eChapters

There are many different methods of extracting DNA bands from an agarose gel. Which one you choose will probably depend on the consumables you have available in your lab. Another important consideration is the yield/ purity of the DNA after extraction. Agarose contains various impurities which may inhibit downstream reactions if not efficiently removed from the DNA. We used to give the generic term ‘hoonose’ to the unspecified carbohydrates that co-purified along with the DNA and inhibited enzyme reactions. Modern spin-column kits are very good at removing impurities, old-fashioned ‘kitchen sink’ methods are less good.

Cutting Out the DNA Band

Most of the methods require you to cut out the band. You must visualise the band, in an ethidium bromide stained gel, in a dark-room on a UV light- box (a trans-illuminator). UV is dangerous, wear gloves, long-sleeves and face protection. Never look at UV with unprotected eyes. If possible, set the trans-illuminator to long-wavelegth UV (or low-power) and minimise the amount of time the DNA is exposed. This is because the UV mutagenises the DNA at a measurable rate. Use a scalpel blade to cut around the band of interest. Switch off the transilluminator, switch on the white light and carefully remove the band from the gel and place it on the glass. It is good to trim off as much empty agarose as possible so go back to UV illumination briefly to do this. Place the excised band in a 1.5mL microfuge tube.

Hunter and Mohler discovered the isozymes. In 1959 Markert and Moler introduced the concept of isozymes, which they defined as the different molecular forms in which proteins may exist with the same enzymatic specificity (Buth 1984). This means that different variants on the same enzymes have identical or similar functions and are present in the same individual. As such, their importance for understanding gene action in development and differentiation was exploited during the1960s in both animals and plants. Nevertheless, isozymes played a minor role in research on plant biochemistry until 1966 when genetic polymorphism for isozymes within the same population was discovered (Stebbins 1989, Wendel 1989). That revealed the possibility for population genetics to make precise quantitative estimates of genetic variability based upon one parameter of the molecular structure of the primary products of the genes themselves.

Plant population genetics were not long in following their zoological colleagues and the investigation of both animals and plants increased explosively. This application, however, necessitated a partitioning of the isozyme concept, since the relevant isozyme subset, defined by Prakash (Buth 1984) as the variant proteins produced by allelic forms of the same locus, to avoid the now common confusion with isozymes which are the various polymer produced from monomers specified by different loci. The development of genetic distance or similarity coefficients (Lukasová 1985) allowed the summarization of isozyme data for intersample comparisons and so quantified isozyme data were soon applied to comparative studies of taxa.

Proteins

Proteins are of central importance in the structure and function of all living organisms. Some proteins serve as structural components while others function in communication, defense, and cell regulation. Some proteins serve as enzymes that act as biological catalysts which control the pace and nature of essentially all biochemical events. Although DNA serves as the genetic blueprint of a cell, none of the life processes would be possible without the proteins.

Electrophoresis of Proteins

Proteins can be separated and purified. Methods for separating proteins take advantage of properties such as charge, size, and solubility, which vary from one protein to the next. Because many proteins bind to other biomolecules, proteins can also be separated on the basis of their binding properties. The source of a protein is generally tissue or microbial cells. The cell must be broken open and the protein must be released into a solution called a crude extract. If necessary, differential centrifugation can be used to prepare subcellular fractions or to isolate organelles. Once the extract or organelle preparation is ready, a variety of methods are available for separation of proteins. Ion-exchange chromatography can be used to separate proteins with different charges (similar to the way amino acids are separated). Other chromatographic methods take advantage of differences in size, binding affinity, and solubility. Nonchromatographic methods include the selective precipitation of proteins with salt, acid, or high temperatures. In addition to chromatography, another important set of methods is available for the separation of proteins, based on the migration of charged proteins in an electric field, a process called (gel) electrophoresis. Gel electrophoresis is especially useful as an analytical method. Its advantage is that proteins can be visualized as well as separated, permitting a researcher to estimate quickly the number of proteins in a mixture or the degree of purity of a particular protein preparation. Also, gel electrophoresis allows determination of crucial properties of a protein such as its isoelectric point and approximate molecular weight.

Polyacrylamide

Polyacrylamide is a cross-linked polymer of acrylamide. The length of the polymer chains is dictated by the concentration of acrylamide used, which is typically between 3.5 and 20%. Polyacrylamide gels are significantly more annoying to prepare than agarose gels. Because oxygen inhibits the polymer- ization process, they must be poured between glass plates (or cylinders).

Two types of molecular weight (MW) standards are routinely used. The Lambda/HindIII and PhiX174/HaeIII MW standards provide a useful reference for calculating molecular weights of large and small DNA fragments, respectively, after electrophoretic separation; the “internal MW standards” provide a means for normalizing fragment migration distances within each lane to facilitate comparisons between lanes on the same or different luminographs in fingerprinting studies.

Electrophoresis is an analytical method frequently used in molecular biology and medicine. It is applied for the separation and characterization of proteins, nucleic acids and subcellular-sized particles like viruses and small organelles. Its principle is that the charged particles of a sample migrate in an applied electrical field. An electric current is passed through a medium containing the molecules, and each molecule travels at a different rate depending on its electrical charge, size and shape. Separation by electrophoresis is based on these differences. In electrophoresis, agarose and acrylamide gels are used for electrophoresis of nucleic acids and proteins.

What is Gel Electrophoresis?

A gel is a solid form. The term electrophoresis describes the migration of charged particle under the influence of an electric field. Electro refers to the energy of electricity. Phoresis, from the Greek verb phoros, means “to carry across.” Thus, gel electrophoresis refers to the technique in which molecules are forced across a span of gel, motivated by an electrical current. Activated electrodes at either end of the gel provide the driving force. A molecule’s properties determine how rapidly an electric field can move the molecule through a gelatinous medium.

Although it can be argued that due to its overall trendiness, they have been a lot of recent developments in making it more fun to work with. As a molecule, it is very similar to DNA (the most notable chemical difference is its flexibility), but suffers because the nucleases (RNases) that can destroy it are notoriously very stable and very difficult to get rid of.

What is DNA

DNA is a simple molecule

DNA is negatively charged

DNA has lots of ring structures; ring structures are generally quite hydrophobic.

DNA is a very robust molecule.

DNA work has lots of toys.

1. pH meter

pH meter/electrode systems are the most common method of measuring pH in the biology laboratory. Although more expensive and more complicated to use than chemical indicators, pH meters provide greater accuracy, sensitivity, and flexibility. When used properly, pH meters can measure the pH of a solution to the nearest 0.1 pH unit or better, and they can be used with a variety of samples.

Abstract

Information and Communication Technology (ICT) played and continue to play pivotal role in better decision making of farmers for plant health management under the climate change scenario. For judicious plant health management by farmers through the use of ICT tools (viz. interactive Kiosk, Mobile Apps, Whatsapp, Webportal), information need and information access network to be assessed. Based on farmers’ information need and network, information delivery tree should be designed in ICT tools. Under this backdrop this research work was carried out among the farmers of Bhagalpur district of Bihar. The sample size was 120. From the study it was noted that the major information needs of the farmers were seed availability, crop disease management, insect pest management, weed management and weather information and pesticide application. Farmers were enthusiastic to improve their knowledge on insect pests and disease identification and management. Specifically, farmers need information on insect pest management of wheat (aphid), maize (stem borer), vegetables (fruit borer), pulses (pod borer) and oilseeds (aphid) and also disease management of wheat (leaf blight and spot blotch disease), maize (Turcicum leaf blight), vegetables (wilt and leaf spot) and pulses (wilt disease). Farmers decision making was positively and significantly correlated with crop disease management(0.196*),insect pest management of crop(0.208*) and weed management(0.239**). Through the Path Analysis it was noted that farmers decision making on disease management was mediated by the variables - Bihar Agricultural University-Kisan Diary, Mobile Apps(Bihar Krishi Apps) and Kisan Salahkar. So, it can be concluded that these much needed information should be provided to the farmers though different ICT tools and extension personnel should be trained to operate these ICT tools for accelerated use of ICT tools by the farmers.

Abstract

Bacterial diseases of solanaceous crops like tomato, brinjal, chilli, capsicum and potato are the most serious and destructive diseases affecting both under field and greenhouse conditions. Under moist field conditions they can cause epidemics to affect total crop loss, quality of the produce and reduce market value. These diseases can be distinguished from each other on the basis of their symptoms appearance of the various parts of the plant as well as by using advanced techniques of diagnosis. Diagnosis of the disease is important step to detect pathogen at early stage of disease and subsequently manage it effectively in efficient manner.

Keywords: Bacterial diseases, solanaceous crops, symptoms, diagnosis.

Introduction

Bacterial diseases of solanaceous crops like tomato, brinjal, chilli, capsicum and potato are the most serious and destructive diseases affecting both under field and greenhouse conditions. Under moist field conditions they can cause epidemics to affect total crop loss, quality of the produce and reduce market value. These diseases can be distinguished from each other on the basis of their symptoms appearanceofthe various parts of the plant as well as by using advanced techniques of diagnosis. Diagnosis of the disease is important step to detect pathogen at early stage of disease and subsequently manage it effectively in efficient manner. Therefore, the early detection of the bacterium from asymptomatic plants, soil, and water and plant residues could facilitate elimination and certainly reduce the risk of crop loss. Powerful tools for the identification and detection of the bacterium in diverse substrates (plant, seed, water and soil) are available for diagnosis of the bacterial diseases. DNA amplification of pathogen offers many advantages over classical techniques; neither purification nor cultivation of the pathogen is required and the specificity, sensitivity and response time of tests are improved. Nevertheless, the PCR method has not yet become a routine diagnostic tool for many laboratories, mainly because of the inhibition of the amplification reaction by compounds contained in crude bacterial extracts, which give false negative results or low detection sensitivity.

Abstract

Onion (Allium cepa L.) and garlic (Allium sativum L.) are important members of the family Amaryllidaceae. Bulbs, which are the edible component in both onion and garlic grow underground and are of nutritional and medicinal importance. These underground bulb vegetables are affected by many diseases caused by different microorganisms like fungi, bacteria, nematode and viruses causing huge economic loss. Stemphylium leaf blight caused by Stemphylium vesicarium, is the major fungal disease affecting their productivity of the edible bulbs adversely.The fungus primarily attacks the leaves but can affect bulbs in storage. Initial infection is characterised by appearance of small greyish white oval spots on the leaves. These spots are often surrounded by a yellowish halo with irregular margins. Fungal pseudothecia may over-winter in contaminated plant material or may survive in the soil having plant debris and ascospores are the primary inoculums in the following season. Spores require rain or persistent dew to cause infection. This disease can spread rapidly when environmental conditions are favourable for spore development. Suitable integration of more efficient treatments like fungicides, bio agents in combination with appropriate cultural control provide better management of the Stemphylium blight disease

Abstract

India ranks first among world’s mango producing countries accounting for about 50 % of the world’s total mango production. In Bihar it is the most important fruit crop as well as occupies a large area affecting socio economic conditions of the region. The crop suffers from over 140 diseases all over the world. From young seedlings to mature trees and fruits in their all stages from start to storage may suffer from a number of biotic and abiotic stresses. All parts of the plant namely, trunk, branch, twigs, leaf, petiole, flower and fruits are prone to attack by a number of pathogens The changes in climatic patterns and shifting towards commercial cultivation of this crop has seen the emergence of some diseases which can cause huge losses to the production of mango over the years. One such problem, decline in mango is reported to be a complex problem involving multitude of factors. Decline disorders are recognized in virtually all mango-producing regions of the world. In most cases, the disease has been characterized by leaf drooping and drying; bark splitting, gum secretion from branches and main trunk, bud necrosis, dieback, vascular discoloration and death of the whole tree. The bark beetle Hypocryphalus mangiferae is closely associated with trees affected by mango sudden decline disease. Report suggests the causal agent to be Botryodiplodia theobromae, Ceratocystis manginecans, Fusarium spp, Colletotrichum sp, Pestalosiopsis maniferae. In most of the cases Botryodiplodia theobromae, Ceratocystis spp are associated with it. Although, none of the pathogens isolated could singly incite the complete decline symptoms under artificial inoculation on mango seedlings indicating towards complex etiology of the disease. Since pathogens are endophytic in nature; mangement of the pathogen after infection is difficult and relies more upon prohytalctic measures. Over all plant health, its nutritional status and abiotic conditions of flood and drought are also the pre disposing factors to decline in Mango. Effective management of decline in mango needs holistic approaches using optimum nutrition, general sanitation, routine crop protection schedule, weed management and appropriate intercropping practices.

Abstract

Fungal rot and wilt caused by Sclerotinia sclerotiorum is a devastating disease of vegetables causing huge loss. The fungus has a broad host of more than 400 species including diverse vegetable crops, viz., carrot, potato, tomato, green pepper, eggplant, lettuce, peas and beans, cole crops, bottle gourd, pointed gourd, etc. Being able to overwinter in the soil in form of sclerotia upto 5 years and having such a wide host range, management of the disease becomes very difficult. Thus development of resistant varieties can be the only long term strategy for control of the pathogen. The screening of the existing genotypes and identification of resistant genotypes forms the base of any resistant breeding programme. The widely available genotypes of different crops have been screened by several researchers and resistant genotypes have been identified in cauliflower, vegetable soybean, peas, tomato, etc. These genotypes are further used in breeding programmes for as donors for resistance to Sclerotinia sclerotiorum.

Keywords: Vegetables, Sclerotinia, Resistance breeding, Genotypic screening

Abstract

Palms are generally grown commercially in tropical climate. But some of the palms like coconut, areca nut and palmyra palm are cultivated successfully in West Bengal, Orissa, Assam, and some pockets of Bihar that lies under sub tropics of India.Due to unsuitable climatic factor their production is more affected by abiotic and biotic stress including different kinds of diseases. The important disease are bud rot, basal stem end rot, nut shedding,stem bleeding in coconut, mahali disease, foot rot, stem breaking in arecanut and palmyra palm in the region have negligible incidence of diseases. These destructive diseases are the major constrain in production and causes severe yield loss in these palm fruits. Therefore effective management strategies are required by continuous monitoring the incidence and severity of these diseases caused by different pathogens.

Integrated disease management is the most accepted strategies for sustainable crop production that involves the holistic approach integrating cultural, physical, mechanical, chemical and biological means to control the disease. However, results of the disease management trials depend upon soil condition, age of the crop, stage of disease development and weather parameters of that particular area. Although it is impossible to manage a field free of pathogen but considerable reduction of the disease can be achieved through a proper integrated disease management system. Thus the causes and remedy of the diseases prevalent mainly in palms particularly in subtropical conditions has been reviewed for better management and sustainable production.

Introduction

The term biological control was first used in scientific literature in 1914. Baker and Cook (1974) described biological control as “reduction of inoculum density or disease producing activities of a pathogen or parasite in its active or dormant state, by one or more organisms, accomplished naturally or thoroughly manipulation of environment, host, or antagonists or by mass introduction of one or more antagonists.”

Certain genera of fungi have greater potential for being effective bio-control agents eg. Trichoderma, Gliocladium, Chaetomium; entomogenous fungi as Entomophthora, Beauveria, Metarrhizium, Hirsutella; Nematophagous viz., Catenaria, Zoopage, Stylopage, Paecilomyces and predaceaus fungi like Arthrobotrys, Dictylella, Dactylaria and Nematoctonus (Narain, 2018). Biopesticide formulations based on these fungi are also called as “Microbial pesticides”.

Abstract

Potato (Solanum tuberosum L.), an important cash crop of India, is prone to many diseases and amongest these, late blight [Phytophthora infestans (Mont) de-Bary] is one of the highly destructive diseases and a major constraint in the profitable cultivation of potato. After realizing the significance of controlling the devastating disease for the sustainability in potato production and thus ensuring more profitability, the present investigation has been conducted with an objective to select the most effective fungicides and botanicals for the management of the disease. The present study was carried out by KVK, Saharsa during rabi season for consecutive two years (2015-16 and 2016-17) as On-Farm Trial mode at farmers fields of an adopted village of Saharsa district in Bihar. In each trial, there were four treatments including one control i.e. farmers’practice. The study concludes that the best treatment option to combat late blight disease is Seed treatment with Trichoderma viride @ 5g per kg of seed and spray of Ridomil @ 2g per litre of water starting from 30 DAP alternating with Dimethomorph 1g + ridomil 2g per litre of water at 10 days intervals. The results of economic analysis reveal that the net return as well as benefit cost ratio was also highest in Seed treatment with Trichoderma viride @ 5g per kg of seed and spray of Ridomil @ 2g per litre of water starting from 30 DAP alternating with Dimethomorph 1g + ridomil 2g per litre of water at 10 days intervals.

Abstract

Mushroom production is emerging as an added dimension in agriculture diversity; its cultivation lies on the utilization of agricultural wastes rich in proteinaceous materials. Five different substrates such as wheat straw, maize cob, paddy straw, banana pseudo stem, and lentil straw were used with suitable supplements to evaluate the average period of spawn run, pin head initiation, time taken for harvesting of flushes, fruit yield during both crop season 2014 and 2015. In 2014, highest fruit yield (380.3 g/ 500g) were recorded when wheat straw supplemented with rice bran (2% of the substrate weight), corn flour @ 25 g/kg prepared straw and gypsum @ 20 g/kg straw among various used substrates. This treatment also resulted better in respect of time taken for spawn run (9.3 days), pin head initiation (11.7 days), time taken for harvesting of flushes (I^st flush- 20.5, II^nd flush-26.7, III^rd flush- 32.9). The second best treatment was paddy straw when supplemented with rice bran (2% of the substrate weight), corn flour @ 25 g/kg prepared straw, and gypsum @ 20 g/kg. The lowest fruit yield (193.6 g/500g), time taken for spawn run (15.9 days), pin head initiation (22.4 days), time taken for harvesting of flushes (I^st flush- 27.9, II^nd flush-37.1, III^rd flush- 44.7) were recorded with banana pseudostem (chopped) when supplemented with rice bran (2 % of the substrate weight), corn flour @ 25 g/kg prepared straw and CaCO₃ gypsum @ 20 g/kg straw. The same experiment was repeated during the year crop season 2015. Surprisingly wheat straw supplemented with rice bran (2% of the substrate weight), corn flour @ 25 g/kg prepared straw and gypsum @ 20 g/kg prepared straw was found again superior over other treatments with more or less similar performance. Similarly more or less same performances were resulted in rest of the treatment too. The incidence of mould infection in mushroom was also noticed in accordance with the kinds of substrates used during both crop season 2014 and 2015. Lowest incidence of mould i.e. green mould (5.13 %), black mould (3.10 %) and blue mould (2.42%) was observed on wheat straw when supplemented with rice bran (2% of the substrate weight), corn flour @ 25 g/kg prepared straw and gypsum @ 20 g/kg straw, green mold (3.13 %), black mold (3.10 %) and blue mold (2.42 %). Similarly more or less same pattern of mould incidence was reported in the crop season 2015.

Abstract

The agricultural scientists particularly those who are in advisory service, irrespective of specialization, are often approached by the farmers with diseased samples of their crops and sought advice on plant protection strategy. The strategy includes components of control measures, doses, mode and time of application. In the present article different steps of formulation of plant disease control strategy are discussed. To further elaborate the contents, examples from integrated management strategy of mango malformation disease is cited.

Keyword: Mango Malformation

Introduction

Mango malformation, disease was first time reported from Darbhanga District of Bihar State in 1891 by G. Watt. The disease is currently prevalent in almost all the mango growing country and is thus designated as the plant disease of International importance and previously considered to be a serious problem in the northern parts of the country, has now started to appear in central India too in severe form. It is well known, the mango malformation is the most serious disease, rendering mango cultivation non-lucrative in subcontinent and other regions of the world (Ahmad et al., 2002; Ploetz, 2001). Mango malformation caused by fungus Fusarium moniliforme var. Subglutinans, are proved (Freeman 1999; Zheng and Ploetz, 2002). Recently, these isolates were described as members of a new species, F. mangiferae Britz, Wingfield and Marasas sp nov. (Britz et al., 2002). The casual organism of mango malformation grows well at lower temperature and higher relative humidity. The disease is quite common in both grafted and the seedling plants (Kumar and Chakrabarti, 1997). The symptoms manifest as bunchy top (BT) in 4-5 month old seedlings in the nursery, vegetative malformation (VM) of twigs arising from axillary buds and malformation of inflorescence (MF). Normally floral malformation is more prevalent in bearing trees than vegetative malformation. Flowers in the malformed panicle are much enlarged and crowded with hypertrophied axes of the panicle. Malformed inflorescences/ panicle of different varieties were variable in respect of their shapes, sizes, growth and compactness, depending upon the cellular structure and physiology under environmental factors (Chakrabarti et al., 1990). Generally malformed inflorescences produce no fruit, or abort already stages (Schloser, 1971) and is directly responsible for reduction in yield (Kumar et al., 1993 and Singh et al., 1998). In the present study the incidence of Floral malformation on a large number of mango cultivars was recorded over a period of 5 years with a view to identify the entries of future resistant breeding programme.