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Bacteria and Viruses

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					Utilizing Soil
Microbes
Objectives of Agriculture

 To meet the food requirements of the
  population
 To generate export earnings
 To provide raw materials for industry
 To eradicate poverty and unsanitary
  conditions of the countryside
      Conventional Agricultural
      Problems
   Over-specialization, monocropping, and
    excessive intensification
   Excessive dependence on external inputs
   Large-scale deforestation
   Salinization, erosion, compaction, and fertility
    loss of soils
   Unsustainable intensive factory farming systems
    of animal production
   Heavy rural-urban migration
    Effects of the Multidimensional
                 Crisis
   State subsidies were reduced
   Purchasing capacity was reduced (up to 40
    percent)
   Fuel price was increased (some even doubled)
   Fertilizers were reduced to 25 percent
   Pesticides were reduced to 40 percent
   Animal feed concentrates were reduced to 30
    percent


        **All agriculture seriously affected
    Strategy to Survive


Shift to a low external input form of
agriculture, while at the same time boosting
production
    Organic Farming
 Alternatives to high input agriculture
 Many traditional practices were
  remembered
 ―Agroecological‖—farms where
  agroecological concepts are applied and
  which promote sustainable production
  systems
    Practices of Organic Farming

 Soil Conservation, and Organic and
  Biofertilization
 Ecological Management of Pests,
  Diseases & weeds
 Crop rotation and polyculture
 Integrated farming systems
Roles of Microbes in Organic
Farming
   Microbes provide nitrogen required for the
    plants through biological nitrogen fixation.
   Phospobacteria provides phosphorus required
    for the plant growth by converting unavailable
    phosphorus to available form.
   Phosphorus uptake enhanced using
    Mycorrhiza fungi.
   Microbes provide plant growth promoting
    substances
   Microbes play an important role in controlling
    diseases of plants as Bio protectors.
Making Microbes Available

 Biofertilizers ,
 Biological Control Agents.
Biofertilizers
 Inputs containing efficient strains of specific
  microorganisms which are capable of
  mobilizing nutritive elements required for the
  plants by fixing atmospheric nitrogen,
  solubilizing and enhancing up take of soil
  phosphorus and stimulating plant growth
  through synthesis of growth promoting
  substances.
 Totally harmless, pollution free and low-cost
  renewable agricultural inputs.
Components of Biofertilizers
   Decomposers
     Reliable  to break down organic waste and dead
      organisms.
     Releases key ions such as nitrates, phosphates and
      sulfates for use by other organisms.
     Composting relies on bacterial action.
     Many types of bacteria participate in the composting
      process, thriving at different temperatures and on
      different materials
          Mesophiles
          Thermophiles (hot)
    Components of Biofertilizers
   Nitrogen Fixing Bacteria
     Contained   in the root nodules of legumes.
     Contained in the root nodules of alders.
     Free living.
     convert nitrogen from the atmosphere into
      ammonium (NH4) or nitrate (NO3) ions.
    Components of Biofertilizers
   Phosphorous Solubilizing Bacteria
     Solubilizingunavailable organic &
      inorganic forms of phosphorus (80%).
     Organic P slowly mineralized by the action
      of phosphatases.
     Inorganic P solubilized by the action of
      organic and inorganic acids.
    Components of Biofertilizers
   Nutrients Up Take Enhancing Fungus
    (Mycorrhizae)
     Maximizing  plants absorption area
      (Mycorrhizae can penetrate smaller
      crevices than root hairs)
     Improve soil texture
    Components of Biofertilizers
   Nutrients Up Take Enhancing Fungus
    (Mycorrhizae)
     Increase water uptake in plant.
     Increase mineral uptake (especially P, Cu
      & Zn).
     Sequester heavy metals (Os, Pb).
     Limits uptake of Al, As, Ti, Ba & Cd).
Mycorrhizae
   Ectomycorrhizae
     Form   a sheath around the root
     The mass of the hyphae =mass of roots
     Hyphae grow between the spaces in the cortical root cell
     No cellular penetration by the hyphae
     Reproductive spores are formed in the soil only
     Members of the basidomycetes or the ascomycetes
     Infect Pinaceae, Fabaceae, and temperate forests
Mycorrhizae
   Endomycorrhizae
     Hyphae   form arbuscles (Bulb structure) where they
      penetrate plant cell
     Were called VAM (vesicular arbuscular mycorrhizae)
     Direct cell to cell exchange of nutrients
     Hyphae grow into intercellular spaces of cortical cells,
      but penetrate cell walls
     Spores can form inside or outside of plant cell
     Belong mainly to Glomalae family
     Infect grasses, most herbaceous dicots, many perennial
      shrubs and 6 genera of gymnosperms
Biological Control
   Pest suppression with biological agents
    operating in a background of integrated
    control that does not depend on host
    resistance, sterilization of the target
    pathogen, or modification of pest behavior
          Biological Control
            Mechanisms
 Competition
 Antibiosis
 Parasitism
 Induced resistance
 Disease agent transfer
Microbial Pesticide
Microbial agent intended for preventing,
destroying, repelling, or mitigating any pest, or
intended for use as a plant regulator, defoliant, or
dessicant, that:
(1) Is a eucaryotic microorganism including, but
not limited to, protozoa, algae, and fungi;
(2) Is a procaryotic microorganism, including, but
not limited to, eubacteria and archaebacteria; or
(3) Is a parasitically replicating microscopic
element, including but not limited to, viruses.
Microbial Pesticides

   May control many different kinds of
    pests, although each separate active
    ingredient is relatively specific for its
    target pest[s].
Finding Biological Control
Organisms
  Suppressive soils
  Old world
  Plant pathogens
  Strange and unusual places
Bacillus thuringiensis
 An insecticidal bacterium for control of many
  important plant pests - mainly caterpillars of
  the Lepidoptera (butterflies and moths) but
  also mosquito larvae, and simuliid blackflies
  that vector river blindness in Africa.
 Bacteria produce a crystalline protein that is
  toxic to a number of insects when ingested
     Paecilomyces lilacinus
A  thermophile fungus
 Parasitize nematodes: attacks
  eggs, juveniles and females
    Other Known Bacteria for
    Biological Control Agents
 Agrobacterium radiobacter
 Bacillus subtilis
 Burkholderia cepacia
 Pseudomonas fluorescens
 Pseudomonas syringae
 Pseudomonas chloroaphis
 Streptomyces griseoviridis
 Ampelomyces quisqualis
Other Known Fungus for
Biological Control Agents
Paecilomyces fumosoroseusTrichoderma harzianum
Candida oleophila        Trichoderma polysporum
Coniothyrium minitans    Trichoderma viride
                          Beauveria bassiana
Fusarium oxysporum
                          Beauveria brogniartii
Gliocladium virens
                          Vertricillium dahliae
Gliocladium catenulatum  Verticillum lecani
Phlebia gigantea         Metarhizium anisopliae
Pythium oligandrum       Phlebiopsis gigantea
    Known Viruses for
    Biological Control Agents
For Insect Control
Granulovirus (Baculoviridae) for controlling
  Adoxophyes orana and Cydia pomonella
Nucleopolyhedrovirus (Baculoviridae) for controlling
  Helicoverpa armigera and Spodoptera exigua

Viruscides (Inoculation)
Potyvirus (Potyviridae) for controlling Zucchini Yellow
  Mosaic Virus
Other Microbial Agents
 Pseudomonas sp. & Bacillus megaterium
 PGPBs or PSBs
 Work through 1 of 2 mechanisms
 Phytohormones secretion improve root
  growth
Bioremediation Agents
Decomposers also:
 Eat toxic compounds like oil, battery acid,
  detergents, pesticides (DDT), some plastics, and
  even radioactive waste products that can
  accumulated in dumps, alongside roads
 Degrade sewage in treatment plants and septic
  tanks, helping to recycle the 11 billion pounds of
  dung that people on earth produce every day,
  and turning it into fertilizer that is then returned
  to the biosphere
Bioremediation
  Natural – using what bacteria exist in the
   soil to break down toxic materials
  ―Nature's Way to a Cleaner Environment‖
  Enhanced – using ―super bugs‖ or merely
   enhancing the environment
Steps to Utilize Soil Microbes
 Isolation
 Identification
 Plant (for biofertilizers) or Target Pest (for
  Biological Control Agents) Test
 Pot Culture Test: Sand test, Sterilized Soil
  test, and Unsterilized Soil test
 Field Trial
Inocula Development

Monocultures
• Processes requiring monocultures
• Sources of monocultures
• Preserving pure cultures
Advantages and disadvantages of pure cultures
• Advantages: easy to obtain (isolate or purchase); better
control of products; can be patented
• Disadvantages: subject to contamination and genetic
change
Processes Requiring Mono
Cultures
PURE CULTURE FERMENTATIONS
 industrial ethanol
 alcoholic beverages
 fermented foods
 pharmaceuticals
 acetone-butanol
 acetic acid
 single cell protein
 industrial enzymes
 biotech products (insulin, growth hormone)
 Biological Control Agents
 Biofertilizers
 Culture Collections Supply of
 Industrial Microorganisms
Abbrev.                    Name                             Location
ATCC      American Type Culture Collection           Rockville, MD, U.S
CBS       Centraalbureau voor                        Baarn, The Netherlands
          Schimmenlculturen
CDDA      Canadian Department of Agriculture         Ottawa, Canada
CMI       Commonwealth Mycological Institute         Kew, United Kingdom
FAT       Faculty of Agriculture, Tokyo University   Tokyo, Japan
IAM       Institute of Applied Microbiology          Tokyo, Japan
          University of Tokyo
NCIB      National Collection of Industrial Bacteria Aberdeen, Scotland
NCTC      National Collection of Type Cultures       London, United Kingdom
NRRL      Northern Regional Research Laboratory Peoria, IL, United States
PCC       Pasteur Culture Collection                 Paris, France
Inocula Development

Mixed cultures
• Processes requiring mixed cultures
• Defined versus enrichment cultures
• Sources of mixed cultures
• Preserving mixed cultures
Advantages and disadvantages of mixed cultures
• Advantages: obtained by enrichment or purchased; can't
  be patented; contamination not as much of problem
• Disadvantages: control of culture and products is less
  definite;
Processes Requiring Mixed
Cultures
MIXED CULTURE FERMENTATIONS
 breads: sour dough, soda cracker
 wines
 vegetables: pickles, sauerkraut
 dairy products: yogurt, sour cream
 composting
 anaerobic digestion
 soil and groundwater remediation
 bioleaching
 microbial enhanced oil recovery
 microbial metals recovery
 waste treatment
INOCULUM PRODUCTION
   DEFINITION OF INOCULUM
     Living  organisms or an amount of material containing
      living organisms (such as bacteria or other
      microorganisms) that is added to initiate or accelerate a
      biological process

   CRITERIA
     Healthy, active state - minimize lag   period
     Available in sufficient quantities
     Suitable morphological form
     Free of contamination
     Stable - retain its product   forming properties
CHOICE OF MICROORGANISM

 Nutritional characteristics - cheap medium
 Optimum environmental conditions
 Productivity - substrate conversion,
  product yield, rates
 Ease of handling and safety (suitability)
SAFETY
   LAMINAR FLOW CABINETS used;
     to limit exposure of operators to aersols and
      other possible infections
     to protect the culture material from
      contamination
 ASEPSIS MUST BE MAINTAINED
 CORRECT STANDARD MUST BE
  APPLIED
SAFETY STANDARD
 CLASS 1 - none or minimal hazard
 CLASS 2 - ordinary potential hazard
 CLASS 3 - Special hazard, require special
  containment
 CLASS 4 - Extremely dangerous, may
  cause epidemic disease
 CLASS 5 - Pathogens excluded by law
  STORAGE AND PRESERVATION

Isolates/cultures should retain desirable characteristics
 over long periods of time
METHODS
    Storage    at reduced temperatures
         Slopes - refrigerator (4 oC), freezer (-20 oC), deep freezer (-80 oC)
         Fungal spores in water (5 oC)
         Liquid nitrogen (-150 to -196 oC)
    Storage    in dehydrated form
         Soil + culture dried. Used for fungi
         Lyophilization\freeze drying. Freezing of culture followed by drying
          under vacuum which results in sublimination of cell water
    QUALITY CONTROL OF
    PRESERVED CULTURES
 Each batch must be routinely tested
 Whatever method is used in preservation of
  stock cultures it is important to assess the
  quality of the stocks
 Each batch of cultures should be routinely
  checked to ensure the propagated strains
  retain the correct growth charatertistics,
  morphology and product forming properties
Stability and performance of a
culture
Influenced by
 Mode of substrate feeding
 Nutrients
 Temperature
 Osmotic pressure
 Oxygen
 Intracellular product accumulation
FUNGAL INOCULA
 Spore  suspension - used at early stages,
  small pellets in subsequent transfers
 Inoculum affected by morphology of
  fungus - can influence size of pellet or floc
Spore Production
 Solidified media e.g. agar media + roll-bottle
  technique
 Solid media e.g. cereal grains, bran, malt,
  flaked maize etc. (amount of water, relative
  humidity of air, temp. are important)
 Submerged culture - influenced by media
Problem with Mycorrhizae
 Mycelium better inoculant in many cases
 Difficult to grow in culture
    Formulation of Inocula
   The ecological competence (the ability of
    microbial cells/inocula to compete and
    survive in nature) of laboratory/bioreactor
    prepared inocula is important to commercial
    exploitation of biotechnological processes
    initiated by the addition of microbial cultures
    to natural habitats.
   It caused by inability to regulate the process
    environment stringently
    Formulation of Inocula
   Inocula systems will require, as a first step, an
    efficient formulation and delivery system, based on
    microenvironmental control, directed at minimizing
    the lag period and maximizing competitive
    advantage to the introduced microorganisms
   The use of polymer gels, for example alginate, to
    immobilize cells has allowed the development of
    spatially organized microenvironments with control
    on the degree of protection afforded, the rate of cell
    release and the positioning of cells with nutrients
    and/or selective agents or chemicals
Types of Inoculant
   Comparison of Sterile and
Nonsterile Peat-based Inoculants
Comparisons of Alginate and Peat
          Inoculant
Comparisons of Alginate and Peat
          Inoculant
   Other
 materials
    as
 potential
  carriers
    for
 bacterial
inoculants
Methods of
inoculation
 with peat-
   based
 inoculants
Methods of inoculation with peat-based inoculants
Factors Influencing
Bacterial Survival in Soils
   Grass Root Colonization by Four
   Bacterial Species




(B. cepacia P2, Flavobacterium sp. strain F4, P. fluorescens, and Alcaligenes sp.
Maximum Abundance Percentage of Actinobacterial
Genera in the Roots of Wheat Grown from Seed
Inoculated with Endophytes



                                   (Microbispora sp. [EN2],
                                   Streptomyces sp.[EN27],
                                   N. albus [EN46])
Maximum Abundance Percentage of Actinobacterial
Genera in the Roots of Wheat Grown from Seed
Inoculated with Bacterial Inoculum (Nutrilife)
           FIELD TRIAL

 Biofertilizers
Rhizobium , Azetobactor ,
Azospirillium and Phosphate
solubilising organisms        A view of untreated (control) field
                              of Pea



 Bio Control Agents
Fungal antagonists
 (AGAINST
 ROOT-ROT FUNGI)
                              A view of biofertilizer &
                              biocontrol agent treated field of
                              pea
Biofertilizers and
Biological Control Agent
 Remember that it is a living organism.
 Living organisms have preferences (Give a
  correct inoculant for a certain crop or pest).
 Organisms may die off in package (Make
  sure it hasn’t expired, fresher better).
 Storage affect lifespan (store in a cool, dark
  place until used).
 Use ASAP after inoculating.
Example of Problem
Reduced Nodulation
   Improper storage of bacteria (heated, dried).
   Fertilizer will kill bacteria if mix granular and
    fertilizer in seeding operation. Fertilizer
    desiccates bacteria.
   Herbicide residue (glyphosate that inhibits root
    hairs will reduce nodulation.
   Plant stress.
   Saline, inadequate soil fertility, low pH.

				
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