Plant Care

Total Plant RNA Isolation Using Trizol Reagent 1. Crush 100 mg of tissues in 1mL of triazole reagent by pestle mortar or micro pestle 1.5 or 2.0 mL RNase free Eppendorf tubes. *Incubate for 2-3 minutes at room temperature. 2. Add 200 uL chloroform and mix by gently inverting the tube. *Incubate for 2-3 minutes at room temperature. 3. Centrifuge at 12000xg at 4oC for 15 minutes. 4. Transfer the supernatant into fresh 1.5 mL RNA’s free tube. 5. Add 500 μL isopropanol mix gently by inverting the tube. *Incubate for 10 minutes at room temperature. 6. Centrifuge at 12000xg at 4oC for 10 minutes. *Discard the supernatant and keep the pallet in the tube. 7. Add 1mL of ice-chilled 75% ethanol to wash the pallet. 8. Centrifuge at 7500xg at 4oC for 5 minutes. *Discard the supernatant and keep the pallet in the tube. *Keep lid opened tube at 4oC for 15 min to air dry the pallet. 9. Add 50-100 uL RNA’s free water in pallet and mix by gentle pipetting. 10. Incubate for 10-15 m

Isolation And Purification Of Plant Pathogens-Citrus Canker Pierce the margin of an isolated canker lesion on a citrus leaf with an isolated alcohol-flamed dissecting needle. From each tomato sample select, young lesions cut out a small piece of the tissue using an alcohol-flamed sharp scalpel. Use alcohol-flamed tweezers to transfer the excised tissue into an empty Petri dish containing a 2.5 µl drop of sterile tap water. Carefully macerate the tissue using an alcohol-flamed sterile scalpel and tweezer to release the Bacteria. Use a flamed inoculating loop to transfer a small amount of the bacterial suspension onto a Petri plate containing NA Medium. Subsequently perform a quadrant streak on the plate making sure to flame and cool the inoculating loop before each streak. Label the plates with your name and sample type and place the plates upside down in the 28Ĉ incubator. Isolation And Purification Of Plant Pathogens-Citrus Canker ----------------------------------

Isolation And Purification Of Plant Pathogens-Bacteria Purification   Xanthomonas spp.   Isolation And Purification Of Plant Pathogens-Bacteria Obtain specimens and record the appearance of the tissue and bacterial lesions for the tomato plants inoculated with Xanthomonas vesicatoria cause the tomato lesions. From each tomato sample select, young lesions cut a small piece of the tissue using an alcohol-flamed sharp scalpel. Use alcohol-flamed tweezers to transfer the excised tissue into an empty Petri dish containing a 2.5 µl drop of sterile tap water. Carefully macerate the tissue using an alcohol-flamed sterile scalpel and tweezer to release the Bacteria. Use a flamed inoculating loop to transfer a small amount of the bacterial suspension onto a Petri plate containing NA Medium. Subsequently perform a quadrant streak on the plate making sure to flame and cool the inoculating loop before each streak. Label the plates with your name and sample type and place the p

GEL Procedure Of Gel Electrophoresis For Separation-DNA/RNA GEL ELECTROPHORESIS GEL Procedure Of Gel Electrophoresis For Separation-DNA/RNA Mix 1 g agarose in 99mL of TAE/TBE buffer and heat it in the oven. Add 10 uL of 10mg/mL ethidium bromide in 100 mL of hand cold agarose gel solution. Pour this mixture into gel caster/tray having comb and sealed from sides. Let it be solidified at room temperature for 30-60 minutes. Remove the comb seals from the gel caster and place it inside the gel tank containing TAE/TBE buffer. Put the DNA samples/PCR product (mixed in 6x loading dye) inside each well and note it in the lab. Notebook. Attach the cathode (towards wells) end of gel and anode (at the bottom) of the power supply to the gel tank. Switch on the power supply giving 80-120 volts and run it for sufficient time allowing the samples to travel in more than half of the gel length. Take the gel out of the gel tank and observe it in a gel doc system/UV transillumin

 Recipes For Semi Selective Media For Pathogens Growth SM-1 medium (18) to TTC medium add the following after autoclaving;  For Litter of Solution (1000ml) Merthiolate tincture               5 to 50 µl Crystal violet                         50mg Polymyxin 𝛽 sulfate              100mg Tyrothricin                            20mg Chloromycetin                      5mg  Cycloheximide                     50mg **Dissolve in 5ml of 75% ethanol 30 min prior to use.  Recipes For Semi Selective Media For Pathogens Growth Modified SMSA medium (12-14) Crystal violet 1%                0.5 ml Polymyxin 𝛽 sulfate  1%    10.0 ml Bacitracin 1%                     2.5 ml Chloromycetin 1%            0.5 ml Penicillin  0.1%                 0.5 ml Cycloheximide 1% in 70% ethanol 2.5 ml  Recipes For Semi Selective Media For Pathogens Growth. ------------------------------------------------------------------------------------------ Here You Can Find Plant Disease and

Media Recipes For Pathogens Growth On Artificial Mediums CPG (1L) CPG (For One Litter Solution) Differential media for Ralstonia from laboratory guide for identification of Plant Pathogenic Bacteria 1.0g      Casamino acid (casein hydrolysate) 10.0g    Peptone 5.0g      Glucose 17.0g    Agar Media Recipes For Pathogens Growth On Artificial Mediums YNA (Yeast extract nutrient agar) - (For 1L) Differential media for Xanthamonas from laboratory guide for identification of Plant Pathogenic Bacteria 5.0g     Yeast extract 23.0g   Nutrient agar NA (Nutrient agar) (1L) Media Recipes For Pathogens Growth On Artificial Mediums Cultivation of a wide variety of Microorganisms from Difco Nutrient Agar container 23.0g      Nutrient Agar 1/4 PDA (QPDA) (Potato Dextrose Agar) (1L) 9.75g   Potato Dextrose Agar 11.25g  Agar PDA (Potato Dextrose Agar) (1L) Media Recipes For Pathogens Growth On Artificial Mediums 39g  Potato Dextrose Agar

GENERALIZED LIFE CYCLE OF BASIDIOMYCOTA Basidiomycota is one of two large phyla that, together with the Ascomycota,  comprise the subkingdom Dikarya (often referred to as the "higher fungi") within the kingdom Fungi. Basically, Basidiomycota are filamentous fungi composed of hyphae (except for yeasts), and reproducing sexually via the formation of specialized club- shaped end cells called basidia that normally bear external meiospores (usually four). These specialized spores are called basidiospores. (Edited from Google images). ------------------------------------------------------------------------------------------ Here You Can Find Plant Disease and Pathogens (Fungi, Bacteria, Molllicutes, Parasitic higher plants, Parasitic Green Algae, Nematodes, Protozoa, Viruses, Viroids,Prions) Diagnosis and Treatment, Control Against Plant Disease. Laboratory Experiments & Work. Plant Pathology Lab Manual. House Plant-Trees Care Guide, Snake Plant Care, Pot

INTRODUCTION TO FUNGI GENERALIZED LIFE CYCLE OF ASCOMYCOTA The Ascomycota are a Division/Phylum of the kingdom Fungi, and subkingdom Dikarya. Its members are commonly known as the sac fungi. They are the largest phylum of Fungi, with over 64,000 species. The defining feature of this fungal group is the "ascus", a microscopic sexual structure in which nonmotile spores, called ascospores, are formed. However, some species of the Ascomycota are asexual, meaning that they do not have a sexual cycle and thus do not form asci or ascospores. (Edited from Google images)  ------------------------------------------------------------------------------------------ Here You Can Find Plant Disease and Pathogens (Fungi, Bacteria, Molllicutes, Parasitic higher plants, Parasitic Green Algae, Nematodes, Protozoa, Viruses, Viroids,Prions) Diagnosis and Treatment, Control Against Plant Disease. Laboratory Experiments & Work. Plant Pathology Lab Manual. House Plant-Trees Care

PREPARATION OF TEMPORARY MOUNT: Temporary mount of diseased specimen is frequently required for microscopic  examination. Such mounts can be made as follows: Take a clean slide and cover slip. Place a drop of water on the slide. Now add the specimen which needs to be examined. Care must be taken that  the specimen should not be of large size. Size of specimen should be in few  millimeters so that it could easily settle down in drop of water. In many cases it may be necessary to tease the specimen with dissecting needle so that it spreads uniformly in a thin layer and does not form a clamp which will  hamper the observation. Then the cover slip is placed on the slide. To avoid the air bubble in mount it is  necessary to place the cover slip carefully. First place the one edge of cover slip in contact with water then with the help of dissecting needle gently and slowly lower the cover slip. Instead of water, lectophenol can also be used for mount preparation b

PREPARATION OF MEDIA FOR CULTURE: Media play an important role not only for the nutrition and growth of the pathogen but also in identification through growth pattern on media. Fungi and Bacteria need synthetic media enriched with essential element for the growth. In general different types of media are prepared to culture fungi and bacteria but Potato dextrose agar (PDA) for fungi and Nutrient agar for bacteria are being used commonly.  RECIPES OF DIFFERENT MEDIA: POTATO DEXTROSE AGAR: Potato Starch 250  mL/15g Dextrose-15g Agar -Agar 15g Distilled water  750 mL/1000mL PROCEDURE TO PREPARE POTATO DEXTROSE AGAR MATERIAL: Petri dish, Flask, test tubes, Agar-agar, Dextrose, Potato starch, Water PROCEDURE: First of all take potatoes to extract potato starch. Peel the potatoes and cut into small pieces. Preferably 200 g of potato pieces are weighed, washed quickly in running water, placed in 1l liter of water and boiled for nearly

GENERALIZED LIFE CYCLE OF PLANT PARASITIC NEMATODES  T he nematodes are invertebrate roundworms that inhabit marine fresh water and  terrestrial environments. The majority of nematodes are microscopic, averaging less than a millimeter in length, but some of the animal parasites are quite large and readily visible to the naked eye. The animal and plant parasites are of direct importance in agriculture, environment, human health; however, most nematodes in the environment are not parasites. Nematodes that feed on other organisms are important participants in the cycling of minerals and nutrients in the ecosystem that is fundamental to other biological activity. Some of these nematodes may have major roles in decomposition, including biodegradation of toxic compounds. Insect-parasitic nematodes can be of importance in regulating insect populations, and are being used in the biological control of insect pests e.g. Steinernema spp. Disease Cycle: The J1 stage resides enti

Generalized Life Cycle Of Plant Pathogenic Bacteria-Plant Care GENERALIZED LIFE CYCLE OF PLANT PATHOGENIC  BACTERIA: Bacteria are the prokaryotes, unicellular microorganisms having DNA as genetic material not bounded by membrane. Plant pathogenic bacteria are rod like; diameter ranging from 0.6-3.5 μm. Different forms of flagella may be present on the body of bacteria. Bacteria reproduce asexually through binary fission or fission at rapid rate. Identification of plant pathogenic bacteria is based on visual observation including symptoms expression, colony pattern on media, microscopic observation, gram staining, serological and molecular techniques. Plant pathogenic bacteria though cause limited number of diseases in plants but all the diseases are of much economic importance. Soft rot, blight and bacterial canker of vegetable and stone fruits are important bacterial diseases. -----------------------------------------------------------------------------------------

LABORATORY BIOSAFETY PROTOCOLS: The protocols emphasize the use of good microbiological work practices,  appropriate containment equipment, proper facility design, operation and maintenance, and administrative considerations to minimize the risk of worker injury or illness.  “Laboratory biosafety” is the term used to describe the containment principles, technologies and practices that are implemented to prevent unintentional exposure to pathogens and toxins, or their accidental release. “Laboratory biosafety” refers to institutional and personal security measures designed to prevent the loss, theft, misuse, diversion or intentional release of pathogens and toxins. MICROBIOLOGICAL RISKS: Pathogenicity of the agent and infectious dose. Potential outcome of exposure. Natural route of infection. Other routes of infection, resulting from laboratory manipulations (parenteral,  airborne, ingestion).

EQUIPMENTS / MISCELLANEOUS LAB ITEMS FREQUENTLY USED IN PLANT PATHOLOGY LABORATORY SR. No. EQUIPMENTS/LAB ITEM PURPOSE 1 Spirit lamp or gas burner For sterilization of inoculation needles, forceps, scalpel, etc. 2 Autoclave For sterilization of heat stable liquids — common culture media, dry bulk materials, heat resistant instruments, glassware. 3 Koch’s steam Used for sterilization of media sterilizer or constituents which are damaged by exposure to temperature above 100°C, namely sugar, gelatin, milk. 4 Oven For sterilization of glassware and metal instruments resistant to high temperature. 5 Gas sterilants For sterilizing heat sensitive equipments and substrates 6 Liquid filters