PAT-101 Manual - Final PDF

UNIVERSITY OF AGRICULTURAL SCIENCES, DHARWAD Laboratory Manual For Management of Plant Diseases PAT 101 (1+1) Prepared by Dr. Shivalingappa Hotkar 0 NAME:...

UNIVERSITY OF AGRICULTURAL SCIENCES, DHARWAD Laboratory Manual For Management of Plant Diseases PAT 101 (1+1) Prepared by Dr. Shivalingappa Hotkar 0 NAME: I.D NO. BATCH: YEAR: CLASS: SEMESTER: DEPARTMENT OF PLANT PATHOLOGY COLLEGE OF AGRICULTURE DHARWAD –580 005 2024-25 CERTIFICATE This is to certify that Mr. / Ms. I.D.No. ------------------ has successfully completed the practical for the partials fulfillment of the course of Management of Plant Diseases (PAT 101)1+1 during first semester of the academic year 2024-25. Course teacher Department of Plant Pathology College of Agriculture Dharwad 1  “Wealth in Plant Health” CONTENTS Sl. Practical Page No. Remarks No. Symptoms of post-harvest diseases caused by fungi, 1. viruses, bacteria, nematodes and mollicutes Diagnosis and Detection of Post harvest diseases 2. Part A: Diagnosis based on disease symptoms Diagnosis and Detection of Post harvest diseases 3. Part B : Detection of plant diseases 4. Seed health testing techniques 5. Seed borne diseases and their management Seed treatment techniques for the management of seed 6. borne diseases Isolation and testing the efficacy of biocontrol agents by 7. dual culture technique 8. Biocontrol agents :Production and application 9. Formulations of Fungicides 2  In vitro and in vivo evaluation of Fungicides and 10. Bactericides 11. Study of pesticide compatibility 12. Methods of application of Plant Protection Chemicals Plant Protection Appliances used in plant disease 13. management 14. Safety measures in Pesticides usage 15. Study of seed storage and packaging techniques Visit to vegetable and fruit markets, Pesticide and Bio- 16. pesticide firms 17. Visit to processing warehouse and testing laboratories Exercise No.1 SYMPTOMS OF POST-HARVEST DISEASES CAUSED BY FUNGI, VIRUSES, BACTERIA, NEMATODES ETC. Plant diseases are associated with various visible changes in the plant, which are generally called as Symptoms. It is the reaction or final result of the reaction in the host plant due to irritation caused by the causal agent in the presence of environment. Whereas, Sign is the visible growth of the causal agent wherein it can be the pathogen itself or its structure. Symptoms may be either confined to definite areas such as leaf spots, cankers, galls, rots etc., or “habitual” (manifested as unnatural) or abnormal habit of the entire plant such as discoloration of foliage, wilting, curling, stunting etc. Further, symptoms are either primary- those occurring on the plant at the point of attack (earliest symptoms) and secondary symptoms- those occurring on some other part as a result of injury or irritation due to the attack or the final visible characteristic symptoms. Normally, they are grouped into three categories: I. NECROSIS: Death of cells or tissues or organs. Various terms are used depending on the habit. Ex: Stripe, Scorch, Scald, Blight, Blast, Anthracnose, Blotch, Scab, Spot, Die-back, Wilting, Damping off, Rot etc. II. HYPERTROPY AND HYPERPLASIA: 3  Abnormal increase in the number of cells leading to the increase in the size of the plant parts like inflorescence, fruits, stem, leaves, root etc. and is known as Hyperplasia and Hypertrophy. Hypertrophy results from an increase in cell size, whereas hyperplasia results from an increase in number of cells. Ex: Galls, Tumors, Witches broom, Cankers etc. III. ATROPY or reduction in size or Hypoplasia (decrease in number of cells). Ex: Suppression, Etiolation, Dwarfing or stunting, Rosetting etc. IV. Other types of symptoms:  Change in colour: Yellowing or chlorosis, Silvering, Anthocyanescence, Mottling or Mosaic etc.  Unusual development or transformation of organs: Mummification, Bleeding, Gummosis, Sugary secretion. Some typical symptoms produced by certain groups of fungi: Downy Mildew, Powdery Mildew, Smut, Rust, White rust or blisters, Moulds (Molds) POST HARVEST DISEASE SYMPTOMS The post-harvest diseases can be grouped into three categories – Seed infection, Seed deterioration and infection of fruits and vegetables. The infections may be caused by fungus, bacteria, virus, nematodes, phytoplasma or other pathogens that are seed borne. Seed infection: A. Fungal: Infection through floral parts / fruits/ natural opening and injuries. B. Bacterial: Infection through floral parts / fruits tissues/ natural opening and injuries. C. Viral: natural opening / injuries/ systemic infection. Seed deterioration: A. Fungal: Effect of seed germination / quality and discoloration of seeds and abnormalities, toxin contamination. B. Bacterial: Effect of seed germination / quality C. Viral: Effect of seed germination / quality Infection of fruits and vegetables: A. Fungal infection process-spore germination, entry into the host tissue, colonization of host tissue, symptom expression B. Bacterial infection process - entry into the host tissue, colonization of host tissue, symptom expression C. Virus infection process - entry into the host tissue, replication of pathogen, symptom expression 4 Exercise No. 2 DIAGNOSIS AND DETECTION OF PLANT DISEASES Part A: DIAGNOSIS BASED ON DISEASE SYMPTOMS Disease diagnosis is very important for developing effective strategies for disease management. Without diagnosis, there can be no disease management. Crop disease diagnosis is an art as well as a science. The diagnostic process involves the recognition of symptoms (which are associated with disease) and signs (which are not outwardly observable) and requires intuitive judgment as well as the use of scientific methods. Several conventional techniques are followed to diagnose disease incidence. These techniques include visual inspection and recognition of symptoms and isolation and examination of crop pathogens using microscopy. Such techniques are time-consuming and may not be able to detect latent infections. Several diagnostic assays have been developed for early and rapid diagnosis. These include immunoassays, nucleic acid probe-based methods, and PCR-based techniques. The use of these techniques in the diagnosis of fungal, bacterial, viral, viroid, and phytoplasma diseases is described here. Methods of diagnosis In the field of human medicine, the doctors invariably use the diagnostic tools (physical, chemical or serological) to be sure of the ailment before starting a line of treatment. The symptoms provide the clues for possibilities, but confirmation comes only after performing diagnostic tests. In plant diseases, visual observations of the infected plant/ plants parts continue 5 to be the dominant method. Several sophisticated techniques are being used for observation, which include microscopy. Isolation and identification of biotic agents associated, besides serology, immunological, bio-chemical and physiological analyses and genome analysis. I. Visual observation (symptoms) Symptoms are the visible expression of host-pathogen interaction. The deviation from normal morphology coupled with presence of pathogen structures form the characteristic symptoms of sign, the basis for preliminary diagnosis. Symptoms induced by parasitic fungi Diseases caused by biotic and mesobiotic agents are identified primarily by symptoms and signs produced on host. The morphological features such as spores, fructifications, or sporophores, which differ from one fungus species to another, form important part of diagnostic programme. If desired structures of the fungus are not readily visible on the infected host surface, the parasite may be induced to sporulate by proper incubation of the infected tissue. Some pathogens produce characteristic symptoms that can be easily recognized in the field. The symptoms of fungus are anthracnose, blights, blast, mildews, rust, smuts, bunts, ergot etc. Symptoms induced by bacterial pathogens Plant pathogenic bacteria induce water soaked lesions in the infected tissues at the initial stages and these lesions turn necrotic late. Formation of bacterial ooze from infected tissues is another distinguishing feature associated with bacterial diseases. As infection progresses, fruit spots, blights, scabs, cankers, tumours, wilts, soft rots, etc., may be the prominent symptoms. Symptoms induced by phytoplasma The phytoplasmas cause general stunting or dwarfing of affected plant parts or whole plants. Chlorosis and reduced leaves are also frequently observed. Phyllody and proliferation of floral tissues-Floral parts are transformed into green leaf-like structures. Partial or total sterility of infected plants may be commonly noted. These symptoms are observed in plants infected by diseases such as aster yellows, little leaf of brinjal, sesame phyllody and witche’s broom disease of potato, peanut and grain legumes. Proliferation of auxiliary buds and formation of a large number of thin shoots are observed prominently in little of brinjal, rice yellow dwarf and sugarcane grassy shoot diseases. Reduction in leaf size and inter nodal length and tendency for the leaves to stand out stiffly, giving a spike like appearance to the infected branches are the distinguishing symptoms of sandal spike disease. Symptoms induced by nematodes Nematodes infect both root and above ground portion of plants. Root symptoms may be appear as root knots, root galls, root lesions, excessive root branching, injured root tips and root rots when nematode infections are accompanied by parasitic or saprophytic fungi and bacteria. These root symptoms are usually accompanied by non-characteristic symptoms in the above ground parts of plants appearing primarily as reduced growth, symptoms of nutrient deficiency, such as yellowing of foliage, excessive wilting in hot or dry weather, reduced yield and poor 6 quality of products. Certain species of nematodes infect the above ground portions of plants. They cause galls, necrotic lesions and rots, twisting and distortion of stem and leaves and abnormal development of floral parts. Certain nematodes attack grains of grasses forming galls full of nematodes in place of seed. Symptoms induced by viruses and viroids Methods of diagnosis of virus include symptomatology, mode of transmission, host- range, particle morphology, antigenicity and electrophoretic mobility in gels. Because some of these techniques require special methodology and equipment for confirmation, the symptoms and distribution patterns frequently suffice for preliminary diagnosis. Plant viruses cause a variety of symptoms, depending on the host plant species and different unrelated viruses may induce similar symptoms in the same host plant species. The viruses induce primary symptoms on inoculated leaves, which exhibit chlorotic or necrotic local lesion or vein clearing. Later when the virus becomes systemic, secondary symptoms developed as colour changes, death or necrosis and abnormal growth form. Colour changes may vary from mosaic on leaves to colour breaking in flowers. Various kinds of changes in size and shape of plant part may be seen as leaf roll, leaf curl, enation, leaf crinkle, galls and tumours. Exercise: Diagnosis of fungal postharvest diseases by visual symptoms, microscopic signs and moist chamber incubation. Fungal leaf spot identification is accomplished by associating a fungus with the symptoms on the foliage. Fungi are usually identified by spore morphology and spore arrangement on conidiophores or by spore morphology and fruiting bodies observed in the diseased plant tissue. If spores are present on symptomatic tissues, then diagnosis may be completed with light microscopy. If spores are not present, then a moist chamber may be prepared to stimulate spore development and maturation. After 1-7 days, tissues are examined for signs of sporulation. If spores are not produced in a moist chamber, then diagnosis proceeds to fungal isolation in culture. After approximately 3-7 days, culture re-examined microscopically to observe fungal spore development. 7  Symptoms of important post-harvest diseases Exercise No. 3 DIAGNOSIS AND DETECTION OF PLANT DISEASES (Contd.,) Part B: Detection of Plant Diseases 8  Conventional plant-pathological techniques need high expertise for routine identification. In the case of latent infections in vegetative planting materials, seeds, and fruit, conventional methods may not be useful to diagnose infection. Detection and identification of diseases in crops could be realized via both direct and indirect methods. Direct detection of diseases includes molecular and serological methods that could be used for high-throughput analysis when large numbers of samples need to be analyzed. In these methods, the disease causing pathogens such as bacteria, fungi and viruses are directly detected to provide accurate identification of the disease/pathogen. On the other hand, In direct detection methods identify the plant diseases through various parameters such as morphological change, temperature change, transpiration rate change and volatile organic compounds released by infected plants. A. DIRECT DETECTION OF DISEASES I. DIAGNOSIS BY IMMUNOLOGICAL TECHNIQUES Immunodiagnostic assays provide a fast method of confirming visible symptoms as well as detecting pathogens that cannot be easily identified by other methods. They permit early detection of plant pathogens and accurate identification of pathogens. Because many fungicides are specific only to certain pathogens or groups of pathogens, immuno diagnosis will be useful in the selection of the most appropriate treatment. Viruses, bacteria, and fungi (especially those spreading as a sterile mycelium) can be readily detected by these methods. Immunoassays depend on the development of antibodies specific to the particular pathogen. Cells of living animals, particularly mammals, have the ability to recognize binding sites on proteins, glycoproteins, lipo polysaccharides, and carbohydrate molecules that are not present in their bodies (i.e., foreign to that animal). Such molecules, known as antigens, stimulate the immune system of the animal and this leads to the production of specific antibodies, each of which specifically recognizes and binds to its complementary antigen. The role of an immunoassay is to reveal the presence of specific complexes between the antibody and an antigen that are unique to the pathogen. The antibodies produced in an animal body can recognize the microbial antigen, which is present on cell walls or found attached with them. In other words, the antibodies can recognize the plant pathogen by recognizing the antigen specific to the pathogen. In principle, immunoassays are based on the fact that antibodies react specifically with the homologous antigen. However, the reaction is not easy to detect. Several techniques have been developed to exploit this reaction in immunoassays as described below. I a. Agglutination Test This test can be carried out in slides or in test tubes. In the slide agglutination test, drops of antigen and diluted antiserum containing antibodies are mixed together on a glass microscope slide. Agglutination is observed by eye or microscope (if ambiguous). In the test-tube agglutination test, the antigens are mixed with antibodies in test tubes, and the aggregation of antigens and antibodies is monitored with a binocular microscope. I b. Precipitation Test 9  In this test, aliquot dilutions of antigen are layered over equal volumes of antiserum diluted in normal serum in capillary, or other small tubes. The test is regarded as positive if there is precipitation at the interface. When the antigens are layered over the antibodies, the antigens are precipitated out of solution by the antiserum when antigen and antibodies are related. I c. Immuno electrophoresis By this method, mixtures of antigens are separated before immuno diffusion. A narrow trough is cut in a layer of thin gel parallel to an electric current that passes close to the antigens along the length of the gel. Each antigen moves in a separate wave at a characteristic rate according to its distinct charge. As a result, proteins separate into bands. Once the proteins have separated sufficiently, the current is switched off and antiserum is added to the trough cut in the gel. Precipitin arcs composed of complexes of antibodies and antigens form where the individual electrophoreses antigens have reached. I d. Direct Sandwich ELISA In the direct sandwich ELISA method, 96-well immunoplates are coated with the specific antibody (polyclonal or preferably monoclonal antibody) and incubated successively with the antigen containing sample followed by a second enzyme labeled specific antibody that is directly conjugated with an enzyme. This leads to a colored product in proportion to concentration of pathogen. I e. Double Antibody Sandwich ELISA In the double antibody sandwich ELISA method, a specific capture antibody is immobilized onto a solid surface, such as the wells of a micro titer plate. The infected plant tissue sample is added, and unbound material is washed away. Bound antigen is detected by the addition of a detecting antibody that has been conjugated with an enzyme, and unbound material is again washed away. The presence of the detecting antibody is determined through the addition of a substrate for the enzyme. The amount of color that develops is proportional to the amount of antigen present in the sample, The intensity of the color can be recorded by automated equipment. I. f. Dot-blot ELISA In this assay system, ELISA reactions are carried out on nitrocellulose membranes. A drop containing the specific monoclonal antibody is absorbed as a “dot” onto which a drop of the test sample is later added and blotted. I. g Immuno fluorescence Two methods of immuno fluorescence are used to diagnose plant diseases. In. the direct immuno fluorescence method, specific antibodies bound to their target antigens are detected by using second antibodies conjugated with fluorescent dyes such as fluorescein isothiocyanate (FITC) or rhodamine isothiocyanate. Fluorescence, indicating the presence of the target antigen, is visualized microscopically. The microscope should have a special device for fluorescence using ultraviolet light (fluorescence microscopy). 10 I. h. Immuno sorbent Electron Microscopy This assay system is mostly used for the diagnosis of virus diseases. Electron microscope grids coated with carbon strongly adsorb protein, and when they are floated on a drop of antiserum containing antibodies to the pathogen, the antibodies become attached. The grids are then floated on a drop of the sap of an infected plant. After staining, the pathogen (particularly virus particles) adsorbed to the antibodies can be seen under a transmission electron microscope. II. NUCLEIC ACID PROBE-BASED METHODS Both DNA and RNA probes are used for crop disease diagnosis. PCR-Based Methodology The polymerase chain reaction provides a powerful and rapid technique to exponentially amplify specific DNA sequences by in vitro DNA synthesis. Three essential steps to a PCR include (1) melting the target DNA, (2) annealing two oligo nucleotides primers to the denatured DNA strands, and (3) extending the primer via a thermostable DNA polymerase. Newly synthesized DNA strands serve as targets for sub- sequent DNA synthesis since the three steps are repeated up to 50 times. The specificity of the method derives from the synthetic oligo nucleotide primers, which base-pair to and define each end of the target sequence to be amplified. PCR uses a thermo stable Thermus quietus (Taq) DNA polymerase to synthesize D A from oligo nucleotide primers and template DNA. The template D A may be genomic, first- strand cDNA, or cloned sequences. Primers are designed to anneal to complementary strands of the template such that DNA synthesis initiated at each primer results in replication of the template region between the primers. The PCR involves three distinct steps governed by temperature. DNA, primers, de oxy nucleotides, buffer, and Taq polymerase are combined in a micro centrifuge and overlaid with mineral oil. The tube is placed in a thermo cycler programmed to repeat a set of short incubations at predetermined temperatures. In the first step, the template DNA is denatured to separate the complementary strands. This is done at 95°C for 5 minutes. In the second step, the mixture is held at an annealing temperature to allow the primers to hybridize to their complementary sequences. This is done at 55°C for 1 min. A PCR primer may comprise two regions, a 3' (priming) region and a 5' (variable) region. The most important region in determining the efficiency of annealing and subsequent DNA synthesis during the PCR is the 3 region, which should be perfectly complementary to the template sequence. The priming region should normally be 20 to 25 bases long. The Taq polymerase stabilizes these base-paired structures and initiates DNA synthesis. In the last step, the reaction is heated to about 72°C for 1 to 5 minutes. This process leads to a Taq polymerase-directed DNA synthesis. The cycle is repeated by keeping the reaction tubes in a thermal cycler for more than 20 times. In the first cycle each template gives rise to a newly synthesized complement. Thus, the number of copies of the target region is doubled. Similarly, in each subsequent cycle, the DNA concentration corresponding to the target region is almost doubled. About 20 cycles of PCR would produce 106-fold amplification of the target DNA. The PCR product is analyzed by agarose gel electrophoresis: The PCR product from a defined band can be recovered from agarose gel. The DNA generated in 11 a PCR can be re-amplified and used for sequencing. III. FLUORESCENCE IN-SITU HYBRIDIZATION Another type of molecular detection technique is fluorescence in-situ hybridization (FISH), which is applied for bacterial detection in combination with microscopy and hybridization of DNA probes and target gene from plant samples. Due to the presence of pathogen-specific ribosomal RNA (rRNA) sequences in plants, recognizing this specific information by FISH can help detect the pathogen infections in plants. In addition to bacterial pathogens, FISH could also be used to detect fungi and viruses and other endo symbiotic bacteria that infect the plant. IV. FLOW CYTOMETRY Flow cytometry (FCM) is a laser-based optical technique widely used for cell counting and sorting, biomarker detection and protein engineering. FCM is used for rapid identification of cells while cells pass through an electronic detection apparatus in a liquid stream. The advantage of this technology is the capability for simultaneous measurement of several parameters. The technique uses an incident laser beam and measures the scattering and fluorescence of the laser beam reflected from the sample B. INDIRECT DETECTION METHODS In addition to the direct methods discussed above, indirect methods based on plant stress profiling and plant volatile profiling have also been used for the identification of biotic and abiotic stresses as well as pathogenic diseases in crops. In this regard, new types of optical sensors that detect biotic and abiotic stresses in plants have been developed. The optical sensors provide detailed information based on different electromagnetic spectra and thus, enable prediction of the plant health. Thermography, fluorescence imaging and hyper spectral techniques are among the most favorable indirect methods for plant disease detection. 1. Thermography Thermography allows imaging the differences in surface temperature of plant leaves and canopies. The emitted infrared radiation can be captured by thermographic cameras and color difference can be analyzed. 2. Fluorescence Imaging In this technique, the chlorophyll fluorescence is measured on the leaves as a function of the incident light and the change in fluorescence parameters can be used to analyze pathogen infections, based on changes in the photosynthetic apparatus and photosynthetic electron transport reactions 3. Hyper spectral Techniques Hyper spectral imaging can be used to obtain useful information about the plant health over a wide range of spectrum between 350 and 2500 nm. Hyper spectral imaging is increasingly 12 being used for plant pheno typing and crop disease identification in large scale agriculture. The technique is highly robust and it provides a rapid analysis of the imaging data. Furthermore, hyper spectral imaging cameras facilitate the data collection in three dimension, with X- and Y- axes for spatial and Z- for spectral, which contributes to more detailed and accurate information about plant health across a large geographic area. 4. Gas Chromatography A completely different non-optical indirect method for plant disease detection involves the profiling of the volatile chemical signature of the infected plants. The pathogen infections of plants could result in the release of specific volatile organic compounds (VOCs) that are highly indicative of the type of stress experienced by plants. Detection of Plant Diseases Using Portable Sensors A wide variety of sensors have been developed and commercialized for various applications including environmental monitoring and medical diagnostics. Depending on the operating principle of the sensor, the analytes could be detected using a sensor based on electrical, chemical, electrochemical, optical, magnetic or vibrational signals. The limit of detection could be enhanced by the use of nano material matrices as transducers and the specificity could be enhanced by the use of bio-recognition elements such as DNA, antibody, enzymes etc. Biosensor Platforms Based on nano materials Recent breakthroughs in nanotechnology enable the preparation of various nano particles and nanostructures with few technical hurdles. Nano particles display fascinating electronic and optical properties and can be synthesized using different types of materials for electronics and sensing applications. 13 14 Exercise No. 4 SEED HEALTH TESTING TECHNIQUES Seed Health Testing is a science of determining the presence or absence of disease causing agents such as fungi, bacteria, viruses etc. Objectives: 1) To detect presence of disease causing 2) To determine health status of a seed lot 3) To decide the grant of certification 4) To check entry of new pathogen during quarantine Testing methods Generalized tests: 1. Examination of dry seed 2. Examination of seed washings 3. Incubation methods: common moist blotter and agar tests 4. Seedling symptom test 5. Grow out test 6. lndexing on indicator test plants 7. Salvaging tests: Fumigation, Hot water treatment, Hot air treatment and irradiation Specialized tests: 1. Insects: X-ray radiography and Seed transparency test 15  2. Nematodes: Sedimentation test, Baerman Funnel Test, Washing test 3. Bacteria: Phage-plague technique. 4. Fungi, bacteria and viruses: Washing test, Serological tests, Indicator test plants. 5. Electron microscopy: Potentially used for identification and characterization of all plant viruses. 6. Serological methods: ELISA, DIBA, ISEM Generalized tests through conventional methods: 1.Visual inspection: Ex: Sclerotia, nematode galls, bunt galls, smuts 2.Microscopic observation: Ex:- Fungi , bacteria and virus 3.X-ray radiography: Hidden infestation of Insects and nematode 4.Washing test: Rusts, Smuts, Downy mildews and a large number of other fungi, eggs of insects adhering to seeds, nematode galls 5. Sedimentation test: Stem and bulb nematode (Ditylenchus dipsaci) (Baerman Funnel Test) 6. Incubation test: Seed borne fungi/ bacteria :Blotter test and agar plate test 7. Grow out test: Seed borne bacteria/viruses/fungi: Embryo count test - internally seed borne IMMUNOLOGICAL TECHNIQUES AGGLUTINATION TEST: 1) Slide agglutination test: Test is designated to observe the direct agglutination reaction of known antibody and unknown particle antigen on slide. Usually used to detect qualitatively the antigen. 2)Tube agglutination test: Antigen mixed with antibody in test tube. Aggregation is observed by using binocular microscope. PRECIPITATION TEST:  Solution of antigen is added to equal volume of antiserum in a test tube.  Test is positive if precipitation is there at the interface. 16 Enzyme-linked Immuno Sorbent Assay (ELISA): Principle- an enzyme-mediated colour change reaction to detect antibody antigen binding. Degree of colour change, usually measured in a computer controlled plate reader - determine the amount of pathogen present. IMMUNO FLUORESCENCE: Antigen- antibody interaction is detected by second antibody conjugated with fluorescent dyes. Fluorescent dyes such as fluorescein isothiocyanate, rhodamine isothiocyanate. Fluorescence indicates the presence of target antigen. Visualized microscopically. 17 Exercise No. 5 SEED BORNE DISEASES AND THEIR MANAGEMENT Introduction  Pathology is the science deals with micro-organism infecting seeds.  Seeds are attached by various fungi, bacteria and virus.  Seeds are attached by various stages,  The mother plant get infected by the pathogen, it attack seed also.  During processing.  At the time of transportation and during storage There are three types of infections: 1) Internally seed borne, 2) Externally Seed borne 3) Fruity bodies/spores Internally Seed borne: Pathogen attacks seed sod, endosperm & embryo Externally seed borne: Pathogens externally carryover on the seeds.  Effects of seed infection: Germination (%) get reduced.  Due to changes is morphology, the market level gets reduced.  Due to infection it induces the changes in the content get reduced. 18   Due to infection it induced the secretion of toxic chemicals ex: Aflatoxin, Rubra toxin, ochre toxin, chitrinin, patulin etc. Important Seed- Borne Fungal Diseases of Major Crops CROPS DISEASES PATHOGEN Wheat Loose smut Ustilago segetum var. tritici Karnal smut Neovassia indica Flag smut Urocystis agropyri Chickpea Ascochyta blight Ascochyta rabiei Wilt Fusarium oxysporum f. sp. ciceri Crucifers Grey and black leaf Alternaria brassicae Spot Brassicicola Rice Bunt Neovossia hordii False Smut Ustilaginoidea virens Stack burn Pyricularia oryzae Trichoconiella padwickii Cotton Anthracnose Colletotrichum indicum Wilt F. oxysporum f.sp.vasinfectum Maize Black kernel rot Botryodiplodia theobromae Cob rot Fusarium Moniliformae Southern leaf blight Drechslera maydis Pearl millet Downy mildew Sclerospora graminicola Smut Tolyposporium penicillariae Sorghum Anthracnose Colletotrichum graminicola Kernel or grain smut Sphacelotheca sorghi Downy mildew Peronosclerospora sorghi Soybean Anthracnose Colletotrichum dematium Pod & stem blight Phomopsis sojae Purple seed stain Cercospora kikuchii Cucumis spp. Anthracnose Colletotrichum lagenarium Brinjal Fruit rot Phomopsis vexans Carrot Black root rot or Alternaria radicina Seedling blight A.dauci Onion Damping off Botrytis allii Downy mildew Peronospora destructor Purple blotch Alternaria porri Stemphylium Blight Stemphylium vesicarium Pepper chilies Anthracnose Colletotrichum capsici Or ripe fruit rot Radish Grey leaf spot Alternaria brassicae Leaf spot A.raphani Tomato Buck eye rot Phytophthora parasitica 19  Damping off Pythium aphanidermatum Early Blight Alternaria solani Late blight or Phytopthora infestans Fruit rot Seed Analysis Report: The Seed Testing Laboratory shall analyze the seed samples in accordance with the prescribed procedure and deliver the Seed Analysis Report to the Certification Agency as soon as may be, but not later than 30 days from the date of receipt of the samples unless the seed is subjected to such tests which require more than 30 days for completion of the test. Seed Treatment: Variety, seed of which is under certification is susceptible to a seed borne disease organism or when seed under certification is carrying a seed borne pathogen The Certification Agency may require such seed to undergo such treatment before Certification If the seeds have been treated, a caution statement such as "Do not Use for Food; Feed or Oil purposes". caution for mercurials and similarly toxic substances shall be the word "POISON" which shall be, in typed and prominently displayed on the label in red Exercise No. 6 SEED TREATMENT TECHNIQUES FOR THE MANAGEMENT OF SEED BORNE DISEASES Seed treatment It is the mixing, coating or soaking of chemicals or protectants, nutrients, hormones or growth regulators into seeds. Objectives of seed treatment: 1. To protect seeds from seed borne diseases and pest attacks. 2. To revive a seed that has been dormant for a long time. 3. Inducing drought tolerance. Purposes of seed treatment Control of Seed borne Pathogens Seed borne disease-causing pathogens may occur on the surface of seed, hidden in cracks or crevices of seed, or as infections deep inside the intact seed. These pathogens may be important for three reasons, 20  1. Some pathogens do not survive in soil or crop residue and are dependent on the seed borne phase for survival between crops, an example is the fungus that causes loose smut of wheat. 2. Even if a pathogen can survive in soil or residue, being seed borne may allow it to get a head start and, thus, result in more severe disease. An example would be the fungus that causes Septoria leaf blotch of wheat. 3. Seed borne pathogens may hitch a ride to new localities in seed shipments (such as the fungus that causes Karnal bunt of wheat or the bacterium that causes black rot of crucifers). Seed treatments can often be used to control pathogens that occur on or in the seed. The choice of seed treatment may be dictated by whether the pathogen is borne externally or internally. For example, both systemic and no systemic (contact) fungicides can eliminate surface contamination of wheat seed by spores of the common bunt fungus. However, the fungus causing loose smut of wheat is borne within the seed embryo and cannot be controlled with a contact fungicide. In that case, a systemic fungicide is required to control the internal pathogen. Pre storage seed treatment Fungicide, insecticide or a combination of both, as well as any other chemical or plant product, are applied to seeds prior to storage. The goal is to keep seeds fresh for longer by disinfecting them against seed borne or seed-storage diseases and storage insects, as well as minimizing seed deterioration directly or indirectly. Conditions under which seed must be treated 1) Injured Seeds Any break in the seed coat of a seed affords an excellent opportunity for fungi to enter the seed and either kill it, or awaken the seedling that will be produced from it. Seeds suffer mechanical injury during combining and threshing operations, or from being dropped from excessive heights. They may also be injured by weather or improper storage. 2) Diseased seed Seed may be infected by disease organisms even at the time of harvest, or may become infected during processing, if processed on contaminated machinery or if stored in contaminated containers or warehouses. 3) Undesirable soil conditions Seeds are sometimes planted under unfavorable soil conditions such as cold and damp soils, or extremely dry soils. Such unfavorable soil conditions may be favorable to the growth and development of certain fungi spores enabling them to attack and damage the seeds. 21 3) Undesirable soil conditions Seeds are sometimes planted under unfavorable soil conditions such as cold and damp soils, or extremely dry soils. Such unfavorable soil conditions may be favorable to the growth and development of certain fungi spores enabling them to attack and damage the seeds. 4) Disease-free seed Seeds are invariably infected, by disease organisms ranging from no economic consequence to severe economic consequences. Seed treatment provides a good insurance against diseases, soil-borne organisms and thus affords protection to weak seeds enabling them to germinate and produce seedlings. Characteristics of ideal chemical seed treatment:  It must be extremely effective in the face of harmful organisms.  Seeds must be somewhat unaffected.  Even if overused, it should be safe for humans, animals and cattle.  During seed storage, it should be relatively stable for a long time.  It should be simple to operate.  It should be economically competitive. Different methods of seed treatment I. A. Dry treatment Mixing the seed with pesticides in powder form. B. Wet treatment Soaking the seeds in a pesticide solution in liquid form. C. Slurry treatment Seeds/ seedlings are dipped in a slurry. II. 1. Seed disinfection It refers to the removal of fungal and bacterial spores that have established themselves in the seed coat or in deeper tissues. Fungicidal treatments must reach the seed to kill the fungus that is there for effective control. 2. Seed disinfestations It refers to the destruction of surface- borne organisms that have contaminated the seed surface but not infected the seed surface. Application of chemicals through chemical dips, soaks, fungicides or pesticides applied as dust have been found successful. 22  3. Seed protection The main purpose is to protect the seed and young seedlings from organisms in the soil, which might otherwise cause decay of the seed before germination. Procedure for seed treatment A. Seed pelleting It is the process of coating seed with enough ingredients to make the seeds larger, heavier and consistent in size for sowing using seed drills. Pesticide pelleting is used to protect from soil organisms and pests, as well as repel birds, ants and rodents. It requires specialized application machinery and techniques and is the most expensive application. B. Seed dressing This is the most common method of seed treatment. The seed with either a dry formulation or wet treated with a slurry or liquid formulation. Dressings can be applied at both farm and industries. Low cost earthen pots can be used for mixing pesticides with seed or seed can be spread on a polythene sheet and required quantity of chemical can be sprinkled on seed lot and mixed mechanically by the farmers. C. Seed coating: A special binder is used with a formulation to enhance adherence to the seed. Coating requires advanced treatment technology, by the industry. Equipment’s used in seed treatment 1. Drum Mixer This equipment is used for different kind of seeds with chemicals in powder form. Seed treatment drum is made up of angle, iron frame and G.I sheet made drum. In one batch 10-15 kg seed can be treated with chemicals. 2. Slurry treaters The slurry treatment principal involves suspension of WP treatment material in water. The treatment material applied as slurry is accurately metered through a simple mechanism composed of a slurry cup and seed drum pan. The cup introduces a given amount of slurry, with each dump of seed, into a mixing where the seeds are mixed thoroughly. The slurry treaters are adoptable to all types of seeds and rates of seed treating. 3. Direct Treaters Direct treaters are the most recent development and incude the Panogen and Mist-o-matic treaters. Mist-o-matic treaters is being used more widely. The Mist-o-matic treater applies chemical as a mist directly to the seed. The treater is equipped with a large treatment tank, a pump and a return that maintains the level in the small reservoir from which the seed is feed. 4. Grain Auger 23  Liquid materials can be dripped on the seed as they enter a grain auger. By the time seeds have left the auger the chemical is spread on the seeds. 5. Shovel Seeds are spread on a clean dry surface 10-15cmin depth. The proper amount of chemical is diluted with water and sprinkled over the seed. Mixing is done with shovel turning the seed at least 20 times Precautions in Seed Treatment Never used for animal or human consumption. The treated seeds must be properly labelled. Avoid use of unsold treated seed for human or animal feed. Treat only the quantity for which sales are assured. Care must be taken to treat the seeds at correct dosage (too low or too high). Seed with very high moisture content is very susceptible to injury when treated with some of the concentrated liquid products. The technique must be economical and practical for the specific crop and above all the materials used should be environmentally safe. If the seeds are to be treated with microbial cultures also, the order in which seed treatments should be done shall be as follows i) Chemical treatments ii) Insecticide and fungicide treatments iii) Special treatments Recommendation of seed treatment for different crops Sl. Crop Disease Seed treatment No. 1. Sugarcane Root rot, wilt Carbendazim @ 2g/ kg seed Trichoderma spp. @ 4-6g/ kg seed 2. Rice Root rot Trichoderma @ 5-10g/ kg seed Bacterial sheath blight Pseudomonas fluorescens @ 10g/ kg 24  Root knot nematode, Soaking seed in 0.2% solution of monocrotophos for 6 hours White tip nematode 3. Chillies Anthracnose spp. Trichoderma viride @ 4g/ kg Damping off Carbendazim @ 1g/ 100g seed Soil borne infection of Trichoderma viride @ 2g/ kg fungal disease Pseudomonas fluorescens @ 10g/ kg Captan 75 WS @ 1.5- 2.5g a. i./ litre for soil drenching. 4. Pigeon Wilt, blight and root rot Trichoderma spp. @ 4g/ kg seed pea 5. Pea Root rot Bacillus subtilis Pseudomonas fluorescens Soil application @ 2.5- 5kg in 100kg FYM or Carbendazim or captan 2g/kg seed White rot Thiram+ carbendazim 2g/ kg seed Carbendazim or captan 2g/ kg seeed 6. Tomato Soil borne infection of T. Viride @ 2g/ 100g seed fungal diseases Captan 75WS @ 1.5-2g a. i./ litre for Early blight soil drenching Damping off Pseudomonas fluorescens and Verticillium clamydosporium @ 10g/kg Wilt as seed dresser. 7. Wheat Bunt/ false smut/ loose Thiram 75% WP smut/ covered smut Carboxin 75% WP Tebuconazole 2 DS @ 1.5- 1.87g a.i. 25  /kg seed. T. viride @ 4g/ kg seed 8. Crucifers Soil/ seed borne Trichoderma viride @ 2g/ 100g seeds diseases Captan 75 WS @ 1.5- 2.5g a. i./ litre for soil drenching. 9. Potato Soil and tuber borne MEMC 3% WS @ 0.25% diseases Boric acid 3% for 20 minutes before storage. 10. Barley Loose smut Carboxin 75% WP Covered smut Thiram 75% WP @ 1.5- 1.87 g a. i./ kg seed Leaf stripe Advantages of Seed Treatment o The following are major advantages of seed treatment o This process protects germinating seeds and seedlings against soil and seed-borne pests and diseases. It improves the germination process and increases the germination percentage. o It enhances the seed viability and vigour which are the two most important factors in agriculture or cultivation practices, o It results in the early and uniform establishment and growth of the crop or plants. o It enhances nodulation in legume crops. o It is better when compared to soil and foliar application in the crop. o It results in uniform crop stand especially in adverse situations. like low moist and high most conditions. Disadvantages  Accidental poisoning Treated seed looks like food to some animals. Hungry livestock Birds, such as pheasants or quail, may consume spilled treated seed. Even young children may find and eat improperly stored treated seed.  Cropping restrictions Just like other pesticides, some seed treatments may have significant grazing or rotation crop restrictions.  Limited dose capacity The amount of pesticide that can be applied limited by how much will actually stick to the seed. Seed coating technologies are helping to overcome this limitation, but phytotoxicity may still be a problem. 26   Limited duration of protection The duration of protection is often short due to the relatively small amount of chemical applied to the seed, dilution of the chemical as the plant grows, and breakdown of the chemical.  Limited shelf life of treated seed Producing excess treated seed is undesirable because the shelf life of treated seed may be limited. Surplus treated seed cannot be sold for grain. This is a particularly serious limitation for seeds such as soybean, where seed germination and vigor decline relatively quickly.  Phytotoxicity Pesticide injury to plant tissues is called phytotoxicity. Since seed treatments must exist in high concentrations on the tender tissues of germinating seeds and seedlings, they generally have very low phytotoxicity. A few seed treatments are partly phototoxic when appl ied at high rates. Lower germination and/or stunting may occur if application rates are not carefully controlled. Cracked, sprouted, and scuffed seeds may be particularly susceptible to toxic effects. A few seed treatments may reduce the length of the sprout and, therefore, affect the choice of planting depth.  Worker exposure. In the course of treating and handling large volumes of seed, workers may be exposed to seed treatment chemicals as aerosols. Inhalation of aerosols and skin contact with seed treatments must be prevented in the seed treatment process.  Contamination of the food supply by accidental mixing of treated seed with food or feed grain.  Accidental contamination of the environment through improper handling of treated seeds or seed treatment chemicals. "All of these risks can be minimized by proper training and proper use of seed treatment pesticides" Exercise No. 7 ISOLATION AND TESTING THE EFFICACY OF BIOCONTROL AGENTS BY DUAL CULTURE TECHNIQUES I. ISOLATION Serial dilution method 27 Principle: The serial dilution agar plating method viable count method is one of the commonly used. procedures for the isolation and enumeration of fungal biocontrol agents which are the most prevalent microorganisms. This method is used on the principle that when material containing microorganisms is cultured each viable microorganism will develop into a colony; hence the number colonies appearing the plates represents the number of living organisms present in the sample. Requirements: Soil sample, test tubes, Rose Bengal agar medium, Potato Dextrose Agar medium, Pseudomonas agar: Fluorescein and Hi-chrome: Bacillus agar) 9 ml of sterile distilled water blanks (7), sterile petri plates, sterile 1ml pipette and colony counter. Composition of medium 1. Potato Dextrose Agar Potato extract 200g Agar-agar 20g Dextrose 20g Distilled water 1000 ml. 2. Rose Bengal Agar Dextrose 15g Papaic Digest of Soybean meal 5g Monopotassium Phosphate 2g Magnesium Sulfate 0.5g Chloramphenicol 0.1g Rose Bengal 0.05g Agar 15g Distilled Water 1000ml 3. Pseudomonas Agar (Fluorescein) Casein enzymic hydrolysate 10g Proteose peptome 10g 28 Dipottasium phosphate 1.5g Magnesium sulphate 1.5g Agar 15g Distilled water 1000ml 4. Hichrome: Bacillus Agar Peptone 10g Meat extract 1g D-Mannitol 10g Sodium chloride 10g chromogenic mixture 3.2g Phenol red 0.025g Agar 15g Distilled water 1000ml Procedure: Fungi  Collect soil samples randomly from five points firm a field.  Remove three Centimetre of the top soil and take the subsamples at random at a depth of 20cm from each site. Then transfer the soil samples into stark polyethylene bags and transported to the laboratory.  Combine all subsamples from one site to yield one composite sample representing the location, exposed to room temperature with 50% humidity & sieve through a mesh.  Label 9ml sterile water blanks culture tubes as 1.2.3.4.5.6 and 7.  Add 1G of soil sample, finely pulverized. into numbered 1 water blank culture tubes to make 1:10 dilution (10-¹).  Vigorously shake the dilution on a magnetic shaker for 20-30 min to obtain uniform suspension. microorganism.  Transfer 1ml of soil suspension from tube number 1 into water blank number 2 with a sterile pipette under aseptic conditions to make 1:100 (10 -2) dilution and shake it well on vertex for about 5 mm.  Make further dilution 10-³ to 10-7 by pipetting 1ml of soil suspension into additional water blanks culture tubes as prepared above.  Transfer 1ml of aliquots each from 2nd dilution to 7th dilution and add approximately 15ml of sterilized cooled medium to the Petri plates.  Incubate all the plates in an inverted position at 25°C for 2-7 days. 29 Observations Observe the plates for number and distribution of colonies of fungi from each dilution. Select plates from the appropriate dilution with colonies in the range of 30 to 300. make plate counts using a colony counter or manually. Viable cells / g dry soil = (Mean plate count x dilution factor)/ Dry weight of soil Bacteria:  Take of rhizosphere soil of plant and mix it with 100ml of sterilized distilled water in 250ml flask.  Mix soil well by agitating it again.  Take 1ml of soil suspension and pour into 9ml sterilized distilled water and further dilute up to 10-7 for each sample separately.  Then take 100μl of dilute suspension at different level as per population of bacteria in the soil (10-3, 10-5 and 10-7) and spread with the help of L-shaped glass rod on to solid Pseudomonas Agar: Fluorescein and Hi-chrome: Bacillus Agar for isolation of Pseudomonas fluorescens and Bacillus subtilis, separately.  Incubate the plate for 24 hrs at 28±1°c in BOD incubator. Observation Creamy flat colonies and greenish flat colonies appear on the Pseudomonas Agar: Fluorescein and Hi-chrome: Bacillus Agar medium respectively, after 24 hrs. II. PURIFICATION OF BIOCONTROL AGENTS Principle: This method is employed to purify particular fungus when it is found mixed with group of fungus and bacteria. In this method. the growth of the spore is allowed on a plain agar as to obtain pare culture. Requirements: Fungal culture, plain agar medium, Petri plates, pipette microscope, slide, inoculation loop, glass marker. 1. Single spore isolation Procedure Prepare spore suspension of the given sample tilt I ml of suspension contains to more than 5-10 spores. Pour the spore suspension (0.5-1ml) aseptically into a Petri plates. Prepare and pour warm plain agar (2x) into Petri plates and mix it thoroughly. Incubate the plates at optimum temperature for 4 hours. Observations 30   Invert the petri plates and examine germinated spores under the microscope and suspected are marked by glass marker on the back of Petri plate.  Cut the marked area containing the spore along with some medium using a sterile cork- borer.  Transfer it with the help of a sterile inoculation needle to agar slant and incubate to obtain single spore culture.  Store the pure culture for further use. 2. Single hyphal tip method  Prepare plain agar (2%) medium and pour into the Petri plates.  Inoculate the mixture culture in the centre of t plates and incubate at 25±1ºC. The fungus grows and put forth hyphae quickly to the periphery of the Petri plates in search of nutrition.  Incubate for 3-4 days at 25 ±1°C. Observations  Invent the Petri plates and examine under the microscope for single hyphal tip in the periphery of the Petri plate.  Mark the single hyphae tip with a glass marker and transfer it to suitable medium as done in single spore isolation for obtaining pure culture.  Store for future use. III. IN VITRO EVALUATION OF BIO CONTROL AGENTS Aim: To study the effective bio control agent in managing the disease effectively, without the use of any chemicals or toxicants. Different methods of evaluation of antagonists. 1. Dual culture technique 2. Poisoned food technique 3. Agar disk-diffusion method 4. Agar well diffusion method. Materials required: Bio control agents, i.e. fungal bio control agents like various Trichoderma species viz., T. harzianum, T. koningii, T. pseudokoningii, T. hamatum, T. virens and T. viride. Bacterial bio control agents like Bacillus subtilis, B. cereus, B. amyloliquefaciens, and Pseudomonas fluorescens etc. Test culture of the required pathogen, Petri plates, different media like PDA, NA, Kings-B, etc. 1. Dual culture technique: 31  The principle involved in this technique is placing the two culture/ mycelial discs, i.e. disc from the test pathogen and the disc of bio control agent on a single Petri plate on the exact opposite side to each other. Procedure: (For fungal bio control agent) 1. Prepare 250ml Potato dextrose agar medium in flasks and sterilize it. 2. Cut small uniform discs (5mm) of the test fungus culture and transfer them under aseptic conditions to one corner of the Petri plates containing the medium. 3. To the exact opposite side of the Petri plate transfer the disc containing fungal bio agent. 4. Maintain suitable checks, transferring the discs of test fungus on to Petri plates containing PDA without the bio control agent. Incubate both the treated and check plates at room temperature. 5. Record the inhibition in the mycelial growth by the bio control agent when the colony growth in control attains maximum. Calculate the efficiency of test bio control agent by using formula C-T I = X 100 C Where, I = Per cent inhibition of the mycelium C = Growth of the mycelium in control T = Growth of the mycelium in treatment 32 Exercise No. 08 BIOCONTROL AGENTS: PRODUCTION AND APPLICATION Biological control is defined as the reduction of inoculum density or disease producing activities of a pathogen or parasite in its active or dormant stage by one or more organisms accomplished naturally or through manipulation of the environment, host or by introduction of one or more antagonists or by mass introduction of one or more antagonists. The chemical methods are uneconomical and cost effective, as seed treatment with chemical may give protection only in the early stages of crop growth 2 weeks. In addition, it is harmful to the beneficial microorganisms in soil and creates residual problems. So, the biological control can be very efficient used for the soil borne and root rot disease management as the biological agent multiply in soil and offer protection throughout the crop growth. The four main mechanisms involved in the biocontrol are (i) the biological agent (antagonist), may parasite the other organism, (ii) antagonist may secrete metabolites (antibiotics) harmful to the pathogens (Antibiosis) (iii) antagonist may compete with the pathogens for nutrients or space (Competition) and (iv) may cause death of the parasite by producing enzymes (Lysis). Parasitism and Lysis The biocontrol against parasitizes the pathogen by coiling around the hyphae. Ex., Trichoderma viride; various bacteria and fungi secrete hydrolytic about the degradation of cell wall of pathogens. Bacillus sp. causes hyphal lysis of Gaeumanornyces graminis The chitnolytic enzymes of Serratia marcescens caused cell wall lysis of Sclerotium rolfsii. Trichoderma sp. produces chitinases and β-1, 3 glucanases which lyses the cell wall of Rhizoctonia solani. Antibiosis The antibiotic compounds secreted by the biocontrol agent suppress the growth of the pathogen. e.g. Phenazine-l-carboxylic acid produced by P fluorescens plays an important role in suppressing the take all disease of wheat. Competition The biocontrol bacteria and fungi compete for food and essential elements with the pathogen thereby displacing and suppressing the growth of pathogen. Ex., the competition for nutrients between Pythium aphanidermatum, P ultimum and bacteria suppress the damping off disease in cucumbers. Fluorescent siderophores (iron chelaters) such as pseudobactinis & pyoverdins produced by P. fluorescons chelates iron available in the soil, thereby depriving the pathogen of Its Fe requirements. MASS PRODUCTION OF BIO CONTROL AGENTS Application of antagonistic fungi to the rhizosphere of crop plants to be protected from 33 any soil borne pathogen requires mass production of antagonist/ antagonists within a short time using a cheap substrate and easy technique. Several attempts have been made in this direction. Wheat bran was used by several workers as a substrate for multiplication of Trichoderma spp. For large scale production of Trichoderma spp. liquid fermentation method is widely followed and the procedure is given as below: 34 Quality Control Specifications 1. Fresh product should contain not less than 28 x 106 cfu / g 2. After 120 days of storage at room temperature, the population should be 10 x 106 cfu / g. 3. Maximum storage period using talc as carrier is 120 days. 4. Size of the carrier (talc) should be 500 microns. 5. Product should be packed in white Polythene bags. 6. Moisture content of the final product should not be more than 20%. 3. Nitrate reduction test 4. Acid and gas production test Mass production of Pseudomonas fluorescens P. fluorescens is multiplied in sterilized Kings ‘B’ broth for 48 hours. The pH of the substrate (Peat soil or talc powder) is adjusted to 7 by adding calcium carbonate @150 g / kg. The substrate is then sterilized at 1.1 kg/cm 2 pressure for 30 minutes for two successive days. Four hundred ml of P. fluorescens suspension is added to 1 kg of substrate containing 5 g of carboxy methyl cellulose and mixed well. The formulation is packed in Polythene covers and can be stored for one month. Quality Control 1. Fresh product should contain 2.5 x 10 cfu / g 2. After 3 months of storage at room temperature, the population should be 8.9 x 107 cfu / g. 3. Storage period is 3-4 months 4. Minimum population load for use is 1.0 x 108 cfu / g. 5. Product should be packed in white Polythene bags. Formulations For the successful development of formulation of biocontrol agents, it is important not only to provide a substrate that will promote the synthesis of the desired enzymes which help in its biocontrol mechanisms but also to provide sufficient substrate, so as not to limit the synthesis of the enzymes at the time when they are required. These include utilization of large number of agro-wastes as substrate for the mass production of Trichoderma, use of a wide variety of solid substrates with less expenditure and higher reproducibility. Different effective substrates for the mass multiplication of antagonists Grain bran, Wheat bran, Wheat straw, Wheat bran-saw dust, Sorghum grain, Wheat bran- saw dust modified medium, Sand and sorghum medium, Tapioca rind, FYM, Press mud, Sand- corn meal, Coconut coir pith, Groundnut shell medium, Peat-bran, Rice bran, Rice straw and Papaya riped fruit. 35 Carrier/food base materials used in Trichoderma formulations Talc, Pyrax, Celatom, Vermiculite and Wheat bran, Diatomaceous earth molasses, Gypsum, Lignite, Kaolin, Peat, Kaolin expanded clay, Vermiculite sodium alginate and CaCl 2. Trichoderma production process must also end up in biomass with excellent shelf-life even under adverse storage conditions. The Department of Plant Pathology, College of Agriculture, Dharwad is producing the talc based good quality formulation and also to study the shelf life of the product. Types of formulations 1. Powder formulation. 2. Encapsulation in organic polymer like sodium alginate. 3. Pelleting biomass and bran with sodium alginate. 4. Wheat bran: saw dust: water for soil application. 5. Molasses enriched clay (Kaolin) granules. 6. Liquid coating formulations bio protectant as powder on which suspension of aqueous binder is sprayed on seeds to form 0.1 mm thick layer. 7. As spray from emulsifiable concentrates. Methods of application There are six methods presently followed for effective management of diseases through bioagents. They are: 1. Broadcast application @125 - 250 kg/ha. 2. Furrow application @ 130 - 160 kg/ha. 3. Root zone application @ l kg/plant. 4. Seed coating or seed treatment @ 4 – 10g/kg seeds. 5. Wound application. 6. Spray application on plant or soil surface @106 - 108 cfu/ml. Some of the biocontrol agents reported against post-harvest diseases Antagonists Disease (pathogen) Fruits/ vegetables Acremonium brevae Gray mold (Botrytis cinerea) Apple Aureobasidium pullulans Monilinia rot (Monilinia laxa) Banana Penicillium rots (Penicillium spp.) Botrytis rot (Botrytis cinerea) Citrus Grape Bacillus subtilis Brown rot (Lasiodiplodia theobromae) Apricot Stem end rot Avocado Bacillus licheniformis Anthracnose (C. gloeosporioides) Mango Burkholderia cepacia Anthracnose (Colletotrichum musae) Banana Candida oleophila Anthracnose (C. gloeosporioides) Papaya 36 Debaryomyces hansenii Rhizopus rot (Rhizopus stolonifer) Peach Metschnikowia fructicola Botrytis rot (Botrytis cinerea) Grape Pseudomonas cepacia Green mold (Penicillium digitatum) Orange Pseudomonas fluorescens Gray mold (Botrytis mali Ruehle) Apple Pseudomonas putida Soft rot Pectobacterium caratovora Potato Trichoderma harzianum Anthracnose (Colletotrichum musae) Banana Gray mold (Botrytis cinerea) Gray mold (Botrytis cinerea) Kiwifruit Gray mold (Botrytis cinerea) Grape Pear Trichoderma viridae Gray mildew (Botrytis cinerea) Grape 37 Exercise No. 9 FORMULATIONS OF FUNGICIDES Commercially available fungicides usually consist of a mixture of active ingredient (a.i.) and other substances including diluents, wetting agents, stickers, emulsifiers, etc. Formulations containing mixtures of different active ingredients (especially mixtures of protectant and systemic fungicides) are also widely used nowadays. Different formulations incorporating the same active ingredient may be used for distinct purposes like seed treatment, foliar application etc. Emulsifiable Concentrates (EC) These are liquid formulations which can be diluted with water before application. The active ingredient is dissolved in a solvent. The fungicides and solvents will often not mix with water, so an emulsifying agent or water dispersible oil is mixed. When these emulsifiable concentrate is added to water, a milky mixture is formed which is a suspension of active ingredient and emulsified solvent in the water. Wettable Powders (WP) Wettable powder is a very common formulation for most of the fungicides, which is used for spray mixtures. The modern wettable powders are water-dispersible which have the quality to wet easily and disperse well in water. They are also called as Water-Dispersible Powders (WDP). The active ingredient is incorporated, usually at the rate of (30-80 %), with a finely ground inert dust (filler) such as Kaolin, a wetting agent and a suspending agent. Dusts (D) Dust formulations usually contain (1-10 %) active ingredient for direct application in dry forms. They are manufactured in such a way that they are light enough to be carried by a slight breeze for a considerable distance. The finely divided particle of active ingredient is carried on a carrier particle. The commonly used carriers (diluents) are attapulgite, kaolin, talc, pyrophylite, diatomaceous earth, bentonite, calcium silicate, hydrated silica, calcium carbonate, magnesium carbonate, gypsum, lime etc. Granules (Pellets) Pellets are the formulations of the fungicide with inert materials formed into particles about the size of coarse sugar. The granules normally contain (3-10%) of the active ingredient. 38 Due to their size, the granules do not drift but have limited application being confined to soil and seed treatments. Granules have the advantage they can be measured in dry form more easily and accurately than dusts or wettable powders. Suspension or slurries These are formulation in which a dry form of the active ingredient is mixed with a liquid. Such formulations usually contain a high percentage of active ingredient similar to wettable powders. They are mixed with water for final use and require agitation. These are mostly used as seed dressers in seed processing companies. Solutions True solutions are formulations in which active ingredient or a combination of active ingredients and a solvent is dissolved in water Solutions have the advantage of requiring no agitation after formulation is added in water. Nowadays, the manufacturers are concentrating to develop new formulations to increase the efficacy of the chemicals. Some new formulations developed are: Soluble Liquid (SL), Soluble Powder (SP), Water Soluble Concentrate (WSC), Suspension Concentrate (SC) and Aqua Flow (AF). Adjuvants In spraying method, the toxicant is made into a suspension in water. In order to increase the efficacy of the water mixed sprays, certain substances like wetting agents, dispersing agents, spreaders, stickers, etc. are added during the formulation of fungicides. These auxillary spray materials are also called adjuvants, which are usually inert materials added to improve the physical characteristics of the toxicant and its carrier. Dispersing agents (Deflocculating agents) These are the substances which keep fine particles away from each other to prevent deflocculation. These materials, when added to formulations, ensure uniform suspension and retard sedimentation of particles in the spray suspension. These are also called as deflocculating agents. E.g. Gelatin, plant gums and milk products. Emulsifying agents Many surface active substances like soap, function as emulsifying agent, which retard the settling out of droplets of water immiscible liquids like oils. This helps in uniform mixing of substances in water suspensions Wetting agent (Wetters) These are the materials which are added to ensure that there will be no layer of air between a solid and a liquid as they reduce the surface tension of the particles. Wetting agents, when added to aqueous fungicidal preparation, help in easy deposition on leaves. E.g. Polyethylene oxide condensat, esters of fatty acids and flour. Spreading agent (Spreaders) 39  Spreaders are the materials added to establish improved contact between the spray materials and plant surface and thus ensuring a good coverage of fungicide. Wetting must precede spreading and this is the only distinction between wetting and spreading. Spreaders also reduce the surface tension and thus improve contact. E.g. Soap, flour, sulphated amines, soapamines, mineral oils, glyceride oil, terpene oil, resinates and petroleum sulphonic acids. Stickers (Adhesives) The materials which are added to spray or dust to improve the adherence to plant surfaces are called as stickers. They increase the tenacity of the fungicidal preparations, thus increasing the residual action. E.g. Polyvinyl acetate, polybutanes, fish oil, linseed oil, milk casein,gelatin, dextrines, polyethylene polysulphide, starch, gum arabic, hydrocarbon oils and bentonite clays; Milk casein, gelatin also act as good spreading and wetting agents besides acting as stickers. Safeners A Chemical which reduces the phytotoxicity of another chemical is called safener. For example, copper sulphate is phytotoxic to plants, but with addition of lime its toxicity is reduced. Lime is, therefore, a safener. Lime is used universally with chemicals to prevent the formation of, or to neutralise arsenic, which is phytotoxic to plants. Glycerine oils are also used as safeners. 40 Exercise No. 10 IN VITRO AND IN VIVO EVALUATION OF FUNGICIDES AND BACTERICIDES Several techniques for evaluating fungicides have been described from time to time by different workers. Some times as in the case-of soil fungicides, it is necessary to follow more than one method to evaluate the chemicals properly. In vitro, laboratory studies on different chemicals will provide basic information concerning the fungi toxicity Methods for testing of fungicides. 1. Food poisoning technique 2. Modified paper disc plate method. 3. Slide germination technique. Standard procedure for preparation of stock solution of test chemical. Concentration of test chemical solution is expressed in percentage or in parts per million (ppm) which is calculated by the following formula. Quantity of chemical (“ppm” required X ‘ml’ required) Required (gm) = X CF 1,000,000 For example to calculate the number of gram of chemical to make a 500 PPM solution in 100 ml, of water, then, 500 gms. of chemical ‘z’ g. = 1,000,000 ml, water 100ml, water 500 X 100 41  Z= = 0.05 g 1,000,000 This means that 0.05 g. of chemical in 100 ml. of water gives a 500 ppm solution provided the chemical employed has 100% active ingredient. But most of the commercial formulation of chemicals do not have 100% a. i. Hence, a correction factor required which is calculated as follows. If the chemical has 50 % a.i. the correction factor has 100/50 = 2. This means that the grams of chemical needed to give specified concentration must be multiplied by ‘2’. Some common correction factors are as follows: Percentage of active ingredient Correction factor 80%` 1.2500 75% 1.3333 70% 1.4286 65% 1.5385 60% 1.6667 55% 1.8188 50% 2.0000 45% 2.2222 40% 2.5000 35% 3.3333 30% 4.0000 25% 6.6666 20% 5.0000 10% 10.0000 5% 20.0000 Food Poisoning Technique: The principle involved in this technique is to poison the nutrient medium with a fungi toxicant and then allowing a test fungus to grow on such a medium. In this technique either a solid or liquid medium can be used. Materials required: 42  Potato dextrose agar (PDA), Test fungus grown on PDA at required temperature; Flat bottom sterilized Petri plates; Sterile cork borer, Glass Marking pencil; inoculation needle etc., Procedure: 1. Prepare 250ml. potato dextrose agar medium in flasks and sterilize it. 2. Add to this medium the required quantity of fungicide based on active ingredient of the chemical in order to get series of concentration of the fungicide. Mix the fungicide thoroughly before the medium is solidified. 3. The medium is then equally distributed into Petri plate and allowed to solidify. Maintain suitable number of replications for each treatment. 4. Take the test fungus grown in Petri plate. 5. Cut small uniform discs of the test fungus culture and transfer them under aseptic conditions on to the center of the Petri plates containing the poisoned medium. 6. Maintain suitable checks, transferring the discs of test fungus on to Petri plates containing PDA without the fungicide. Incubate both the treated and check plates at room temperature. 7. Take the mean colony diameter of the fungal mycelial growth, when the colony growth in control attains maximum, and compare the results. Slide germination technique : Principle: Look for inhibition of spore germination in the treated slide and compare per cent spore germination with control. Material required: Glass slides, Petri plates, glass blotting paper, Test fungicides, spore culture of fungus etc., Procedure: I 1. Spray the glass slide with fungicidal suspension concentration and allow it to dry. 2. Coat the spore suspension on this glass slide. 3. Incubate them in most chambers for 24 h. Procedure : II 1. Mix spore and fungicidal suspension in a known quantity of water. 2. Place a drop of the above suspension on a glass 3. Incubate them in moist chambers for 24 h. In both cases, keep a control without fungicidal suspension Observation: a) Measure the colony diameter at an interval of 24 hr until maximum growth is attained in control. b) Calculate the efficiency of test fungicide by using formula. C - T X 100 43  C Where C = Growth of fungus in control T = Growth of fungus in test fungicide c) Calculate percentage germination of spores in different treatments and compare with control. Exercise No.11 STUDY OF PESTICIDE COMPATIBILITY Compatibility Compatibility is the ability of two or more components of a pesticide mixture to be used in combinations without impairment of toxicity, physical properties or plant safety of either of the component. Possible effects of mixing incompatible chemicals Reduced effectiveness of one or both compounds. Precipitate in the tank, clogging screens and nozzles in the sprayer. Plant phytotoxicity, stunting or reducing seed germination. Excessive residues. Excessive runoff. Types of Interactions 1. Additive effects: Mixing of two pesticides provides the same response as the combined effects of each material when applied alone. The products neither hurt nor enhance each other. Such mixes save time, labor and equipment use. 2. Synergism: When pesticides provide a greater response than the added effects of each material when applied separately. 3. Antagonism: When two pesticides applied together produce less control than if each material is applied separately. Fenobucarb mixed with tridemorph exhibit antagonistic effect against Spodotera litura 44 4. Enhancement: It is not between two pesticides and happens with some adjuvants. When pesticide is mixed with an additive to provide a greater response than if pesticide is applied alone. Types of Incompatibility  Chemical: When two or more pesticides are mixed together the resultant loss or reduction of effectiveness of one or all components. Deactivation of active ingredient often occurs. Chemical compatibility is Common in insecticides especially organophosphate E.C. formulations. Chemical incompatibility is affected by temperature, tank pH and length of time the mixture is held before use. Avoid tank mix combinations for strongly acid and alkaline materials which can result in chemical incompatibility.  Mechanical Reasons for mechanical incompatibility were different pesticides may require different droplet size to be most effective, spray volumes may vary, adjuvant recommendations may vary, over agitation may cause foaming in some older products.  Phytotoxic: When two or more pesticides used in combination result in injury to the host plants. Pesticides are perfectly safe when used alone, but injurious in certain combination. Symptoms include chlorotic spots, darkened shallow pits on fruits, scorching and bleaching of foliage and reduced growth.  Physical: When two or more pesticides are mixed together and the result is an unstable mixture or soapy flocculate. Usually involves inert ingredients of a formulation. Sometimes a problem in EC formulations which don’t have a good stabilizing agent. They may cause due to improper mixing and sometimes avoided with jar test. Factors that affect the compatibility  Difference in composition of products  Sequence of mixing  Degree of agitation  Quality of water used in mixing and preparing the fluid  Spraying of equipment and its design  Temperature  Crop and its variety  Quality of different components of the mixture and their concentrations Tank Mixing Guidelines Recommendations, labels and compatibility charts are certainly helpful but the pesticide user should take additional precautions. Check the pesticide label for tank mix recommendations and note any restraints. No label recommendations exist, do a jar test prior to spraying. Mix all pesticides properly and according to labels. Make sure all components of your spraying have the correct filters. If you have a spray blockage, try to retrieve the mix, before disposing of your tank mix. Test pH many incompatibilities result from excessively alkaline (sometimes acidic) pH in the tank. The addition of buffering adjuvants can help. 45  Make a test application to expose any phytotoxicity or antagonism before you make a large-scale application. If you overlap a few strips, this also can show you how much of a margin of safety you have. Wait a few days for symptoms to become visible. Take care with fertilizers. If you add fertilizers, be aware that they can have substantial effects on the chemistry of a tank mix, especially PH. Read the pesticide label for any fertilizer restrictions. Mix no more than one soluble or emulsifiable chemical with any insoluble products such as wettable powders or flowables. Avoid mixing strongly acid materials with strongly alkaline materials. Apply sprays soon after mixing. Mixes that sit for several hours or longer are prone to degrade, especially if the pH is alkaline. Proper Mixing Procedures  Mixing Order: Pesticide labels usually provide directions for mixing different materials, often describing the sequence of mixing. In general, follow the W-A-L-E-S plan when adding herbicides to a tank mix.  Wettable Powders (WP) then Flowables (F, DF)  Agitate then add adjuvants such as anti-foaming compounds, buffers  Liquid and Soluble products  Emulsifiable concentrates (EC)  Surfactants Jar test for compatibility: 46  The physical compatibility of pesticides will be evaluated with jar compatibility test. In this test, initially 500ml of standard hard water (0.304g calcium chloride and 0.139g of magnesium chloride hexa-hydrate in one litre of double distilled water) will be taken in one litre jar. Two pesticides whose compatibility has to be tested will be added in the jar. The volume of pesticide mixture will be made up to one litre with hard water, agitated by shaking the jar and will be kept in lab without disturbing for 30 minutes. Observations will be recorded after 30 and 60 minutes with respect to foaming and sedimentation. The pH of pesticides alone and in combinations will be recorded and adjusted to neutral. Exercise No. 12 METHODS OF APPLICATION OF PLANT PROTECTION CHEMICALS Plant protection chemical formulation is a mixture of active ingredient (a.i) and other substances including carriers, stickers, softeners, spreaders, wetting etc., Most common chemical formulation are dusts, wettable powder, water dispersible powder, oil emulsifiers, granules, slurries, paste, aerosols. Chemicals for plant disease control can be applied in the following ways. 1. Foliar sprays and dusts: Many inorganic and fungicides including systemics and antibiotics are generally applied to foliage by spraying with either high volume or low volume sprayers. The insoluble or non-suspendable materials are normally dusted on the foliage by using different types of dusters. 2. Treatment of tubers, bulbs, fruits and other plant parts: Formaldehyde, Mercuric chloride, Semesan etc. are used as dip treatments of tubers and bulbs. Thiobendazole and Benomyl are used as dip or spray on harvested fruits and vegetables. Normally Bordeaux paste, chaubattia paste etc. are used as wound dressers. 3. Seed dressing: Most of the chemicals used for seed treatment are protectants and disinfectants. Organo merucurials are commonly used for seed treatments. On these chemicals some are readily absorbed by plants soon after germination because they are systemic and get translocated in plant sap and keeps the plant free from infections for a considerable period of time. Seeds are normally treated by dry or wet treatment method. a) DRY SEED DRESSING: Materials required: 1. Seeds, 2. Captan, 3. Container. Procedure: 1. Weigh one kg of seeds and put them in a container. 47 2. Weigh 2 gm of captan and add to the container having the seeds. 3. Close the mouth with a lid and mix them so as to get a uniform coating of the fungicide on the seed coat. b) WET SEED DRESSING: Materials required: 1. Seeds, 2. Bavistin, 3. Plastic basin, 4. Glass rod. Procedure: 1. Take 1 litre of clean water in a plastic bucket. 2. Add 2 gm of Bavistin to the water and thoroughly mix it by constant stirring with a glass rod. 3. Take 1 kg of seeds and soak them for 15 minutes in the above fungicide suspension taken in the plastic basin. 4. Remove the seeds from the suspension and dry them in shade on a paper. 5. Sow a known number of treated seeds in soil taken in plastic pots. 6. Similarly soak 100 g of seeds in sterile water for 15 minutes. Dry them on paper and sow an equal number of seeds in sterile soil in plastic pots. This will serve as check. Observation: 1. Record the percentage of germination of seeds in the treated and check pots. 2. Observe for the associated pathogens. 1. Furrow Application: Whenever the crops are planted in furrows, this method of application is used either in dust or liquid form. The fungicide are applied about 2-3 feet away from the main root of the plants. 2. Broadcasting: The non-volatile chemicals like nematicides are mixed with soil or fertilizers and broadcasted on the soil surface. Light ploughing or harrowing is done to mix the chemical to sufficient depths. These are applied during crop free period or well in advance of sowing or planting. 3. Drenching: A suspension of the chemical either before sowing or after the emergence of seedlings is applied to the soil surface in quantities sufficient to wet soil to a depth of 10-15 cm. Usually at 0.2 % concentration. 48 Exercise No. 13 PLANT PROTECTION APPLIANCES IN PLANT DISEASE MANAGEMENT The common plant protection appliances are sprayers and dusters A. Sprayer: Sprayer is a mechanical device in which the liquids are broken up in to tine droplets and discharged with some force so as to distribute on the required sites as quickly as possible. It consists of following essential parts Tank or container: It is a metallic container plastic bucket! cement tank to hold the spray liquid. Pump: It is meant for creating pressure in the tank and it may be pneumatic or hydraulic type Discharge line or delivery line: It. is meant for discharging the spray liquid and consists of delivery hose, lance and nozzle (which consists of swirl plate, swirl chamber washer, jet bearing apparatus) and agitators. Types of sprayers: I. Manually operated sprayers: It works on the system of air compression. Hand sprayer: a. Automiser: specially for kitchen gardens b. Pneumatic knapsack or back sprayer: A shoulder mounted sprayer for regular spraying in small sized plots. Hydraulic sprayer: It works on principle of hydraulic pressure a. Foot sprayers: Pedal type pump designed for field crops and fruit crops. b.Bucket sprayers: Pump barrel with single or double barrel, good for all kinds of small scale spraying. 49  c. Rocking sprayers: Well suited for field orchard, plantations and used for general spraying. d. Knapsack sprayers: Ideal for small scale or spot spraying in vegetable gardens or vine gardens etc. e. Mist Blowers: These are low volume sprayers carried on the back of the operator. The force of the air blast produced by the fan dispenses the spray in very fine droplets of 50 to 100 µ (microns) in size. Ideal for field crops, orchards etc. II. Power operated sprayers: It consists of a petrol engine and a frame work in addition to other standard components of sprayer- small portable hydraulic energy power sprayer, tractor mounted large sprayers, compression sprayers. motorized knapsack sprayer, knapsack mist blower. B. Soil injectors: It is used to inject volatile chemicals in to the soil C. Dusters: Appliances used for distributing fine particles of dry dust formulations. The essential parts are, 1. Container 2. Blower: to create an air current for ejecting out dust. 3. Operating system: to work the equipment on a system of gears 4. Agitator: to stir the powder or dust in the container so as to make it flow uniformly 5. Feed mechanism and level regulator: to control the rate of flow of dust or powder 6. Discharge line: consists of a metal lance and nozzle Types of dusters I. Hand dusters: Plunger type duster consisting of a metal air pump dust chamber and a discharge assembly suited for dusting small areas like kitchen gardens. Racking or container dusters, hand pumps, shoulder mounted knapsack, bellow type dusters. rotor hand dusters. II. Power dusters: Excellent for quick coverage of large areas, good for all types of crops, orchards, plantation areas. Motorized power dusters, motorized knapsack duster. III. Hand Rotary duster: Can be used for small to medium scale crops - vegetable plots, nurseries etc. The essential parts are (a) tank (b) agitator (c) fan and blower (d) flow regulators and (e) distribution system. The rate of flow can be regulated from 2-20 lbs/minute. D. Seed treatment mercenaries: 50  They consist of drum fitted on stand which can be rotated with the help of handle provided at one end of the drum. The drum has a large opening to introduce the seeds and chemical for treatment and to take the seeds after treatment. Ex. Motorized seed dresser, gravity seed dresser, slurry seed dresser, grain treating machinery. 51 Exercise No. 14 SAFETY MEASURES IN PESTICIDES USAGE A. Purchase 1. Purchase only JUST required quantity e.g. 100, 250, 500 or 1000 g/ml for single application in specified area. 2. Do not purchase leaking containers, loose, unsealed or torn bags. 3. Do not purchase pesticides without proper/approved labels. B. Storage 1. Avoid storage of pesticides in the house premises. 2. Keep only in original container with intact seal. 3. Do not transfer pesticides to other container. 4. Never keep them together with food or feed/fodder. 5. Keep away from the reach of children and livestock. 6. Do not expose to sun-light or rain water. 7. Do not store weedicides along with other pesticides. C. Handling 1. Never carry/transport pesticides along with food materials. 2. Avoid carrying bulk - pesticides (dusts / granules) on head, shoulders or on the back. D. Precautions for Preparing Spray Solution 1. Use clean water. 2. Always protect your NOSE, EYES, MOUTH, EARS and HANDS. 52 3. Use hand gloves, face mask and cover your head with cap. 4. Use polyethylene bags as hand gloves, handkerchiefs or piece of clean cloth as mask and a cap or towel to cover the head (Do not use polyethylene bag contaminated with pesticides). 5. Read the label on the container before preparing spray solution. 6. Prepare spray solution as per requirement. 7. Do not mix granules with water. 8. Concentrated pesticides must not fall on hands etc. while opening sealed containers. Do not smell the sprayer tank. 9. Avoid spilling of pesticide solution while filling the sprayer tank. 10. Do not eat, drink, smoke or chew while preparing solution. 11. The operator should protect his bare feet and hands with polyethylene bags. E. Equipments 1. Select right kind of equipment. 2. Do not use leaky, defective equipment. 3. Select right kind of nozzle. 4. Don’t blow/clean clogged- nozzle with mouth. Use old tooth- brushes tied with the sprayer and clean with water. 5. Do not use same sprayer for weedicide and insecticide. F. Precautions for applying pesticides 1. Apply only at recommended dose and dilution. 2. Do not apply on hot sunny day or strong windy condition. 3. Do not apply just before the rains and also after the rains. 4. Do not apply against the wind direction. 5. Emulsifiable concentrate formulations should not be used for spraying with battery operated ULV sprayer. 6. Wash the sprayer and bucket etc with soap water after spraying. 7. Containers, buckets etc. used for mixing pesticides should not be used for domestic purposes. 8. Avoid entry of animals and workers in the fields immediately after the spraying. G. Disposal 1. Left over spray solution should not be drained in ponds or water lines etc. Throw it in barren isolated area, if possible. 2. The used/empty containers should be crushed with a stone / stick and burned deep into soil away from water source. 3. Never re-use empty pesticide container for any purpose. 53 Exercise No. 15 STUDY OF SEED STORAGE AND PACKAGING TECHNIQUES Seeds should have a good storage facility to protect them from seed-borne diseases. They should be stored in such a manner, that its germination capacity and vigour should not decline. It is important to package seed in dry containers for proper storage. For small quantities of seed, these containers may be tin cans, jars, or pots that are glazed on the inside; even reinforced boxes or bags can be suitable. Metal or plastic jerricans, or drums are often used to package large quantities of seed. Regardless of the type of container employed, it should be of standard size and shape, if poss