Molecular Biology in Plant Pathogenesis and Disease Management:: Disease Management, Volume 3

Printed book. Hardcover Molecular Biology in Plant Pathogenesis and Disease Explores the use of molecular methods in measuring and managing disease.
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Get to Know Us. English Choose a language for shopping. Amazon Music Stream millions of songs. Amazon Drive Cloud storage from Amazon. Alexa Actionable Analytics for the Web. AmazonGlobal Ship Orders Internationally. Amazon Inspire Digital Educational Resources. Amazon Rapids Fun stories for kids on the go. Amazon Restaurants Food delivery from local restaurants. Rajakumari, made it possible to devote my undivided attention for the preparation of this book.

To all my family members Mr. Varun Karthik, I am glad to express my thanks for their affectionate encouragement to heighten the level of my involvement in academic endeavors. Finally it is with a sense of gratitude, I extend my thanks to Mr. Pappa Vidyaakar, founder of Udavum Karangal, for his enduring encouragement and appreciation for my academic and humanistic activities. A later assessment by Pimentel et al. The imperative need to save the crops from the attack of potential pathogens was realized and research efforts were intensified in the later half of the twentieth century.

Various aspects of the pathogens and the diseases induced by them were investigated. The conventional methods involving cultural and microscopic methods provided the basic knowledge on plant-pathogen interactions leading to development of disease symptoms on compatible host plant species or cultivar. In contrast, the pathogen development was partially or entirely inhibited depending on the levels of resistance in incompatible interaction.

Furthermore, epidemiological factors favoring disease incidence and spread under experimental and natural conditions were determined to develop disease prediction models and forewarning systems. Short-term and long-term strategies were planned based on the results of conventional techniques. However, the inherent limitations of the conventional methods necessitated to look for techniques with higher levels of precision and reliability Narayanasamy , The importance of discovery that Tobacco mosaic virus TMV causing tobacco mosaic disease could be crystallized, was duly recognized by the award of Nobel Prize to Stanley and this work signaled the commencement P.

Microbial Plant Pathogens, Vol. Equally important was the finding of Bawden et al. The presentation of crucial evidence for gene-for-gene interaction in flax-rust pathosystem by Flor , established the concept that the genes conditioning the reaction of the host may be identified by their interaction with specific strains of the pathogen. On the other hand, those genes that condition pathogenicity may be identified by their interaction with specific host varieties.

The concept of Flor, demonstrated to be true in several pathosystems, was hailed as one of the most important contributions during the last century, providing a firm footing for studying the host plant-pathogen interactions more critically. Development of techniques for isolation, cloning and sequencing the deoxyribose nucleic acid DNA of various organisms including plants and pathogens marked the period of significant revolution in biological sciences.

Braun and Pringle demonstrated that the crown gall bacterium Agrobacterium tumefaciens induced permanent transformation of plant host cells resulting in the autonomous and rapid growth of the transformed cells in culture. Arabidopsis thaliana, a small dicotyledonous cruciferous plant species has been adopted as a model species for most aspects of plant biology, because of its small genome size, short life cycle, small stature and ability to produce large number of progenies. These attributes have made this plant species ideally suited for genetic and mutational analyses. Arabidopsis has been shown to have a relatively high proportion of genes involved in metabolism and defense.

Many of the physiological processes investigated in Arabidopsis and crop plants appear to share many common features, especially related to disease and pest resistance and salt tolerance.


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As the genome of A. However, the conclusions arrived at based on experiments performed on A. Application of genomics to molecular resistance breeding is considered as the most important area of promise, leading to development of crop cultivars with built-in resistance to one or more diseases Slater et al. Some of these early breakthrough-findings obtained by applying the molecular techniques revealed the effectiveness and potential of these approaches. Evidences in support of numerous suggestions and hypotheses proposed earlier have been obtained by using molecular methods.

These methods have been shown to be very useful to investigate various aspects of plant-pathogen interaction resulting in either disease progress or restriction due to effective elicitation of host defense responses nullifying the adverse effects of the pathogen-derived products. Interaction between avirulent strains of pathogens and resistant hosts generally results in hypersensitive response HR.

Bacterial pathogens and their host plant species have been shown to be preferable systems for molecular approaches. An avirulence gene avrA from a race 6 strain of Pseudomonas syringae pv. By cloning of virulence and hrp genes from bacterial pathogens, significant progress was made in understanding various phenomena relating to pathogenesis such as virulence, plant recognition and host range in many pathosystems. The discovery of genes involved in the production of host-specific toxins HSTs by fungal pathogens elucidated the molecular bases of symptom induction and resistance to the pathogens producing the HSTs Durbin ; Baker et al.

Furthermore, the effectiveness and reliability of the molecular techniques in hastening the application of different disease management strategies particularly for the development of cultivars with built-in resistance to diseases caused by microbial pathogens has been well recognized as a significant advantage over conventional methods. Biochemical studies are also required for classifying bacterial pathogens, since the variations in the morphological features alone are not enough to differentiate genera and species. As the viral pathogens are extremely small in size, the virus particle morphology offers no dependable basis for differentiation, necessitating the use of molecular biological approaches for their detection and differentiation.

Even in the case of fungal pathogens differentiation based on morphological characteristics may not be possible, if their development is affected by environmental conditions and the presence of other fast-growing saprophytic microorganisms. The effectiveness and applicability of molecular techniques in studying the pathogen characteristics, disease development and formulation of suitable crop disease management systems are discussed in three volumes that include ten chapters, in addition to this introductory chapter. In Volume 1, the information on the molecular techniques to study the characteristics of microbial pathogens is presented.

Rapid detection, precise identification and unambiguous differentiation of various microbial pathogens or variants of a pathogen species are of paramount importance to initiate effective strategies of crop disease management. The effectiveness of molecular techniques to meet this requirement is discussed in Chapter 2. The genetic diversity of plant pathogens has to be assessed to understand the different levels of pathogenic potential virulence of strains, races or biotypes of a pathogen species, so that the occurrence of more virulent strain s can be detected, identified and quantified rapidly Volume 1, Chapter 3.

Various phases of disease development in susceptible plants under in vitro and factors influencing disease incidence and spread under in vivo resulting in occurrence of epidemics have been examined in detail by using molecular techniques. It has been possible to visualize and monitor various steps from pathogen adhesion to tissue colonization and symptom expression during different phases of pathogenesis by applying suitable and sensitive molecular methods Volume 2, Chapter 2.

Factors influencing plant disease incidence and spread have been studied using conventional methods for identification and quantification of pathogen populations in epidemiological investigations. Management of crop diseases successfully is the ultimate aim to provide reasonable margin for the grower for his efforts to produce food to meet the requirements of the consumers who need food free of pathogens and their toxic metabolites or chemical residues.

Volume 3 encloses six chapters which provide information on the short- and long-term disease management strategies that can be made effective by application of appropriate molecular methods. It has been well demonstrated that molecular assays are highly efficient in detecting and identifying pathogens present in plant materials that may or may not exhibit symptoms of infection by pathogens of quarantine importance.

Application of these techniques can effectively prevent introduction of exotic pathogen s that may find suitable conditions for development and spread in new geographical location s Volume 3, Chapter 2. Use of crop cultivars with built-in resistance to different diseases is considered as the economical and ecologically safe strategy of disease management. Locating disease resistance genes not only in plants, but also in diverse sources including insects and rapid selection of resistant genotypes or lines using genetic markers at early growth stage, have been possible due to the application of appropriate molecular methods.

Furthermore, the visual scoring methods to assess the disease intensities exhibited by different genotypes have not provided consistent results, because environmental factors are likely to influence the symptom expression. In contrast, molecular methods employed to determine the quantum of pathogen DNA have proved to be more accurate, reliable and rapid Volume 3, Chapter 3.

It is very difficult to transfer resistance gene s into cultivars from distantly related or unrelated plant species, because of the sterility of progenies associated with interspecific or intergeneric crosses. But biotechnological approaches have offered the possibility of transferring resistance gene s from plants and also from diverse sources such as fungi, insects, and frogs. Genetic engineering techniques have provided wide options which are not available, if conventional breeding procedures are to be followed.

Pathogen-derived resistance PDR approach has been shown to be successful in developing virus disease resistant crop cultivars Volume 3, Chapter 4. Development of disease-resistant cultivars through genetic engineering technology has been attempted in the case of a few crops such as tomato, tobacco, 1. Nevertheless, the possibility of enhancing the levels of resistance of desired cultivars by the application of effective inducers of resistance has been demonstrated to be a practical proposition.

Both biotic and abiotic inducers have been tested on a wide range of agricultural and horticultural crops. As resistance inducers activate the natural disease resistance NDR mechanisms existing in the plants, it is apparent that the plants treated with inducers, behave like genetically resistant plants. Furthermore, the inducers act on plants, but not on the pathogens.

Hence, the chances of development of resistance to these agents are remote, indicating the usefulness and practicability of this disease management strategy Volume 3, Chapter 5. Various fungal and bacterial species existing in the rhizosphere, phyllosphere or spermosphere have been found to have the potential of protecting plants against microbial pathogens. The molecular bases of protection to plants against microbial pathogens by the activities of biocontrol agents BCAs have been investigated. The genes controlling the production of antibiotics and enzymes capable of inhibiting the growth and development of the pathogen have been isolated, cloned and characterized.

Efforts have been made to enhance the biocontrol potential of the selected BCAs by transferring genes from other microorganisms. In addition, monitoring the spread, persistence and survival of the introduced BCAs has been effectively carried out by using appropriate molecular techniques Volume 3, Chapter 6. Various kinds of chemicals have been used at different stages of crop growth and storage for the control of microbial pathogens causing different diseases in agricultural and horticultural crops and the produce.

Although chemicals are able to reduce the disease incidence and spread significantly, the emergence of resistant or less sensitive strains of fungal and bacterial pathogens has been of great concern for the growers and the industry. Further, the growing awareness of the general public of the possible effects of pollution and persistence of residues, due to chemical application resulted in considerable difficulty in marketing the produce with higher levels of chemical residues. Rapid identification and differentiation of resistant and sensitive strains and monitoring of appearance of new strains resistant to fungicides may be possible using molecular techniques that can detect the changes in the sequences of specific gene s of the pathogens Volume 3, Chapter 7.

This book aims to provide the latest information to gain comprehensive knowledge on various aspects of plant-pathogen interaction leading either to development of symptoms induced by the pathogen or restriction of development and consequent elimination of the pathogen. The study of plant-pathogen interaction at cellular and molecular levels needs no emphasis, since molecular biological investigations have opened up the avenues that could not be accessed through conventional procedures.

The information presented in this volume is expected to be useful for researchers, teachers and upper level graduate students, pursuing investigations in biological sciences in the Departments of Plant Pathology, Molecular Biology and Biotechnology, Microbiology, Biochemistry, Plant Physiology and Plant Breeding and Genetics and also personnel of Plant Quarantine and Certification Programs. Mol Plant Microbe Interact J Agric Res Pimentel D ed Techniques for reducing pesticide use.

The conventional cultural methods involving isolation and studying the morphological characteristics using microscope are labor intensive and cumbersome, yielding sometimes, inconclusive results. The molecular techniques, on the other hand, are able to provide precise, reliable and reproducible results rapidly, facilitating early disease management decisions. Biochemical, immunological and nucleic acid-based assays have been preferred, because of the distinct advantages over the conventional methods.

The molecular techniques have been very useful in the identification of obligate pathogens causing diseases such as downy mildews, powdery mildews and rusts and also fungi that grow very slowly in the culture media, taking several weeks to produce spore forms that can be used for identification. Furthermore, molecular techniques have been demonstrated to be useful in breeding programs to identify sources of resistance to disease s.

Several protocols for molecular techniques, useful for researchers and students are presented in the Appendix. In a compatible susceptible interaction, development of symptoms characteristic of the disease may be observed, facilitating the identification of the pathogen inducing such symptoms. In another type of interaction, the plant species is susceptible to the pathogen in question, but no symptom of infection may be discernible.

This type of interaction known as latent infection is frequently seen P. These plants are known as symptomless carriers, posing potential danger for crop production. In yet another type of interaction designated quiescent infection, the pathogen remains dormant in infected immature fruits till they ripen. Such quiescent infections are seen in bananas and mangoes frequently. Nevertheless, the characteristic symptom may provide, however inaccurate it may be, a basis for the identification and differentiation of some microbial pathogens. Disease diagnosis relates to the identification of the nature and cause of the disease, whereas detection deals with establishing the presence of causative target organism with the test sample.

It has been found to be very difficult, frequently unsuccessful, if the symptom expression in a pathosystem is not clear. Further, if a host plant is subject to attack by many pathogens simultaneously or successively one after another, the symptom picture may be very different from those symptoms induced by pathogens individually. Reliability on symptoms alone may lead to erroneous identification of the causative agent s.

Rapid and precise identification of the cause of the disease is the basic requirement for the development of effective disease management systems. A wide range of diagnostic procedures has been employed for identification, differentiation and quantification of microbial plant pathogens. The sensitivity, reliability and reproducibility of the techniques have been constantly improved by the intensive research efforts in different countries.

Oomycetes lack taxonomic affinity with true fungi. The taxonomic position of the oomycetes with a unique lineage of eukaryotes unrelated to true fungi, but closely related to brown algae and diatoms has been established based on molecular phylogenetic and biochemical investigations. Oomycetes are included in the Kingdom Stramenopila, group Stramenopiles which encloses golden-brown algae, diatoms and brown algae such as kelp Sogin and Silberman ; Baldauf et al. The fungi-like oomycetes are included under different sections for fungal pathogens for discussion of various aspects.

Tests to determine physiological and biochemical characteristics were the basic tools used to identify and differentiate between bacterial pathogens for several decades from s. These tests were occasionally applied for the identification of filamentous fungi which in general exhibit greater phenotypic plasticity than bacteria Bridge The physiological and biochemical tests applied for the bacterial and fungal pathogens cannot be employed for the identification and differentiation of plant viruses, since they are extremely small and do not have any detectable physiological activity.

The physiological and biochemical properties vary widely between different groups of bacterial pathogens. No single standard set of tests can be used for all bacteria. Hence, different sets of tests have to be employed to identify isolates of different bacteria. However, commercial kits have been developed for Gram-positive and Gram-negative bacteria based on assimilation tests by Biolog Inc. As some metabolites like mycotoxins aflatoxin, ochratoxin are produced only by a narrow range of fungal species, this property may be of significance in the systematics of 2.

Production of mycotoxins may be more precisely detected by using serological methods Narayanasamy The conventional methods depending on the isolation of microbial pathogens from infected plant tissues, multiplication on suitable media and determination of morphological characteristics and physiological and biochemical features require substantially long periods. Furthermore, the results are significantly influenced by cultural conditions and the interpretation of observations requires considerably long experience.

Attempts were made to develop methods that depend on the intrinsic characteristics of the microbial pathogens. This will facilitate initiation of measures to prevent or restrict further spread of the disease s. Diagnostic techniques based on the molecular characteristics of the microbial pathogens have been demonstrated to fulfill these requirements. During the three decades and more, rapid advances made in the study of molecular biology of microbial pathogens have provided adequate information for the development of several sensitive and rapid methods for characterization of microbial pathogens and determination of molecular variability of and relationship between fungal, bacterial and viral pathogens.

Molecular techniques have been shown to be very useful in studying various aspects of plant-pathogen interactions, epidemiology of crop diseases and to assess the effectiveness of different disease management strategies. A wide range of techniques has been employed to suit the pathosystem Narayanasamy , The relative usefulness of some of the basic molecular techniques widely applied, are presented in this chapter. Manipulating and analyzing DNA are fundamental procedures in the study of molecular biology of organisms.

DNA is isolated intact and treated with restriction enzymes to generate pieces small enough to be resolved by electrophoresis in polyacrylamide or agarose. Separating complex mixtures of DNA into different sized fragments by electrophoresis has been a well established method for over three decades. Analysis of total protein profiles generated by separating whole cell protein extracts by electrophoresis has been shown to be useful.

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Isozyme electrophoresis of enzymes such as esterases, phosphatases and dehydrogenases of fungal and bacterial origin provides different patterns according to their relative mobility. Each band is considered as an allele of a specific locus in the pathogen genome. The bands are labeled alphabetically from the slowest to the fastest.

Based on the analysis of isozyme patterns, Phytophthora cambivora, P. Further, it was possible to further subdivide each species into electrophoretic types ETs.

Molecular Biology in Plant Pathogenesis and Disease Management: Microbial Plant Pathogens

By using cellulose acetate electrophoresis CAE for fractionation of phosphoglucose isomerase malate dehydrogenase and lactate dehydrogenase, intraspecific diversity and interspecific relatedness of different papillate species of Phytophthora were assessed. A very close genetic relatedness between P. Within a morphologic species of Leptosphaeria maculans, highly virulent and weakly virulent strains inducing black leg or stem canker disease in canola Brassica napus were differentiated based on the fast or slow movement of isozymes of glucose phosphate isomerase GPI.

The highly virulent strains contained fast isozyme and they were placed in electrophoresis Type 1 ET1. The isozymes of weakly virulent strains moved only short distance and they formed a distinct group ET2 Sippell and Hall The presence of GPI in leaf lesions induced by L. In addition to ET1 and ET2 electrophoretic patterns of highly virulent and weakly virulent strain, ET3 allozyme was present in a few typical and atypical lesions caused by L. The lesions induced by P. The isolates of Phytophthora infestans, causing late blight disease of potato and tomato collected in Canada were classified into eight genotypes depending on the allozyme patterns at two loci GPI and peptidase Pep with markers for mating type, metalaxyl sensitivities and cultural morphology.

Differences in the banding patterns for the allozymes of the GPI locus were significant leading to differentiation of seven of the eight genotypes Peters et al. Isozyme polymorphisms among different isolates of closely related species of Fusarium such as F. However, PEPD alone was useful as a marker to distinguish the four taxa studied, providing a rapid and simple CAE-based diagnostic protocol. The results based 2. The morphological similarity of F. Silver staining of SDS-lysed cells of Xanthomonas campestris pv. Silver staining of protein profiles and testing for amylolytic activity of Xcv may be useful to assign uncharacterized strains of Xcv to appropriate phenotypic group Bouzar et al.

In the case of Pseudomonas syringae pv.

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Psp strains showed three unique protein bands 60, 65 and kDa which were absent in 29 strains of P. Some of the bacterial pathogens have been identified using their isozyme profiles. The suitability of applying the patterns of enzymes of esterase EST and superoxide dismutase SOD for the identification, differentiation of strains and diagnosis of diseases caused by Psp was assessed. Two EST zymotypes specifically present in the profiles could be used for the identification of Psp. Furthermore, there was significant correlation between these EST patterns and race structure of this pathovar.


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    6. Races 2, 3, 4 and 6 exhibited patterns similar to zymotype 1, whereas races 1, 5 and 7 were included in zymotype 2 based on the similarity of isozyme profiles Malandrin and Samson It was possible to detect and identify the strains 75 based on the presence, absence or intensity of 10 protein bands and assign them to 6 different groups. This technique has been shown to be a rapid and consistent method of identifying the strains of X.

    With further studies, PFGE has reached a level for routine application and commercial pulsed field units have been manufactured for large scale use. Now PFGE permits cloning and analysis of a small number of very large pieces of a genome. The genomic analysis of strains of Erwinia amylovora, causing fire blight disease , from the Mediterranean region and European countries was performed.

    The strains from the eastern Europe and Mediterranean region were placed in the second group Pt2. Italy has strains showing patterns of all three types Zhang et al. The isolates of Acidovorax avenae subsp. The isolates were grouped into two classes. The cellular nucleic acids from both healthy and infected plants were extracted and separated by PAGE. The causative agent Potato spindle viroid PSTVd appeared as a distinct band only in the samples from infected tissues and it was absent in the comparable healthy potato tissues.

    The mild and severe strains of PSTVd could be detected and differentiated. Further, PAGE was successfully employed to free several elite or basic seed stocks of potato in certification programs Morris and Wright The differences in the electrophoretic mobility of isolates of Coconut cadang-cadang viroid CCCvd were used as the basis of differentiating the different isolates Randles Coconut tinangaja viroid TiVd in coconut leaf samples was detected by analytical agarose gel electrophoresis Hodgson et al.

    After staining the gel with ethidium bromide, a segment 1. After silver staining, the viroid bands can be viewed Barbosa et al. Plant virus suspensions in suitable buffers are layered into appropriate buffered density gradients of gels formed in a tube and separation of components of the suspension takes places over a period of several hours in a zonal density gradient. Other methods based on the principle of electrophoresis, using of pH gradient or paper curtains have been used for the purification of Southern bean mosaic virus and for separation of Tobacco mosaic virus strains.

    For unstable viruses like Citrus infectious variegation virus, electrophoretic technique is especially valuable Narayanasamy and Doraiswamy The chitinase isozymes 2.

    Molecular Biology in Plant Pathogenesis and Disease Management Disease Management Vol 3

    There were eight dominant chitinase isozymes detected in tobacco extracts. One of them was present only in the TMV-infected leaves, while another accumulated at a greater concentration in TMV-infected than in mock-inoculated leaves Pan et al. This protein was not present in extracts from healthy maize leaves [Appendix 2].


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    The amino acid sequences of their protein was most closely related to the nucleoprotein of Rice hoja blanca virus, indicating that WYHV is a tenuivirus Seifers et al. The dissociated CP migrated as a single band with an estimated size of ca 50 kDa Ghanen-Sabanadzovic et al. SDS-PAGE technique was applied to determine the molecular masses of coat proteins in purified preparation of Peanut chlorotic streak virus that has two polypeptides with approximate relative molecular masses of 51 and 58 kDa Reddy et al. The virus coat proteins may be expressed in the bacterium Escherichia coli.

    Molecular markers [albumin egg MW 45, and albumin bovine MW 60,00 ] are placed in lane 3. They have been found to be highly specific, sensitive, simple, rapid and cost-effective and can be automated for large scale applications. Hence, the immunoassays have largely replaced conventional analytical methods which are time-consuming, cumbersome and expensive. The comparative effectiveness and usefulness of several immuno-assays have been discussed earlier Narayanasamy , Improvements and modifications, however, have been made to suit different host-pathogen combinations. The advancements during the recent years have focused the attention of the researchers on the need for applying rapid, reliable and sensitive methods for the detection of microbial pathogens and diagnosis of diseases caused by them.

    Immunoassays primarily depend on the visualization of the specific binding of antibody to the antigen, directly or indirectly. The formation of precipitate or precipitin lines indicating the binding between reactants, may be seen. These tests require large volumes of the reactants and longer time to provide results which may be inconclusive.

    On the other hand, the assays requiring labeling of antibodies are more sensitive and rapid with possibility of automation for large scale application. The antibodies may be labeled with enzymes such as alkaline phosphatase or fluorescent dyes. Among the techniques using labeled antibodies, enzyme-linked immunosorbent assay ELISA has been most widely applied for studying various aspects of plantpathogen interactions, in addition to detection and characterization of microbial pathogens. The development of monoclonal antibody technology has remarkably enhanced the specificity of immunoassays, resulting in the differentiation of strains, biotypes or races of microbial pathogens more precisely than by using polyclonal antibodies.

    For certification programs depending on the biological indexing by grafting onto indicator hosts, it was very difficult to ensure freedom from viruses in the plant materials. Indexing required 2—3 years for symptom development on indicators. Application of immunoassays for the detection and resolution of etiology of leaf roll and rugose wood complexes of grapevine progressively increased due to the availability of results rapidly Martin et al.

    Tospoviruses, belonging to the family Bunyaviridae, cause spotted wilt diseases that are agriculturally important because of the significant losses in different crops and flowers. ELISA has been frequently employed using PABs specific against viral nucleoprotein, due to the ease of the purification step, in preference to other methods that rely on detection of complete virus particle with envelope which require a different enveloped particle preparation Hsu et al.

    No significant cross-reaction could be noted for TYRV with antisera of other tospoviruses tested and vice-versa, suggesting that TYRV was serologically distinct from the viruses compared Hassani-Mehraban et al. Calla lilies Zantedeschia spp. From time to time, infection by viruses on host species which are not known as hosts, has been observed later. Three distinct potyviruses Vanilla mosaic virus, Watermelon mosaic virus and a virus related to Bean common mosaic virus were detected and differentiated by this technique Grisoni et al.

    Among the PVY-positive samples , three samples reacted positively with the MAB 1F5 and they induced veinal necrosis symptoms in tobacco. Two of these isolates caused tuber necrosis in the potato cv.

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    The disease incidence was observed in 11 of 21 leek and 2 of 26 onion plantings. As the distribution of IYSV in leek was irregular, it was suggested that samples should include tissue subsection from the top and middle portions of several leaves from each plant for obtaining realistic estimate of disease incidence Smith et al. Polyclonal antibodies generated against the purified P10 protein, specifically recognized RBSDV from infected plant tissue and a planthopper vector in Western blotting assays. Development of monoclonal antibody MAB technology has led to substantial enhancement of the sensitivity and specificity of immunoassays including ELISA tests.

    All the three assay procedures detected CPsV readily in young shoots and leaves.

    Molecular Biology in Plant Pathogenesis and Disease Management Vol.2 - Disease Development

    There was good correlation between results of immunoassays and that of biological indexing Martin et al. The results showed that CPsV in infected trees was clearly detected in old hardened leaves in winter. By using a minimum of four leaves taken from around the trees, high reliability can be achieved in any season Cambra et al. Furthermore, tissue prints can be prepared in the field and stored for long periods without loss of reactivity, making DTBIA more useful and 18 2 Molecular Techniques for Detection of Microbial Pathogens convenient technique for epidemiological studies.

    In addition, several membranes can be printed with the same shoots for future processing with new antibodies and the tissue-printed membranes may be mailed to a laboratory where the required antibodies are available Martin et al. The background reactions with control non-infected tissues were very weak, similar to those produced by monoclonal antibodies, indicating the usefulness of the approach of using polyclonal antibodies in place of monoclonal antibodies which require long period of time and expertise for production Kumari et al.

    Tissue immunoblot analysis of tobacco cv. Xanthi nc inoculated with Potato Virus Y T01 isolate alone and also in combination with Cucumber mosaic virus CMV was made to visualize the distribution of viral antigens in cross-sections of stems or petioles. PVY was detected at high levels in almost all sections in stems, petioles and apical tissues in doubly infected plants. But in singly infected plants, PVY was not detected in shoot apex and the reaction was weaker in tissues from middle or young stems and petioles.

    However, CMV was detected in almost all sections of both singly and doubly infected plants Fig. Tissue immunoblot technique was employed to study the distribution of Turnip mosaic virus TuMV and its chimeraic viruses in the cotyledons and leaves of cabbage and radish at 12 days post inoculation. Chimeric viruses showed very low systemic infectivity Suehiro et al. An improved DTBIA protocol for the detection of Citrus tristeza virus CTV involves printing of fresh young stems of healthy and infected plants by gently and evenly pressing the fresh cut surface of the stems onto a nitrocellulose membrane.

    After rinsing the blots in water for a few seconds to stop the reactions, results are recorded by observation under a light microscope. This TBIA was found to be as reliable as PCR for detecting CTV in field samples and this procedure could be completed within 1 h by having a pre-reaction of CTV-specific antibodies and labeled secondary bodies in solution prior to their application to the tissue blots Lin et al.

    Simplification increased the feasibility of application of immunoassays. Courtesy of Ryang et al. Development of a purple color indicating a positive reaction for the presence of viral antigen, can be visualized by light microscopy. Hammer blotting is another simple technique for detecting virus infection in plants. Kits for detection of several viruses, are available. Certification of plant materials represents the first line of virus disease management systems. ELISA is considered to be the technique of choice for certification or field surveys to assess the disease incidence levels for the detection of Prune dwarf virus PDV , a major virus affecting stone fruits.

    Variants of ELISA have been demonstrated to be efficient in the detection of viruses in various host plant species. Seeds infected by viruses are the most important sources of infection spreading the viruses into new areas or countries. Transgenic crops can come up through normal flow of genes among species, via cross-pollination dependent hybridization among assorted plant species which is a standard occasion in flowering plant evolution , or by means of laboratory manipulations through man made insertion of genes from one other species.

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