Bacillus Biocontrol ability modes of biocontrol

1. PRODUCTION OF BIOCONTROL AGENTS
Bacillus, Pseudomonas
PHOSHATE SOLUBILIZING BACTERIA Psudomonas
SUBMITTED TO
Dr.R.Subhash Reddy
Professor & Head
Agril microbiology & bioenergy
SUBMITTED BY
K.MANASA
M.Sc Ag 1st yr
RAM/13-75

2. CONTENT
• Definition of Biological control
• Advantages &Disadvantages
• Bacillus isolation
• Mass multiplication
• Pseudomonas isolation
• Morphological characters
• Mass multiplication
• Quality contol parameters
• Posphhate solubilizing bacteria
• Mass multiplication

3. BIOLOGICAL CONTROL
• Biological control is the use of natural enemies to reduce
densities of insect pests and weeds.
• The reason biological control is so effective and safe is that a
high degree of host-specificity for the targets is sought before
a potential control organism can be released into the
environment.
• The control of pests by interference with their ecological
status, as by introducing a natural enemy or a pathogen into
the environment.Also called biocontrol.

4.  Biological control is a component of an integrated
pest management strategy.
 It is defined as the reduction of pest populations
by natural enemies and typically involves an
active human role
 This is frequently referred to as natural control.
This guide emphasizes the biological control of
insects but biological control of weeds and plant
diseases is also included
 Biological control agents of plant diseases are
most often referred to as antagonists.

5. ADVANTAGES
 The most important advantage to the use of biological control
is that it typically offers longer term management than the
more traditional technology areas.
 Longer term control is achieved because biocontrol agents act
as if a host specific control method is continualy present and
impacting the target plant.
 for example, once an agent is released and well established,
insect population levels cycle proportionately with the
population of the plant.
 That is when plant population levels are high, there will be a
corresponding increase in the population levels of the
biocontrol gents.
 When plant levels decrease, there is a corresponding
decrease in the numbers of the biocontrol agents persist and
continually exert controlling or regulatory pressure on the

6.  Another advantage is that the cost for control is typically
lower to relative to more traditional control procedures
 Typically, biocontrol agents are released in relatively low
numbers for only a short time in the beginning of the
program unlike more traditional methods of control which
are used continually over many years.
 After the releases are discontinued, the agent population
increases, if successful and begings to damage the target
population.
 selectivity,it does not intestify create new pest problems.
 no manufacturing of new chemicals, the organisms are
already available.
 control organisms will increase in number and spread.
 the pest is unable ( or very slow) to develop a resistance.

7.  free of side effects
 safe to handle or use
 occurs naturally
 high degree of host specificity
 cost effective
 self perpetuation
 searching ability
 survive of low host density
 pesicides which are harmful to all parts of the food chain,
are not needed.
 biological control is self- perpetuating
 suitable biological control organisms do not attack other
species
 usually a large proportion of the pest population is
destroyed.

8. DISADVANTAGES
• control is slow
• it will notthe pest
• it is often unpredictable
• it is difficult and expensive to develop and supply
• it requires expert supervision
• slow to achieve results
• impact often not dramatic
• partial success
• can be complex
• disruption of food chains
• the need for environmentally unfriendly follow up operations
to ensure that the populations does not build up resistance to
the biological control agent.

9. Bacillus
 Bacillus spp. having potent plant growth promoting traits
such as IAA production, phosphate solubilization, nitrogen
fixation, and biocontrol attributes like production of HCN,
siderophore, hydrolytic enzymes and antibiotics
 We have earlier reported PGP attributes (for chir-pine) and
biocontrol potential in B. subtilis against M. phaseolina
associated with root rot disease of the same plant .
 Several others have also found the biocontrol activities of
Bacillus against many common phytopathogens

10. MORPHOLOGY
• Bacillus subtilis, known also as the hay bacillus or grass bacillus, is
a Gram-positive, catalase-positive bacterium.
• A member of the genus Bacillus, B. subtilis is rod-shaped, and has
the ability to form a tough, protective endospore, allowing the
organism to tolerate extreme environmental conditions.
• B. subtilis has historically been classified as an obligate aerobe,
though recent research has demonstrated that this is not strictly
correct
• Although this species is commonly found in soil, more evidence
suggests that B. subtilis is a normal gut commensal in humans.

11. • Soil samples (10 g) from rhizosphere of soil
heat-treated (80 °C), transferred to 90 ml
sterile distilled water and mixed thoroughly
by shaking the flask on a rotatory shaker for
5 min.
• After serial dilution 0.1 ml suspension was
spread over pre-sterilized and cooled down
nutrient agar plates in triplicates.
• The inoculated plates were incubated at 30 ±
1 °C for 24–48 h.
• Rough and abundant colonies with waxy
growth (1–4 mm diam) and irregular
spreading edge were obtained.
ISOLATION OF BACILLUS

12. Mass multiplication of Bacillus subtilis
Preparation of mother culture
The nutrient broth medium is prepared as detailed below
• Glucose : 5.0 g
• Peptone : 5.0 g
• Beef extract : 3.0 g
• Sodium chloride : 3.0 g
• Distilled water : 1000 ml
• The above medium is dispensed in conical flasks and
autoclaved at 15 lb pressure for 15 mts.
• A loop of B.subtilis is inoculated into the medium and
incubated for 2 days. This serve as the mother culture

13. Mass multiplication:
• The nutrient broth is prepared in fermentor and sterilized at
15 lb pressure for 15 mts.
• Then the mother culture is added @ 1 lit / 100 lit of the
medium and incubated at room temperature for 2 days.
• The medium containing the bacterial growth of B.subtilis is
used for mixing with talc powder.
• Bacillus subtilis :
• The broth containing the bacteria is collected from fermentors
and mixed with 250 kgs of sterilized neat soil for 100 lit of
broth.
• Then 37 kgs if calcium carbonate is added thoroughly mixed,
dried is shade and packed in polythene bags. This can be
stored upto 6 months.

14. Bacillus Subtilis
 It is antagonistic bacterial biological agent.
 This bacteria controls many and air borne diseases of
rice, groundnut, Cotton, Vegetables etc.
 Foliar application of B.subtillus with P.fluoresence can
control root and leaf diseases of many crops.
 It protects plants from seed and root diseases.
 The bacterium colonies the root and leaf system of the
plant and completes and there by suppresses the growth
of the plant diseases causing organisms.
 It is suitable for Paddy, Millets, Oil seeds, Fruits,
Vegetables, Coffee, Lime, and Banana etc.
 Dosage: 500-700gms/acre as foliar spray.

15. Bacillus thuringiensis (Bt) as a biocontrol agent
 Bacillus thuringiensis (Bt) is a common soil bacterium that
produces proteins that are toxic to specific classes of insects,
including certain moths, beetles, and flies.
 It has been used as a biological control agent against these
insects, which are agricultural pests, for at least 40 years.
 It is only effective against young insects (larvae).
 BT proteins are transformed into toxins by specific digestive
enzymes present in susceptible insects; these toxins cause the
eventual death of the insect within a few days.
 The digestive enzymes are present only in certain insect
species. Thus, Bt proteins and Bt-treated crops are generally
safe for humans, animals and most other beneficial insect
species.

16. USE OF Bt IN AGRICULTURE
• Traditionally, spores (reproductive cells) of the Bt bacterium
were used in spray formulations for insect control.
• Modern genetic engineering allows the genes for Bt proteins
to be inserted directly into crops, so that the plants
themselves produce the insecticidal proteins.
• This has led to the development of crops such as Bt corn and
Bt cotton

17. Advantages
 Controlling insects reduces crop damage, and improves crop
yields and quality.
 Bt insecticides are biodegradable and non-toxic, an
improvement over strong chemical pesticides. These spores
are approved for use in organic agriculture as natural
pesticides.
 Genetically engineered Bt crops have the increased
advantages of:
 Requiring less insecticide application with machines, leading
to reduced labour and fuel costs;
 Greater effectiveness in insect control because the crops
continuously produce the Bt proteins within their own tissues;
 Control of molds and fungi that can infect holes in the crops
left by burrowing insects.

18. • All Bacillus isolates formed clear halos around spot inoculation.
Pikovskaya's medium having bromothymol blue (BTB) changed its
colour from blue to yellow due to decrease in pH of growth medium
• The same experiment was performed by replacing TCP in Pikovskaya
agar medium with di-calcium phosphate (DCP) and zinc phosphate
(ZP).
• None of the isolates solubilized DCP, while isolates of Bacillus
solubilized ZP as indicated by formation of clear halo around spot
inoculation.
Phosphate solubilization

19. • P. fluorescens has multiple flagella. It has an extremely
versatile metabolism, and can be found in the soil and in
water.
• It is an obligate aerobe, but certain strains are capable of
using nitrate instead of oxygen as a final electron acceptor
during cellular respiration.
• Optimal temperatures for growth of Pseudomonas
fluorescens are 25-30 degrees Celsius.
• It tests positive for the oxidase test. Pseudomonas
fluorescens is also a nonsaccharolytic bacteria.
• Heat-stable lipases and proteases are produced by
Pseudomonas fluorescens and other similar pseudomonas.
• These enzymes cause milk to spoil, by causing bitterness,
casein breakdown, and ropiness due to production of slime
and coagulation of proteins.
PSEUDOMONAS

20. MORPHOLOGY
 Pseudomonas is a genus of Gram-negative aerobic
gammaproteobacteria, belonging to the family Pseudomonadaceae
 The best studied species include P. aeruginosa in its role as an
opportunistic human pathogen, the plant pathogen P. syringae, the
soil bacterium P. putida, and the plant growth promoting P.
fluorescens
 Gram-negative, rod-shaped and polar-flagella bacteria
 The optimum growth temperature is between 25-30 degrees Celsius

21. Isolation and identification
• Collect rhizosphere soil particles loosely adhering to the roots
and make fine powder
• Add 10 gm sample of finely pulverized, air dried soil into 90 ml
sterilized distill water to make 1:10 dilution
• Vigorously shake the dilution on a magnetic shaker for 20-30
minutes to obtain uniform suspension.
• Transfer 1 ml of suspension (1:10 dilution) to another 9 ml
sterilized distill water to make 1:100 dilution
• Prepare serial dilution 10-3 to 10-7 as outlined earlier.
• Use King’s B medium for isolation of P. fluorescens.
• Pour 20 ml sterilized, melted and cooled (450 C) King’s B
medium in each Petri plate.

22. • Transfer 1 ml of soil suspension from aliquot dilution to sterilized
Petri plates containing King’s B medium.
• Incubate Petri plates at 28 + 20C for 24 hours.
• Detect and mark individual colonies with yellow-green and blue
white pigments by viewing under UV light.
• Pick up individual colony with sterilized loop and transfer on to
fresh king’s B medium.
• Transfer single colonies to King’s B medium slants to obtain pure
culture and store in refrigerator at 400C.
Pseudomonas fluorescens under U.V light.

23. Mass production of Pseudomonas fluorescens
Preparation of mother culture
• Mother culture is prepared by using the king’s B medium
• Peptone : 20.0 g
• K2HPO4 : 1.5 g
• Mg SO4 : 1.5 g
• Glycerol : 10 ml
• Distilled water : 1000 ml
• The above broth is dispersed into conical flasks and
autoclaved at 15 lb pressure for 15 minutes and cooled and
inoculated with a loop of P.fluorescens and incubated for 2
days.

24. • The kings B medium is prepared and poured into the
fermentor and sterilized at 15 lb pressure for 15
minutes.
• After the broth has cooled below the mother culture
of P.fluorescens is added to the king’s B medium in
the fermentor at the rate of 3 lit for 40 lit of the
broth.
• Then it is incubated in the fermentor for 2 days with
frequent mixing of the broth by operating the stirrer.
• Then the broth containing the bacterial growth is
collected in plastic buckets and used for mixing with
talc powder for commercial formulation.
Mass multiplication

25. • The broth containing the bacterial growth is collected from
fermentor and added @ 400 ml / kg of talc powder.
• Then CMC (carboxy methyl cellulose in substrate ) is added @
5 g /kg mixed well air dried to 20% moisture level and packed
in polythene bags.
Quality control parameters:
Fresh produce should contain 2.5 x 108 cfu/g
• After 3 months of storage at room temperature the population
should be 8-9 x 107 cfu/g
• Storage period is 3-4 months
• Minimum population load should be 1.0 x108 cfu /g
• Moisture content should not exceed 20% in the final product
• Population per ml of the broth should be 2 x 108 cfu /g

26. Pseudomonas Fluorescens
• It is systemic Bio-control argument.
• This bacteria effectively controls wilt and root rot diseases of Ground nut,
Cotton, banana, Soybean, Tomato, Pigeon Pea etc.
• It also controls rice blast and sheath blight of paddy.
• It secretes certain enzymes that has the capability to destroy the cell wall
of fungal pathogens.
• It secretes Hydrogen Cyanide and antibiotics such as pycocyanine and
phenazine which inhibits the growth of disease causing organisms.
• It also produces sideropores which chelate with Iron in the soil, and make
and pathogen proliferation difficult.
• It secretes growth harmones like gibberellins compounds and increase the
plants growth vigorously.
• It is suitable for all crops and has no phytotoxicity Can be used along with
biofertilizers.
• Seed Treatment: -10gm/1kg seed or 4-5 ml/1 kg seed.
• Dosage: 500-750 gms/acre;250ml/hectare as foliar spray

27. • Some P. fluorescens strains present biocontrol properties,
protecting the roots of some plant species against parasitic
fungi such as Fusarium or Pythium, as well as some
phytophagous nematodes
• The bacteria might induce systemic resistance in the host plant,
so it can better resist attack by a true pathogen
• The bacteria might outcompete other (pathogenic) soil
microbes, e.g., by siderophores, giving a competitive advantage
at scavenging for iron
• The bacteria might produce compounds antagonistic to other
soil microbes, such as phenazine-type antibiotics or hydrogen
cyanide

28. P. fluorescens as biocontrol agent:
 The bacteria P. fluorescens possess many traits that
make them well suited as biocontrol and growth-
promoting agents.These include the ability to-
 Grow rapidly in vitro and to be mass produced.
 Rapidly utilize seed and root exudates.
 Colonize and multiply in the rhizosphere and
spermosphere environments and in the interior of the
plants.
 Produce a wide spectrum of bioactive metabolites.
 Compete aggressively with other microorganisms.
 Adapt to environmental stresses and,
 Inexpensive

29. POTENTIAL OF PSEUDOMONAS FLUORESCENS AS BIOCONTROL
AGENT OF FUNGAL PATHOGENS
• Plant diseases cause 13 - 20 % losses in crop production
worldwide. Control of plant disease by chemical can be
spectacular but the accumulation of harmful chemical residue
sometimes causes serious ecological problems.
• Biocontrol agents are economical, suppress the inoculum load
of the target pathogen, long lasting and free from residual side
effect.
• Fungi in the genus Trichoderma and Bacteria in the genera of
Pseudomonas and Bacillus are increasing interest as
bioprotectants against plant diseases.
• Biocontrol agents in general and Pseudomonas fluorescens in
particularhave gained importance as a component of
Integrated Pest Management for sustainable agriculture

30. Phosphate solubilization
 Phosphorus (P) is one of the major essential macronutrients
required for plants growth
 Soils are generally low in P readily available for plant growth.
Therefore, a large quantity of soluble forms of P fertilizer is
applied to achieve maximum plant productivity.
 However, the applied P fertilizers are easily precipitated into
insoluble forms – CaHPO4, Ca3(PO4)2, FePO4 and AlPO4– and
are not efficiently taken up by the plants, which lead to an excess
application of P fertilizer to crop land .
 Excessive application of P causes environmental and economic
problems.
 That is, the overfertilization of P leads to pollution due to soil
erosion and runoff water containing large amounts of soluble
 Furthermore, use of P fertilizers has become a costly and there is
a need for alternative sources.

31. Pseudomonas Mechanism for Phosphate
Solubilization
• The principal mechanism for mineral phosphate solubilization
of Pseudomonas is its production of organic acids and acid
phosphatases which play a major role in the mineralization of
organic phosphorous.
• Although several phosphate solubilizing bacteria occur in soil,
usually their numbers are not high enough to compete with
other bacteria commonly established in the rhizosphere.
• Thus, the amount of P liberated by them is generally not
sufficient for a substantial increase in plant growth.
• Therefore, inoculation of plants by a target microorganism at
a much higher concentration than that normally found in soil
is necessary to take advantage of the property of phosphate
solubilization for plant yield enhancement.

32. • It is generally accepted that the major mechanism of
mineral phosphate solubilization is the action of organic
acids synthesized by soil microorganisms
• Production of organic acids results in acidification of the
microbial cell and its surroundings.
• Consequently, Pi may be released from a mineral
phosphate by proton substitution for Ca.
• The production of organic acids by phosphate solubilizing
bacteria has been well documented.
• Among them, gluconic acid seems to be the most frequent
agent of mineral phosphate solubilization.
• As the principal organic acid produced by phosphate
solubilizing bacteria such as Pseudomonas sp.

33. • It has been shown how phosphate solubilizing bacteria help
mycorrhizal fungus help plants Several studies have shown that
P solubilizing bacteria interact with vesicular arbuscular
mycorrhizae by liberating phosphate ions in the substrate.
• This causes a synergistic interaction that allows better use of
insoluble phosphate sources
• The P solubilized by Pseudomonas fluorescens is more easily
taken up by the plants through a mycorrhizae mediated
channel between roots and surrounding soil.
• This would allow nutrient transfer from soil to plants
• phosphate- solubilizing bacteria associated with mycorrhizae
improved mineral accumulation of phosphorus and nitrogen in
plants.
• Inoculated rhizobacteria could have released phosphate ions
from insoluble rock phosphate and/or other P sources, which
were then taken up by the external mycorrhizal mycelium.

34. • Processing of soil samples for isolation of Phosphate solubilizing
microrganisms soil samples by dilution plate technique using Pikovskaya’s
medium (Pikovskaya 1948) containing tri-calcium phosphate (TCP).
• Appropriate soil dilutions were plated on Pikovskaya’s agar medium by
spread plate technique and incubated at 30 ± 1 ºC for 2-3 days.
• The colonies forming halo zone of clearance (Pikovskaya’s medium)
around them were counted as P-solubilizers.
• All the bacterial colonies exhibiting halo zones were selected, purified and
maintained on nutrient agar slants for further studies.

35. • P solubilization by using the National Botanical Research
Institute's phosphate solubilization-bromophenol blue media
(NBRIP) and rhizospheric phosphate solubilizing medium
(PSM), separately.
• This method of screening of phosphate solubilization gave
comparatively quick results compared to Pikovskaya's agar
plate assay as the zones were visible overnight due to pH
change, while in Pikovskaya's agar-plate based assay it took 48
h or more.

36. Qualitative assay of phosphate solubilizing activity
• Pure cultures of phosphate solubilizing bacteria were spot inoculated at
the centre of already prepared plates of Pikovskaya’s agar medium.
• The plates were incubated for 7-10 days. The colonies forming more than
5.0 mm zone of solubilization were stocked.
• The zone of phosphate solubilization (mm) formed around colonies was
recorded after every 24 hours for 10 days.
• The solubilizing efficiency of the microorganisms was calculated using
following
• Formula Z – C
• Solubilizing efficiency (% S.E) = C x 100
• Z = Solubilization zone (mm)
• C = Colony diameter (mm)

37. Advantages
 The effective strain of Phosphate Solubilized Bacteria used, increase the
level of available P2O5 in the soil. With the increase in available P2O5 level,
overall plant growth can be increased.
 In certain condition they also exhibit anti-fungal activities and thereby
fungal diseases may be controlled indirectly.
 About 10 to 15% increase of crop yield can be achieved with the use of
this culture.
 Phosphate Solubilizing Bacteria are useful for all the crops i.e. Cereals,
Cash crops. Leguminous crops. Horticultural crops. Vegetables