisms.
➢ It provides food to entire soil life.
➢ It regulate the soil temperature.
➢ It reduces alkalinity of soil.
➢ It increases soil texture and structure.
➢ It imports dark colour to soil which absorb more light and hence the warmer.
➢ It is a good source of micro-nutrients and harmones.
➢ It improves seed germination, root growth, nutrient uptake by plants and desirable
physiological effects on plants.
➢ It improves enzymatic activity and amino acid activity.
➢ It improves synthesis of nucleic acid.
LECTURE 19 & 20 : IMPORTANCE OF TRICHODERMA SPP., PSEUDOMONAS SPP. AND
BACILLUS SPP. AS A BIOCONTROL AGENT.
A] Trichoderma spp :-
➢ Seed and soil borne plant pathogens cause diseases at seed germination, seedling
development and later stages of crop growth reducing the crop stand and economic
value.
➢ Most of the soil borne diseases are very difficult to control.
➢ Various species of Trichoderma have proved useful in controlling seed and soil borne
pathogens.
➢ Mechanism of action like antibiosis, competition for nutrients and space and
mycoparasitism have been reported.
➢ The species of Trichoderma are widely distributed in soil and other natural habitats
containing organic matter.
➢ The concept of biological control of soil borne pathogen was advanced on the
antagonisms by T. viride against soil fungi like Pythium, Phytopthera, Rhizoctonia and
Sclerotium.
Antagonism may be due to the three types of activities :
➢ Antibiosis : The pathogen is suppressed due to production of metabolic substances
from Trichoderma having an inhibiting effect.
➢ Competition : It is due to active demand for space and nutrient being greater than
availability due to the antagonist as a result of which pathogens being poor
competitors and suppression of their normal activities due to starvation.
Mycoparasitism :
Hyphae of Trichoderma grow adjacent to the hyphae of the pathogenic fungus and
produce haustoria in them and grow inside. Trichoderma species first soften the hyphal wall
of pathogenic fungi and then produces enzymes like cellulose, 1 – 3 Beta glucanose
chitinose and lipase which results in lysis of cell wall of pathogen hyphae.
Method of Application :
1) Seed Treatment – 5 gm/kg of seed.
2) Soil Treatment – 5 kg in 100 litres of water and apply one litre of solution to per tree
and 15 to 20 ml solution to seedling.
3) Seed and Soil Treatment – Seed Treatment with T. viride and soil treatment with T.
harzianum has proved better than seed or soil treatment alone in development of
maximum stores in rhizosphere and maximum density in soil.
4) Root dip treatment of transplanted crops.
Efficiency of Trichoderma with fungicides :
Several fungicides have no adverse effect on the activity of Trichoderma, rather their
combined treatment is more beneficial in the control of seed and soil borne pathogens.
Following are the some examples.
1) Trichoderma hamatum + 0.1 % Drenching of Bavistin –
Effectively controlled Root Rot of Black Gram caused by Macrophomina
phaseolina.
2) Trichoderma harzianum + Bavistin –
Effectively controlled Rhizoctonia seed rot and fusarium wilt in many crops.
3) Trichoderma + PCNB –
Effectively controlled cotton wilt and sugarbeet wilt caused by Rhizoctonia
solani.
4) Trichoderma + Bonomyl –
Cotton wilt.
5) Trichoderma + Metaloxyl –
Tomato and Brinjal wilt caused by Pythium aphanidermatum.
6) Trichoderma harzianum + Bordeaux Mixture –
Effectively control Root Rot of Betelvine, Papaya caused by phytopthera.
B] Bacillus Spp. :-
Among the different species of entomo-pathogenic bacteria only B. thuringiensis and
B. popilliae have used extensively in biological pest control.
➢ Bacillus thuringiensis is effective against large number of lepidopterous foliage
feeders and many other insects belonging the orders Coleoptera, Diptera,
Hymenoptera and Orthoptera.
➢ B. popilliae is effective against white grubs.
➢ Both the bacteria are Gram positive, spore formers and hence ideal for production of
different type of formulation.
➢ There are reports that B. thuringiensis survive in environment even upto 10 years
after application while B. popilliae to be carried to new environment by the infected
adults.
➢ B. thuringiensis produces several types of insecticidal toxins.
➢ One of the most interesting among these are proteinaceous toxins often called delta
endotoxins/ crystal toxins.
➢ These toxins are produced in large amount during sporulation and accumulate
crystals.
➢ The gene coding for this proteins is normally found on plasmids and chromosomes of
bacteria.
➢ The crystalline toxin when injested by insects, a combination of high pH and
proteinases of the insect mid gut is believed to be responsible for the solubilisation
of the crystals and the rapid cleavage of protoxin to active toxins.
➢ The effect of toxin occus within minutes of ingestion beginning with midgut paralysis
and ending with disruption of midgut cells which finally affect the feeding of insects
and kill due to starvation.
Following are the B. thuringiensis (Bt) products are available in India.
Sr. No.
Bt Variety Target Pest Crop Trade Name and Manufacturer
1. Bt. Kurstaki Cotton Bollworm Cotton Biosapo Biotech International Ltd. New Delhi.
2. Bt. Kurstaki Cotton Bollworm and Fruit Borer
Cotton, Tomato, Brinjal, Okra
Lupin “Dipe18L” Agrochemicals.
3. Bt. Gulleriae American Bollworm, Leaf Folder, Diamond Black Moth
Cotton, Cabbage, Cauliflower, Chilies
“Spicturin” Tuticorin Alkalli Chemicals Ltd. And Chennai Fertilizers.
4. Bt. Kurstaki Lepidopterous Caterpillars
Several Crops
Rallis India Ltd. Bangalore
LECTURE 28 : ESTABLISHING INSECTORY FOR HOST INSECT AND NATURAL ENEMIES.
This topic mainly focused on the development of commercial insectory with an
attempt to highlight the basic requirement of equipments, glasswares, chemicals and
labortary hosts. An Ideal Insectory have the following characteristics :
➢ Ideally located in temperate zones, creating warm conditions is easier and cost
effective than cold conditions.
➢ A minimum distance 200 to 250 meter should be maintained from the farm area to
avoid pesticidal effect and minimize the entry of harmful natural enemies like Bracon
spp.
➢ Preferably away from industrial area because the coccinellid predators are prone ill-
effects of air pollutants.
➢ In order to avoid field contamination, exclude plantation within the immediate
vicinity of the insectory.
➢ Insectory building face in east-west direction, especially in areas with severe summer
because natural lighting aids inn uniformity.
➢ Regular water supply is very essential.
➢ An area of 288 sq. m. is essential in order to accommodate all equipments, generator
and staff.
➢ Insectory should dust/ ant and rat proof.
➢ Easy assess to facilitate handling of material and disposed of waste material.
➢ It should be well equipped with required equipments glasswares, chemicals for mass
production.
➢ Ceiling height should not be more than 2.5 meter, so as to easy for insect trapping
and reduce the load of air conditioners.
➢ A good and low cost insectory using indigenous is with efficient security system.
Equipments Required for Insectory :
Sr. No. Equipments Purpose Quantity
1 Diesel Generator 40 K.V. To manage electricity failure and maintain desire environmental conditions.
1
2 Air Conditioners 1.5 M.T. To regulate the temperature in the rooms depending on the kind of organisms handled.
2
3 B.O.D. (Biological Oxygen Demand) with stabilizer and humidity control.
To maintain predators, parasitoids, microbes or their host and study their biological requirements.
1
4 Advanced Research Microscope To carry out advanced 1
with Image Analysis System studies in biocontrol because high magnification and documentation are vital.
5 Binocular Microscope having provision to fit the Occular Micrometer.
For general purpose such as dissections and measurements.
1
6 Hot Air Oven To sterilize glasswares and food materials.
1
7 Humidifiers with humidistat To maintain and observed relative humidity of a particular rooms where cultures are maintained.
4
8 Desert Cooler To maintain cool temperature during extreme summer and to increase humidity.
4
9 Refrigerator To maintain natural enemies, pure culture of microbes and certain media.
1
10 Deep freezer To store chemicals and cell fractions.
1
11 Environmental Simulation Chamber
To conduct advanced ecological and semi-chemical studies.
1
12 Autoclave To sterilize media and plastic wares
2
13 Inline Stabilizer To stabilize voltage, fluctuations and run equipment uninterruptedly.
1
14 Analytical Single Pan Balance To weigh the ingredient for different media/ artificial died.
1
15 High Speed Centrifuge To isolate semi-chemical microbial work.
1
16 Homogenizer To develop media for 1
17 P. C. with printer To store and analyse the data.
1
18 Gas Limited Chromatography To analyse the constituents of insects required for developing the diets.
1
LECTURE 26 & 27 : IMPORTANCE OF TRICHOGRAMMA, CRYPTOLAEMUS , CHRYSOPERLA,
NPV (NUCLEAR POLYHEDROSIS VIRUS) AND EMP (ENTOMOFUNGAL PATHOGENS).
I] Trichogramma :-
➢ It is an parasites, most used for management of borer pest in crops like sugarcane,
cotton, sorghum, maize and paddy.
➢ Trichogramma parasites eggs of lepidopterous and also reported to parasite eggs of
Coleoptera, Neuroptera and Diptera.
➢ It is most widely used natural enemies in India.
➢ Generally 1 CC eggs (18000 to 22000) frozen host egg glued on Trichocard (size 15 x
75 cm).
➢ The card is further divided into six strips each of 7.5 cm X 2.5 cm in size which can
easily pressed and separated.
Rearing of Trichogramma –
➢ A strip dated and named after Trichogramma spp. Containing glued eggs is inserted
into another glass tube having newly emerged adults of the same parasitoid species
for parasitisation.
➢ The adult parasitoids are fed with honey applied on the innerside of tune that is
secured tightly secured with cloth and rubber bands.
➢ Thus cards changed after 24 hours and replaced with fresh card.
➢ Thus cards are changed for 3-4 days or till the female oviposits in the host egg, which
turns black after 3 days of parasitisation.
➢ The parasitoid complete its life cycle in 7 to 9 days at 27˚ C to 75 % Relative
Humidity.
II] Chrysoperla :-
➢ Green Lace Wing is well distributed all over world and In India.
➢ Larvae are important predators of insect pest viz. – aphids, mealy bugs, eggs and
smaller larvae of various insects of agricultural importance.
➢ On an average each larvae has potential to feed 12 aphids/ day or about 120 aphids
during the entire life cycle.
Rearing of Chrysoperla –
➢ In India, it is advisable to culture predator larvae (chrysoperla) on frozen eggs of
chrysopa due to economic advantage and it is easy to mass produce chrysoperla
without any determinant effect on the predators (chrysoperla) progeny.
➢ Newly hatched larvae of chrysopa are reared in individual vials.
➢ On an average 1 CC eggs (20000 to 22000 eggs) of the host (chrysopa) are required
for rearing 5 to 6 larvae the predator (chrysoperla).
➢ Then each predators larva placed in separate vial with sufficient food and covered
with white paper and finally cage is covered with lid and shifted in room at 27˚ C
temperature and 60 to 65 % relative humidity for further development.
➢ After 3-4 days, if food is required add in to the each vial from the top cocoon
formation.
➢ At the end of the week these cacoons are collected and kept in separate jars for
emergence.
➢ Newly emerged 10 pairs of chrysoperla are caged in a jar with swab of 20 % honey,
pollen grains of castor if available.
➢ Then jar are covered with wet black cloth because predator prefer to lay eggs on
black cloth.
➢ In recent studies it is proved that the predator larvae (chrysoperla) can be easily and
economically mass multiplied on the frozen grubs of Tribolium castaneum.
III] Cryptolaemus :- (Lady Bird Beetle)
➢ In India, it was introduced during 1898 to control Mealy Bugs.
➢ It is known to prey on various stages of mealy bugs, scale insects and on Aphids.
➢ It is reported that cryptolaemus have feeding potential and development on the
grape, mealy bugs in India.
➢ A grub of cryptolaemus could prey on 880 eggs or 260 nymphs or 27 adults of grape
mealy bugs.
Rearing of Cryptolaemus –
In India, it was reared on pseudococcids by several workess, since its introduction at
I.A.R.I. New Delhi. It is reared on Ferrisia virgata by inoculation so newly hatched bugs in a
glass jar or releasing 10 pairs of cryptolaemus per jar and removing them after 2 to 3 days.
IV] NPV (Nuclear Polyhedrosis Virus) :-
➢ Viral diseases are common in insect populations.
➢ Most of the insect viruses are differ than plant viruses.
➢ Insect viruses have an inclusion body around virions.
➢ Insect viruses being obligate pathogens need to be cultivated by live insect hosts.
➢ Since live insects are needed to multiply the viruses, production of these in large
quantities is a limitation.
➢ These viruses look quick knock down effect and take about a week time for causing
insect mortality.
➢ They are highly host specific and each species of insects requires specific virus of its
own.
➢ In general earlier instars of insects are more susceptible to viruses hence require
correct timing of application.
➢ Viruses are ultramicroscopic, obligate, intracellular and pathogenic entities.
➢ Among the several viruses isolated so far, only baculoviridae comprising NPV and GV
(Granulosis Virus) have been extensively applied under field conditions.
Mass Multiplication of Viruses :
NPV of S. litura was recorded for the first time in India in the year 1969. The
Polyhedrosis Inclusion Bodies (PIB) are irregular in shape and majority of these appear in
hexagonal outline. This virus is specific to host S. litura only. It is multiplied by two methods
–
1. Leaf Dip Method –
✓ The initial concentration of virus (PIB) were determined through
haempcytometer counts which was subsequently diluted to have desire
concentrations.
✓ Castor leaves were dipped in such diluted concentration and air dried.
✓ The treated leaves offer to S. litura larvae were allowed to feed for 24 hours.
✓ By then entire treated leaves were consume by the larvae.
✓ It is considered that upto fourth instar was fed in groups where as fifth instar
larvae were fed in singly.
✓ The virus fed larvae started showing disease symptoms on 6th day after
treatment.
✓ By about 7th and 8th day approximately 90 % of the larvae started drying in
higher concentration.
✓ These diseased larvae were collected, just before death in stopbered conical
flasks and kept for purification under condition.
✓ The purified material was homogenized and then filtered thorugh double
layered muslin cloth.
✓ The PIBs were resuspended in water and counted through Neubauer
Haemocytometer.
✓ The count was expressed on larval basis i.e. Number of Larvae as well as per
unit of larval weight basis.
✓ The PIBs were dried over CaCl2 and further formulated as dust or wettable
powder.
2. Spot Feeding Method –
✓ In spot feeding method, known number of PIBs were directly placed over
circular disc of castor leaves.
✓ The treated castor leaf disc after drying under fan were offered to 4th and 5th
instar larvae of S. litura.
✓ The rest of the procedure were similar to as Leaf Dip Method.
Mass Multiplication of NPV on Helicoverpa :
The larvae of these species are individually reared on artificial diet. Then six day old
larvae weighing 179 ± 1 – 2 mg/ larvae were allowed to feed on treated surface of the diet.
The diet surface was treated with 0.1 ml of the virus suspension containing 1.8 X 107 PIB/
ml. The larvae were further reared at 24˚ C. More than 99 % larvae were dead after 10 days
of incubation.
Diseased larvae collected after 5 – 7 days were frozen and kept at 20˚ C. Inclusion
bodies were recovered by blending with water and differential centrifugation. The
concentrated virus was freeze dried or stored in water till use.
Advantages of Viral Pesticides :
➢ These viruses are safe to human beings and other organisms as these kill specific
insects only.
➢ Resistance towards insect is not observed as it is appeared with chemical
insecticides.
➢ This method can be integrated with other methods of insect control including
chemical pesticides.
➢ Maintaining period of harvesting of crops after treatment is not needed.
➢ The biocontrol agent can be produced by pomshnel or at farmer level.
➢ The biocontrol agents are self perpetuating in nature and once introduced in the
area is likely to keep the pest population under check.
➢ These viruses may not replace the chemical insecticides but will definitely reduce the
use of chemical insecticides as thus helps in minimizing the environmental solution.
Entomofungal Pathogen :
➢ Fungi belonging to all four group namely Phycomycetes, Ascomycetes,
Basidiomycetes and Deuteromycetes attacks different species of Insects.
➢ The insects pathogenic fungi in general have a wide host range.
➢ The fungal pathogens mainly infect the insects through their integument.
➢ The infected insects are characterized by the presence of fungal matramigying the
entire insect body.
➢ Usually high relative humidity is needed for the successful germination of fungal
spores and development of Mycelial Mat.
➢ There is a wide scope for utilization of insect pathogenic fungi in India.
➢ Fungal pathogens helps to supress the increasing population of chilli aphids, banana
aphids, mustard aphids, cotton aphids, cotton mealy bugs, papaya mealy bugs,
sugarcane pyrilla, white grub, etc.
Some commercial fungal biopesticides available in India as below.
Sr. No. Fungal Pathogen Insect Pest Commercial Name
1. Beauveria brassiana Potato Beetle Boverin
2. Verticillium lecanni Aphids and White flies VetaLec / Mycotal
3. Metarhizium anisopliae Sugarcane pyrilla/ white grubs
Metaquino
Entomofungal pathogens also producing toxins which are harmful to insect pest and
also to vertebrates and human beings.
Toxins Produce by Fungi
1. Destraxin A and B Metarhizium anisopliae.
2. Beauverian Beauveria brassina.
3. Aflotoxins Aspergillus spp.
Mass Multiplication of Beauveria spp., Verticillium and Metarhizium on Solid Media :-
➢ For mass production on solid media, we can use cereal grains like rice, wheat, maize,
sorghum and ragi.
➢ First take 1000 gm/ 1 kg raw grains and soaked for 12 hours in tap water and excess
water drained out completely.
➢ Soaked raw grains 100 gm placed in a autoclavable polypropylene bags.
➢ In each bag add 2 gm of calcium sulphate and mixed thoroughly out to get uniform
coating of these over grains.
➢ This process helped in preventing the grain particles sticking together and there by
providing more surface area for the growth of fungus.
➢ Then bags were sterilized twice at 121˚C for 20 minutes in autoclave.
➢ Then grain media was inoculated with 1 ml of fungal suspension (1 X 107 spores per
ml) under laminar air flow cabinet (aseptic condition).
➢ The bags were seeded manually and incubated for 14 days at 25˚ C and 90 % relative
humidity in growth chamber.
➢ After 14 days of incubation, grains with fungal growth dried under aseptic condition
at 30˚ c until the moisture content was reduced to 8 %.
➢ After drying spore production was estimated by Neubauer’s haemocytometer.
➢ Then grains with fungal growth were sieved with vigorous agitation.
➢ Then coarse dust thus collected further sieved through a sterile 105 mm sieve to get
fine spore dust for further use.
Mass Production in Jowar (Sorghum) Medium using PDA :-
In this method –
➢ First culture of fungal species is inoculated into PDA medium in petri-dish.
➢ Then prepare jowar medium. Jowar gains are crushed into pieces, which is then
filled in a conical flask to the tune of 250 gms. In each flask so as to jowar seed
pieces are softened.
➢ Each flask is plugged with cotton and sterilized in autoclave.
Incubation of Sterilized Flask :
➢ From petri-dish small bit of fungal species with the help of cork borer added into
conical flask under Laminar Air Flow Cabinet just to avoid contamination.
➢ Then conical flasks are placed in an incubator for abundant growth of fungus.
➢ Once the fungus covers the entire surface and turns completely white.
➢ Then spores are collected by crushing in hand mill, pulverized in grinding mill and
sieved/ strained through the fine mesh sieve.
➢ The fine powder is then collected in sterilized screw capped tubes for further use.