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Dr. V. K. Yadav, V. K. Yadav
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This Laboratory Manual has been designed for students for easy understanding of basic plant pathological laboratory techniques related with Isolation of pathogen. Preservation of disease sample, Demonstration of Koch.s postutlates. Study of different groups of fungicides and antibiotics. Preparation of fungicides. Methods of application of fungicides. Bio-assay of fungicides, Bio control of plant pathogens and Identification of some important fungal pathogens. The book is fully colour book with digitized images have been made to identify diseases and pathogens with explanations of new terminologies to enhance students understanding about the subject. The book will be useful to beginners, students, instructors, scientists and research workers in the field of Plant Pathology and Agricultural Microbiology.

0 Start Pages

Preface This Laboratory Manual has been designed for students for easy understanding of basic plant pathological laboratory techniques related with Isolation of pathogen, Preservation of disease samples, Demonstration of Koch‘s postulates, Study of different groups of fungicides and antibiotics, Preparation of fungicides, Methods of application of fungicides, Bio-assay of fungicides, Bio control of plant pathogens and Identification of some important fungal pathogens. It will be useful for students of Plant Pathology and Agricultural Microbiology. In general a number of constraints often combine to reduce crop production and it is necessary to quantify all of the main constraints. As an assumption about 85 percent of plant diseases are caused by fungi and they are ever- present and are significant constraints in reducing plant growth and crop production. Fungi are multicelled and many species of fungi produce spore, which are reproductive structures that help in dispersal survival and its identification. There are several groups of fungi that are important in causing plant diseases in the nursery. In this manual description of some important causal organisms are also given to make identification easier for students. This manual is designed to help plant pathologists and to enhance learning capacity of students in significant ways. Hopefully, the technical information on fundamental concepts and fungal identification contained herein will certainly useful to the students, scientists and research workers and others, particularly in those areas where access to expert help and advanced facilities is limited. The accurate diagnosis of the cause of a disease helps researchers to recommend successful control measures. In the past, probably every plant pathology instructor has felt limited supply of images of diseases and pathogens, especially for the introductory courses. Now extensive collections of digitized images are available with each instructor for instructional purposes. Keys used by professionals to identify diseases and pathogens can be modified to include diagrams and photographs along with explanations of new terminologies to enhance students understanding about the subject. Without these enhancements, many key are unusable except by experienced pathologists, who are already familiar with the organisms and their features. Keeping in view all the above points, this manual was prepared with illustrations and it is expected that beginners, students, instructors, scientists and research workers will be benefited from this manual.

1 Acquaintance with Different Plant Pathological Appliances in the Laboratory

The students in batches will visit the laboratory of Plant Pathology for their acquaintance with different appliances, tools, glassware‘s and other miscellaneous items, which they will use for various exercises and experiments to be conducted. A list of some of them is given below:

1 - 2 (2 Pages)
2 Preparation of Culture Media for Fungi and Bacteria

Procedure 1.Peeled Potato slices are cooked in 500 ml of water by the time till they are easily penetrated by a glass rod. 2.Filtered with the help of muslin cloth. 3.Agar-agar is melted in another 500 ml of water. 4.Potato extract is added to the melted agar. 5.Dextrose is added in this mixture and stir well with the help of glass rod. 6.Make the volume to 1000 ml, by adding required water. 7.Medium is sterilized in an autoclave at 15 lbs pressure for 20 minutes at temperature of 121.6oC.

3 - 4 (2 Pages)
3 Isolation Techniques

Isolation of fungal pathogens a. From leaves •Choose leaves with young lesions. •The lesions along with some healthy portion are cut in small pieces. •The leaf portion is surface sterilized with 0.1% HgCl2 for 10-15 seconds and washed with sterilized water 4-6 times. •After washing, infected leaf pieces are placed on PDA plates with a sterilized forcep. •After two days of incubation at optimum temperature, bits of mycelium from the margins of leaf pieces are transferred to fresh PDA slants. •In certain cases the leaves are incubated in moist chamber and growth •from infected portion is transferred directly to culture media.

5 - 8 (4 Pages)
4 Preservation of Disease Samples

Collection and dry preservation of disease samples Materials : Iron knife, hand lens, vasculum, pruning scissors, paper envelops and plant press with blotters and news papers. Method •Collect infected plant parts and place them in paper envelops (use separate envelops for different kinds of diseased specimensxsxs). •Place envelops in vasculum and bring to the lab. •Arrange leaves and herbaceous stems inside the folder newspaper sheet. •Place the newspaper between the blotters in plant press and tighten the bolts. •After proper drying remove dried specimen from the press and keep them in separate folders. •Label each folder giving the following informations. a.Name of the organism b.Habitat c.Locality d.Date of collection e.Name of collector f.Collection number g.Other notes

9 - 10 (2 Pages)
5 Demonstration of Koch‘s Postulates

Koch‘s Postulates (Pathogenicity test) Robert Koch developed the following postulates in 1883 on the basis of which an organism could be attributed to be the cause of a specific disease. i)Recognition: The suspected organism must be found in every case of disease (Presence of organism in the diseased tissues). ii)Isolation: The organism must be isolated in pure culture from every case of disease (Isolation in pure form). iii)Inoculation: The isolated pure culture when introduced into the same species or variety of susceptible host must be capable of reproducing the original disease with its typical symptoms (Pathogenicity). iv)Re-isolation: The organism must be re-isolated from the artificially infected host and its characteristics must be exactly like those observed in step 2 (This step was added by E.F. Smith). If all the postulates are proved true, then the isolated pathogen is identified as the actual causal organism responsible for the disease.

11 - 14 (4 Pages)
6 Study of Different Groups of Fungicides and Antibiotics

Introduction Chemicals that kill or retard fungi are fungicides. Most chemicals used to control plant diseases are ”protectants“ and must be applied before infection to protect the plant from invasion by a pathogen. Protectant fungicides are usually effective against a broad range of fungi and protect the plant against infection on the surfaces of the plant to which they are applied. Often, they require multiple applications during the growing season to maintain coverage as new growth emerges and weathering removes past coverage. A few chemicals are termed ”eradicants“ since they can eliminate an established infection. Some newer fungicides are systemic, meaning that they are absorbed by the foliage or roots and move within the plant to the site of infection. Systemic chemicals may be protectants, eradicants, or both systemic fungicides can be absorbed by the plant without harming it, and transported to other tissues where they are toxic to fungi. These compounds can control and eradicate established infections, but they are also vulnerable to fungi developing resistance, as they generally only target one step in a biosynthetic pathway to kill the fungus. To minimize the development of resistance by chemical overuse, fungicides are classified into groups based on their chemical activity.

15 - 18 (4 Pages)
7 Preparation of Fungicides (Bordeaux mixture, Bordeaux paste, Cheshunt compound)

Preparation of Bordeaux mixture Materials: Iron, beaker, copper sulphate, lime, weighing balance, plastic container, wooden stick and muslin cloth. Bordeaux mixture derives its name from the place of its discovery, Bordeaux, France. Ingredients required for the preparation of 100 litre of 1 percent elicoid mixture are: 1.Copper sulphate (blue vitriol or bluestone) : 1 kg 2.Quick lime (unslaked) : 1 kg 3.Water 100 litres Method •Dissolve 1kg powdered copper sulphate crystals in 50 litres of water in a mudpot or plastic bucket. •In another 50 litres of water dissolve 1kg of quicklime. •Stir thoroughly with the help of wooden stick. •Put a filter cloth on the mouth of 3rd container. •Pour the contents of both the containers in a 3rd container simultaneously and stir it thoroughly. •Make up the desire volume (ie. 100 litres). •Test the mixture before use for the presence of free copper, which is harmful to the plant.

19 - 22 (4 Pages)
8 Methods of Application of Fungicides

Introduction Diseases are a common occurrence on plants, often having a significant economic impact on yield and quality, thus managing diseases is an essential component of production for most crops. These fungicides are used because of three main reasons: a.To control a disease during the establishment and development of a crop. b.To increase productivity of a crop and to reduce blemishes. c.To improve the storage life and quality of harvested plants and produce. Application Methods To protect the crop from fungal pathogens, fungicides are applied in various formulations (as dust, granules, gas and most commonly, liquid). They are applied to: 1.Seed treatment 2.Soil treatment 3.Protection of standing crop

23 - 26 (4 Pages)
9 Bio-assay of Fungicides

Poisoned food technique The principle involved is to poison the medium with fungi toxicants and then allow a test fungus to grow on such medium. The test may be performed both on solid and liquid medium. Procedure On solid medium •Prepare potato dextrose agar medium. •Add required amount of fungicide and thoroughly mixed. •The medium is poured into sterilized Petri-plates. •A culture of test fungus is grown on PDA for a certain period (generally seven days) at the optimum temperature for growth. •Small disc (7 mm) of fungus culture is cut with sterile cork borer and each Petri plates are centrally inoculated aseptically containing the medium with desired fungicidal concentration. •Suitable checks are kept where the culture discs are grown under the same conditions on PDA without fungicide. •The fungal colony diameter is measured at every 24 hr.

27 - 30 (4 Pages)
10 Bio-Control of Plant Pathogens

Biological control of disease employs natural enemies of pests or pathogens to eradicate or control their population. Interactions that lead to biocontrol can include antibiosis, competition, induction of host resistance, and predation. Some of the microbial taxa that have been successfully commercialized and are currently marketed include bacteria belonging to the genera Agrobacterium, Bacillus, Pseudomonas and Streptomyces and fungi belonging to the genera Ampelomyces, Candida, Coniothyrium and Trichoderma. •Dual Culture Technique: For in vitro screening, performance of antagonist isolates in dual culture (Morton and Stroube, 1955) with test pathogen can be tested. •Pour 15-20 ml of sterilized melted PDA in 9 cm Petriplates and allowed to solidify. •Place the bits (5 mm) of the test pathogen and antagonist on the PDA plates opposite to each other from 1.0 cm from the periphery of plates (if both are fast growing) or place it 2-3 cm apart (if both are slow growing). •The Petriplates are incubated (at 28 ± 2oC) for desired duration. •Periodical observation on the growth of the antagonists and the ability of the antagonist to colonise the pathogen are recorded.

31 - 32 (2 Pages)
11 Study of Some Important Fungal Pathogens

Some basic informations about genera of Family Pythiaceae, Peronosporaceae and Albuginaceae; Pythiaceae Indeterminate (grows continuously) sporangiophores, have sporangia that are borne variously, but not in chains, on somatic hyphae or on sporangiophores. This family includes the genera Pythium and Phytophthora. Peronosporaceae Determinate sporangiophores, form sporangia on well-defined, branched sporangiophores of determinate growth. There are seven genera in the family including Basidiophora, Bremia, Bremiella, Peronospora, Plasmopara, Pseudoperonospora and Sclerospora. Albuginaceae Club shaped sporangiophores, catenate sporangia, produces chains of sporangia on club-shaped sporangiophores of indeterminate growth. The Albuginaceae contains a single genus Albugo.

33 - 76 (44 Pages)
12 End Pages



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