Biocontrol biopesticide
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The document discusses the preparation of biocontrol agents (biopesticides) from microorganisms. It describes the process of isolating beneficial microbes from soil samples, including Trichoderma fungi, Bacillus bacteria, and Pseudomonas bacteria. The microbes are purified into pure cultures and their antagonistic effects against pathogens are tested. The microbes are mass cultivated in nutrient broth, then formulated into talc-based powders or with molasses yeast broth. The biopesticide formulations are tested for shelf life and plant treatment effects. In summary, the document outlines the process of isolating beneficial soil microbes, producing pure cultures, testing their effects against plant pathogens, mass cultivating them, and formulating
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Application of biocontrol agents
Biological control agents can be applied using three main techniques: introduction, augmentation, and conservation. Introduction involves deliberately introducing natural enemies into areas where they are not native. Augmentation involves mass rearing and releasing natural enemies to supplement existing populations. Conservation preserves and increases natural enemies through environmental manipulation. Specific application methods depend on the type of agent. For insects, techniques include seed treatment, soil application, and foliar application. Release methods aim to maximize agent effectiveness against target pests.
Trichoderma
Trichoderma is a common fungus that lives in soil and suppresses plant pathogens through competition, parasitism, and antibiosis. It has been used for over 75 years as a biocontrol agent to reduce diseases caused by fungi and bacteria. Trichoderma works against pathogens by competing for space and nutrients, parasitizing other fungi through coiling around and lysing their hyphae, and secreting metabolites that have harmful effects. It also promotes plant growth. Trichoderma is mass produced on molasses medium and formulated into powders to apply as seed treatments, soil drenches, or amendments to improve soil health and provide natural disease control for crops.
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BAU-Biofungicide is a biofungicide formulated from the fungus Trichoderma, which protects crops from diseases caused by fungi. It was developed by Professor Ismail Hossain at Bangladesh Agricultural University. Trichoderma species are common soil fungi that act as biocontrol agents against plant pathogens through several mechanisms. BAU-Biofungicide can be used to treat seeds of many vegetables, pulses, oilseeds, and cereals, providing benefits like increased germination, plant growth, and yields. It is effective against seed-borne and soil-borne diseases as well as nematodes. Being environmentally friendly and easy to produce, BAU-Biofungicide provides farmers an affordable means
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Nitrogen is a key nutrient for plants but it exists primarily as inert nitrogen gas in the atmosphere. The nitrogen cycle describes the process by which nitrogen is converted between various chemical forms as it moves between the atmosphere, soil, organisms, and various environmental reservoirs. Key steps in the nitrogen cycle include nitrogen fixation by bacteria, nitrification of ammonia to nitrites then nitrates, assimilation of nitrogen by plants and animals, ammonification of organic nitrogen waste by decomposers, and denitrification of nitrates back to nitrogen gas. The nitrogen cycle is essential for providing plants with nitrogen needed for growth while enriching soils with nutrients.
Psychobiotics
Psychobiotics refers to live bacteria that may provide mental health benefits by affecting the microbiota in the host's gut-brain axis. Common psychobiotics used in research include Bifidobacterium and Lactobacillus families of gram-positive bacteria, which are less likely to cause an immunological response. Specific psychobiotics like Lactobacillus rhamnosus have been shown to lower anxiety and reduce obsessive-compulsive disorder. Probiotic foods high in beneficial bacteria include yogurt, kimchi, kefir and sauerkraut. Research suggests psychobiotics may reduce stress hormones, stimulate the vagus nerve, and increase neurotrophic factors to help treat anxiety disorders.
Measuring RNA concentration
The document describes how to calculate the concentration of an RNA stock solution given absorbance measurements of a diluted sample. It explains that an absorbance of 1.0 at 260nm corresponds to 40 μg/mL of RNA. It then shows the calculations to determine that a diluted sample with an absorbance of 0.142 corresponds to 5.7 μg/mL, and that multiplying this by the dilution factor of 1000/40 gives a stock concentration of 142.5 μg/mL.
Burst size
The burst size experiment involved infecting 2 x 108 cells with phage at an MOI of 0.1. Unadsorbed phage were removed and infected cells were incubated for 90 minutes. 200 infective centers (ICs) were counted initially. After incubation, 150 plaques were counted, corresponding to 3 x 109 PFU/ml. The burst size was calculated as the PFUs released (3 x 109 PFU/ml) divided by the original ICs (2 x 107 ICs/ml), which is 150 PFUs/IC.
Diluting bacteriophage
A phage stock with a concentration of 2.5 x 109 PFU/ml needs to be diluted to give approximately 500 plaques on a plate. To calculate the required dilution factor, the researcher sets up an equation relating the original phage concentration, desired plaque count, and dilution factor. Solving the equation reveals the stock must be diluted 2 x 10-7 times. The document then shows example dilutions and volumes that would achieve this dilution factor.
Measuring phage titer
A series of dilutions were performed on a bacteriophage stock to determine its titer. The stock was diluted through four successive 10-fold dilutions. After plating 0.1 mL from the final dilution, 180 plaques were counted. Using the dilution factor of 1x10-8 mL and the formula that divides the number of plaques by the dilution factor, the titer of the original bacteriophage stock was calculated to be 1.8 x 1010 PFU/mL.
Ssdna calci
This document provides instructions for calculating the concentration of single-stranded DNA (ssDNA) in micrograms per milliliter (μg/mL). It explains that an optical density (OD) reading of 1 at 260nm corresponds to 33 μg/mL of ssDNA. It then works through an example calculation where M13mp18 DNA diluted 10 μL into 1000 μL has an OD of 0.125, corresponding to a concentration of 4.125 μg/mL. To determine the original stock concentration, this value is multiplied by the dilution factor of 1000 μL/10 μL, giving a concentration of 412.5 μg/mL for the M13mp18 DNA stock solution.
Calculation of ds DNA Concentration
This document summarizes the formula for calculating the concentration of double-stranded DNA (dsDNA) based on its absorption at 260nm. The formula, known as Beer's Law, states that absorption at 260nm (A260) equals the extinction coefficient at 260nm (E260) multiplied by the concentration (c) and light path length (l). For dsDNA, the extinction coefficient is 20 for a 1mg/mL solution and 1cm light path length. Two examples are provided to demonstrate calculating DNA concentration in μg/mL from a given A260 value using this formula and extinction coefficient.
Moi ii
The document describes how to calculate the multiplicity of infection (MOI) for an experiment using E. coli and bacteriophage. It shows that a 0.25 mL aliquot of E. coli culture containing 2 x 108 cells requires 0.05 mL of a bacteriophage stock with a concentration of 2 x 109 phage/mL to achieve an MOI of 0.5. The calculation involves determining the number of cells, calculating the number of phage needed based on the MOI and cell count, and then calculating the volume of stock solution required to provide that number of phage.
Multiplicity of infection
The document discusses the calculation of multiplicity of infection (moi), which is the ratio of bacteriophages to host cells at the initial stage of infection. Too high or too low of an moi can impact the infection process. The document then provides an example calculation where 0.1 mL of a phage stock containing 4 x 109 phage/mL is added to 0.5 mL of E. coli cells at 2 x 108 cells/mL. It calculates the total number of phage and cells, then divides the phage by the cells to determine the moi is 4 phage/cell.
Stock solution calculation
To prepare 300ml of 1x TAE buffer solution from a 50x stock solution, calculate the volume of stock solution needed using the formula C1V1=C2V2. Plugging in 50x for C1, 1x for C2, 300ml for V2 gives a value of 6ml for V1, the volume of 50x stock solution required. Take 6ml of the 50x TAE stock solution and dilute it with deionized water to a total volume of 300ml to obtain the desired 1x TAE buffer solution.
Transposons
Transposons are segments of DNA that can move from one location in the genome to another. There are two main types: insertion sequences (IS), which are short DNA sequences that code for transposase enzymes; and transposons, which can carry additional genes and are flanked by IS elements or inverted repeats. Transposition occurs when the transposase enzyme cuts and inserts the transposon into a new target site, sometimes duplicating flanking DNA. This movement allows transposons to generate genetic diversity and influence genome evolution over generations.
Ara operon
The L-arabinose operon contains three structural genes - araB, araA, and araD - that encode enzymes required for the metabolism of L-arabinose in E. coli. The expression of the operon is regulated by the AraC protein and the CAP-cAMP complex. In the absence of L-arabinose, AraC acts as a repressor by forming a DNA loop, while in the presence of L-arabinose, AraC binds to different DNA sites and activates transcription. CAP facilitates activation when glucose is absent. The operon is also used in protein expression systems by fusing genes of interest to the araBAD promoter.
Trp operon
The document discusses the tryptophan (trp) operon in bacteria. The trp operon contains five structural genes that encode enzymes for tryptophan biosynthesis. Expression of the structural genes is regulated by a trp promoter, operator sequence, and repressor protein. In the absence of tryptophan, the repressor cannot bind to the operator and the structural genes are transcribed. However, in the presence of tryptophan, it binds to the repressor and prevents transcription of the structural genes. The trp operon is also regulated by attenuation - a mechanism where tryptophan availability affects the formation of transcription terminator stem loops in the mRNA leader sequence.
Lac operon
The document describes the lac operon in E. coli, which regulates genes involved in lactose metabolism. The lac operon contains three structural genes - lacZ, lacY, and lacA - that are regulated by the lac repressor and catabolite activator protein (CAP). The lac repressor normally binds to the operator region and blocks transcription. In the presence of lactose, allolactose binds to the lac repressor and causes it to detach from DNA. CAP promotes transcription when glucose levels are low, allowing the cell to preferentially metabolize lactose over glucose. Together, these regulatory mechanisms ensure the lac genes are only expressed when lactose is available and glucose is not.
Classification of culture media
This document discusses the classification and preparation of culture media in microbiology. It begins by explaining the different types of nutritional requirements in microbes and aseptic techniques used in the lab. It then summarizes various forms of culture media like broths, slopes, plates and semi-solid media. Media can be classified based on agar concentration, ingredients used, and oxygen requirements of microbes. Simple and complex media types are described as well as enrichment and selective media. Special media like Robertson's meat broth and thioglycollate are used for isolating anaerobic bacteria.
Different forms of media
This document discusses different forms of culture media used in microbiology. It describes nutrient broth as a liquid culture medium used to grow fastidious microorganisms. Preparation of nutrient broth involves adding broth powder to water, mixing, pouring into containers, and autoclaving. Agar slopes are prepared by pouring molten agar into test tubes at an angle to solidify in a slanted form. Agar deeps allow observation of bacterial growth patterns in agar due to oxygen gradients. Solid and semisolid media contain varying concentrations of agar to control consistency and allow different growth applications.
Phage typing
Phage typing is a phenotypic method used to differentiate between bacterial strains using bacteriophages (viruses that infect bacteria). Certain bacteriophages can only infect specific bacterial strains. Phage typing involves growing bacterial cultures and spotting them with different bacteriophages - susceptible strains will show clearings where the bacteria have been lysed. While a complex method, phage typing has been useful for epidemiological surveillance and tracing outbreaks of pathogens like Salmonella typhimurium and Staphylococcus aureus. Improved standardization is needed to ensure reliable comparison of results between laboratories.
Production of biodegradable. plastic
The document discusses production of biodegradable plastics. It notes that over 140 million tons of plastic are consumed annually, most of which are not biodegradable. Bioplastics are a feasible alternative as they are made from renewable resources like vegetable oils and can biodegrade. Common types of bioplastics include polylactic acid, poly-3-hydroxybutyrate, and thermoplastic starch. The production process for polylactic acid and uses of bioplastics in packaging, catering, gardening, medical, and sanitary products are described.
Lytic and lysogeny
The document summarizes the lytic and lysogenic cycles of bacteriophage λ (lambda), which infects E. coli bacteria. It describes the key genes involved in each cycle and how they regulate viral replication and integration into the host chromosome. The lytic cycle results in viral assembly and lysis of the host cell, while the lysogenic cycle involves integration of the viral DNA into the host genome and replication with the host. The document outlines the complex interplay between viral and host genes that determines whether the lytic or lysogenic cycle occurs.
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Chapter 2
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Chapter 3
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Biocontrol biopesticide
- 1. Y5C10 ENTREPRENEURSHIP IN MICROBIOLOGY Unit – IV Preparation of Biocontrol agent (Biopesticide) Dr. S. Sivasankara Narayani Assistant Professor Department of Microbiology Ayya Nadar Janaki Ammal College Sivakasi, TamilNadu, India 01-09-2020Dr.SS
- 2. Biopesticides • Biopesticides are certain types of pesticides derived from such natural materials as animals, plants, bacteria, and certain minerals. For example, canola oil and baking soda have pesticidal applications and are considered biopesticides 01-09-2020Dr.SS
- 3. Classes • Biochemical pesticides • Microbial pesticides • Plant-Incorporated-Protectants 01-09-2020Dr.SS
- 4. Preparation • Culture isolation • Purification • Mass cultivation • Preparation of Talc based formulation • Packaging 01-09-2020Dr.SS
- 5. Culture isolation • Maize crop – pull out – root – soil sample • Nutrient agar media • Trichoderma selective media • King’s B Medium • Actinomycetes isolation agar 01-09-2020Dr.SS
- 6. Pure culture technique – Pour plate technique • Dilution preparation upto 10-7 • Serial dilution • 10-3 for the isolation of fungi Trichoderma – Pour plate technique – add pinch of streptomycin sulfate in media before pouring onto petri plate. • Three replicates 01-09-2020Dr.SS
- 7. Cont.. • 10 -6 dilution for isolation of Bacillus – transfer 1ml of suspension to a sterile petriplate – pour Nutrient agar media - solidfy • 10-6 dilution for isolation of Pseudomonas - transfer 1ml of suspension to a sterile petriplate – pour kings’ B media – solidfy • Wrap the petriplates and incubate at RT 01-09-2020Dr.SS
- 8. Cont… • 7 days – fungi • 2-3 days – bacteria • 7 – 14 days – actinomycetes 01-09-2020Dr.SS
- 9. Cont… • Bacteria observed after incubation • Trichoderma – white cottony growth • Subcultured the colonies using PDA for culture maintenance and further studies 01-09-2020Dr.SS
- 10. Cont… • Pick single colony (bacteria) from plate – using buds - simple streak on media ( Three replicates) • Sterilize the inoculation needle – take Trichoderma colony using loop – place on PDA center spot. • Pure colonies - Bacillus, Pseudomonas • Pseudomonas - fluoresce • Bacillus -straighted margins 01-09-2020Dr.SS
- 11. Cont… • Pure colonies of Trichoderma observed after – 3- 4 days • After 6- 7 days - full growth • Actinomycetes after 2 days growth starts • 7 days – clear growth 01-09-2020Dr.SS
- 12. Antagonistic activity • Dual plate technique • Make 8mm mycelial disk by using sterile cork borer • Place mycelial disk Pythium on one side of petriplate – 1cm away from side margins • Make 8mm mycelial disk of Macrophomina • Fusarium • Take biocontrol agent –using buds – streak biocontrol agent – opposite side of the petriplate at the opposite side – perpendicular to the pathogen 01-09-2020Dr.SS
- 13. Cont.. • Bacillus inhibit Pythium • Pseudomonas – Fusarium • Trichoderma – Fusarium • Bacillus, Pseudomonas, Actinomycetes- Macrophomina 01-09-2020Dr.SS
- 14. Mass Multiplication • Nutrient agar broth • King’s broth • Sterilize loop – Bacillus culture – inoculate into nutrient broth – 2 – three times • Pseudomonas culture – inoculate into King’s B broth • Incubate broth in a rotary shaker at 150 rpm for 72 hours at RT • After 72 hours – 9 x 108 CFU/ml 01-09-2020Dr.SS
- 15. Talc based formulation • 1 Kg talc powder – add 10g Carboxy methyl cellulose – 400 ml of bacterial suspension – mix well • Shade dry the product – reduce moisture content - < 20% 01-09-2020Dr.SS
- 16. Molasses yeast broth • Trichoderma – mycelial mat of Trichoderma observed after inoculation • Homogenised mycelial mat of Trichoderma – talc powder – mix – refined product • Commercial product 01-09-2020Dr.SS
- 17. Assessing shelf life of Bioformulation • Serial dilution method • 1g of Trichoderma talc powder – serial dilution upto 10-7 • take 1ml of 10-3 dilution – add plate – pour Trichoderma selective medium • Test Pseudomonas - Kings’B medium and Bacillus - Nutient agar Talc Powder • Colonies were observed 01-09-2020Dr.SS
- 18. Test • Treat with plants 01-09-2020Dr.SS
- 19. Questions to think • Biopesticide • Mechanism behind biopesticide? • Name the ingredients of media which is used for Biopesticide production? • Applications of Biopesticide • What are types of organisms comes under biopesticides? 01-09-2020Dr.SS
- 21. 01-09-2020Dr.SS