Diseases and Management of Crops under Protected Cultivation

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(Diseases and Management of Crops under Protected Cultivation) Use of Biolog for Identification of Bacterial Plant Pathogens R.P. Singh and Smita Puri Department of Plant Pathology, G.B.P.U.A&T., Pantnagar- 263 145 (UK) In recent years, popular methods for studying plant pathogens community structure were based on the analysis of phospholipid fatty acids (PLFA) or denaturing/temperature gradient gel electrophoresis (DGGE/TGGE) etc. The above-mentioned methods are based on molecular assays. Measurements of physiological activity of microorganisms represent another approach, allowing the study of different characteristics of microbial communities. The Biolog technique is one of the methods which rely on measurements of utilizing different carbon substrates by microorganisms. Measurements of substrate use enable qualifying microbial metabolic capabilities and hence functional diversity of a microbial community. The Principle of the Biolog Method - The Biolog plate’s method was first used to compare metabolic activity of heterotrophic microbial communities from different habitats such as water, soil and wheat rhizosphere. The technique is based on a redox system. Various types of plates are used, but Biolog GN plates for gram-negative bacteria are the most popular ones. Biolog Microbial Identification System is based on metabolic phenotypes. Biolog plates are plastic microtiter plates containing 95 different carbon substrates in wells, and no substrate in one well which is used as control. Among the 95 substrates one can distinguish a few groups of chemical compounds, for example, carbohydrates, amino acids, carboxylic acids, amines, amides and polymers. Biolog is based on the theory that a species of bacteria develops a unique metabolic finger-print on a set of carbon sources and biochemicals. The cultured bacteria are tested for utilization of different carbon sources and biochemicals, which are pre-filled and dried into a 96 well test plate. Additionally, each well contains a colourless Tetrazolium redox dye, used to colorimetrically indicate utilization of the substrates. Cells utilizing nutrient, respire and release energy which reduces proprietary Tetrazolium dye to form a distinct purple colour. Biolog data collection software is used to record the unique metabolic profile into the computer which can be compared with thousands of profiles (corresponding to thousands of species) stored in the Biolog databases. If the profile is matched, computer displays the identified species. Biolog has designed proprietary microplates for identification of a wide range of microbes up to species level, such as Gen III plate (for gram negative and gram positive aerobic bacteria), AN plate (for anaerobic bacteria), YT plate (for yeast) and FF plate (for filamentous fungi). Nearly 2550 species are covered by Biolog for identification. Application- It has been used for clean room analysis of microbial identification prevalent in environment, industrial quality control in analysis of food and/or agricultural products, plant disease diagnosis, veterinary, analysis of clinical samples including dangerous pathogens of human, animal and plant origin, education and research involving general and applied microbiology. Different types of Biolog plates- The Biolog GNplates have been designed for identification of - 255 -
(Diseases and Management of Crops under Protected Cultivation) gram negative bacteria and contain substrates appropriate for this group of microorganisms. Analogically, the GP plates are adapted for identification of gram positive bacteria. Two types of Biolog plates are available i.e. GEN II and GEN III. Microbial identification for GEN II MicroPlates involves five basic steps (identification process for AN, YT, or FF) while GEN III plates involves in four basic steps. These steps apply to all identifications. A small number of species have peculiarities that may require an extra step or special handling techniques. The Microbial Identification Process for GEN II Micro Plates Step 1: Isolation of a pure culture - As a first step to accurate microbe identification, streak agar plates using correct techniques to generate well isolated colonies. Always use Biologrecommended culture media and growth conditions. Step 2: Gram staining and determining test protocol- For bacteria, proper Gram stain technique and interpretation are the important second step in the ID process identification; use the wet prep test as necessary to differentiate yeasts from filamentous fungi. Step 3: Prepare inoculum at specified cell density- Determine cell density using Colorimeter. Cell density describes oxygen concentration a key parameter to control when testing microorganisms in Micro Plates. Additionally, Biolog has carefully optimized the required inoculating fluids and standards. Step 4: Inoculate and incubate MicroPlate- Pipette the specified amount of cell suspension into the Micro Plate, put the lid on, and incubate under the same conditions of temperature and atmosphere used to culture the microorganism. Biolog Micro Plates do not need oil overlays or color-developing chemicals. Step 5: Read MicroPlate and determine ID- After an appropriate incubation time, read Micro Plates either by eye or using the Micro Station Reader. In either case, the pattern formed in the wells is entered into the software, which searches the database and provides identification in seconds. Data is fed into a software enabled computer which performs analysis and reports the species of the isolated micro-organism. ‣ A large sized database is comprised of ~2550 species of which ~700 are of clinical importance. ‣ Gen II Microplate can be used to identify 1350 species of aerobic bacteria. ‣ AN Microplate can be used to identify 361 species of anaerobic bacteria. ‣ YT Microplate can be used to identify 267 species of yeast. ‣ FF Microplate can be used to identify 619 species of filamentous fungi (619 species). ‣ GN2 Microplate can be used to identify species of gram negative bacteria. ‣ GP2 Microplate can be used to identify species of gram positive bacteria. Technology can be used in manual, semi-automatically or fully automatically as per the researcher’s variable needs and budget. - 256 -
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CENTRE OF ADVANCED FACULTY TRAINING
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CONTENTS Sl. No. Title Speaker Page
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REMARKS by Dr. K.S. Dubey Director
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and extension literature that have
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discussions. No doubt the course is
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Introduction (Diseases and Manageme
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Loads For Greenhouse: (Diseases and
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Chemical Control Chemical Abamectin
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Water Requirement (lpd/plant) (Dise
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clear (Boulard et al, 1989). (Disea
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Risks of biocontrol agents: (Diseas
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Diseases and Management of Crops under Protected Cultivation

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