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Topics to cover : Importance of disease in crop production. Plant resistance mechanisms. Disease control methods. Biotechnological approaches to plant disease management . Topics to cover Slide 4: 1 2 3 Plants diseases : Plants diseases Disease: major limiting factor to improved crop productivity Slide 6: Clare Kenag (1974): Plant disease as “infectious” or “non-infectious” Infectious plant disease: an abnormal plant condition caused by biological agencies eg fungi, bacteria, viruses and nematodes and are capable of being transmitted from diseased plants to health plants to cause disease on the latter under favourable environmental conditions. Non-infectious plant disease: an abnormality on plant cause by agencies such as nutrient deficiencies, weather conditions, mechanical injury, chemical injury, and effects by hail, wing, frost lightning and other external factors which are non infectious and their effects are not transmissible from affected to health plants. Plant Disease: Stackman and Harrar (1957) definition: Any deviation from normal growth or structure of plants that is sufficiently pronounced and permanent to produce visible symptoms or to impair quality and economic value. Slide 7: Agrios, G.N. 1998 Slide 8: Agrios, G.N. 1998 Different Pathogens Causing diseases Slide 9: Agrios, G.N. 1998 Slide 10: Advance leaf symptoms caused by bacterial Xanthomonas campstries pv. vesicatoria on tomato affected leaf carry little or no photosynthesis Agrios, G.N. 1998 Introduction: Molecular plant pathology : Introduction: Molecular plant pathology Molecular basis of plant pathology and crop protection to improve productivity. Plant disease resistance mechanisms. Apply knowledge to enhance plant disease resistance and productivity. Slide 12: In 1940s, H.H. Flor studied inheritance of both, plant resistance and pathogen virulence using flax (Linum usitatissimum) and its fungal rust pathogen Melampsora lini. His work revealed the classic “Gene-for-Gene Model’’. (Flor, 1971) Gene-For-Gene Model: For each gene of resistance in the host there is a corresponding gene for avirulence in the pathogen and for each virulence in the pathogen there is a gene for susceptibility in host plant . A loss or aulteration to either the plant resistance (R) gene or the pathogen arirulence (Avr) gene leads to diseases (compatability). (B) Interactions involved in R gene-Avr gene incompatibility Various types of genetic interactions between plants and pathogenic microbes (A) Interactions involved in toxin-dependent compatibility I = incompatable i.e. resistance, C = compatable i.e. susceptible Molecular Basis for Disease Resistance Slide 13: Plant resistance mechanisms Two broad mechanism Race-specific or gene-for-gene resistance Non-specific or broadspecrtum resistance: Systemic acquired resistance (SAR): Induced systemic resistance: Slide 14: Identification of resistance (R) genes through genetics Most R-genes are dominant, as are their cognate pathogen avirulence (avr) genes.; Plants possess many R genes, active against many different pathogens; R genes are often found clustered on chromosomal loci. Plant breeders have successfully introduced disease resistance through introgression of foreign R-genes. Sources of new R-genes are sought. Elicitation of race-specific resistance; genetic incompatibility H. Flor described the genetics that underlie race-specific or gene-for-gene resistance of flax rust with its host flax (Flor, 1956); Many pathogens exhibit gene-for-gene resistance on their hosts; Non specific or broad spectrum resistance : Systemic acquired resistance. Induced systemic resistance Non specific or broad spectrum resistance Slide 16: SYSTEMIC ACQUIRED RESISTANCE What is SAR? SAR confers quantitative protection against an broad spectrum of microorganisms in a manner comparable to immunization in mammals although the underfying mechanisms differ. Resistance is expressed locally at the site of primary inoculation but also systemically in tissues remotely located from the initial treatment. This form of induced resistance called systemic acquired (SAR) Ryals et al. 1992) Slide 17: THE MECHANISM INVOLVED IN SYSTEMIC ACQUIRED RESISTANCE Lignifications and other structural barriers Pathogenesis Related proteins Conditioning 1. Lignification and other structural Barriers Deposition of lignin in cell wall is called as lignification It observed in many plants It is an important mechanism for disease resistance Lignin incerporation strengthen plant mechanically. Lignified cell starved the pathogen Lignin pre cursors may might exert toxic effect Mycelia of colletotrichum lagenarium become lignified in vitro Glycine rich proteins accumulate systemically in cell wall of tobacco plants infected with TMV and virus infected rice plants Slide 18: 2. Pathogenesis related proteins Pathogenesis related protein (PRs) were first described in 1970 in tobacco leaves infected with TMV (Van and Van, 1970) PRs have been defined as plant proteins that accumulate after pathogen attack or related situation, (Van et al. 1994) Several PRs including PR-1, β -1, 3- glucanases (PR-2) Chitinases (PR-3), PR-4 and Osmotin (PR-5). PRs referred as SAR Proteins The nature and level of expression of SAR protein vary among Plant species In tobacco over expression of PR-1 significantly increase resistance against infection by Perenospora tabacina and Phytophthora parasitica var. nicotianae (Alexander et al. 1993) 3. Conditioning When plants are pretreated with necrotizing pathogen or a synthetic inducer of SAR, the systemically protected leaves react more rapidly and more efficiently to challenge infection with a virulent pathogen. This phenomenon is known as conditioning or sensitizing. Skipp and Daverall (1973) induced resistance in bean hypocotyls though localized HR caused by Colletotrichum lindemathianum. Slide 19: General features of SAR Controlled studies by Frank Ross at Cornell showed reproducible systemic induction of resistance. Ross showed that TMV and other viruses in tobacco could induce local and systemic resistance against the same and other pathogens; he coined the terms "LAR" and "SAR" to describe this (Ross, 1961). Resistance was later shown by others to be induced by many pathogens, -most often those eliciting a necrotic reaction Activation of SAR leads to broad-spectrum resistance that acts against many pathogens beyond that triggering the response (Dean and Kuc, 1985; Hecht and Bateman, 1964; Kuc, 1982) Resistance can persist for weeks after elicitation. Some reports describe season-long protection after activation of SAR Biochemical studies showed that many new proteins accumulate after induction of SAR; (Van Loon and Antoniw, 1982; van Loon et al., 1994) Approx. 13 families of SAR-associated genes are now known in tobacco (van Loon et al., 1994; Ward et al., 1991) Slide 20: INDUCE SYSTEMIC RESISTANCE Other forms of Induced resistance (Pieterse et al., 1996; Pieterse et al., 1998; Van Wees et al., 1997) Some soil bacteria, such as P. fluorescens p417 can activate an IR response. Van Loon and colleagues call this induced systemic resistance (ISR). ISR is not be associated with typical SAR genes, suggesting that SA accumulation is not required for ISR. Genetic evidence shows that other IR pathways exist outside of SAR and ISR. To identify genes required for or associated with SAR-independent resistance (SIR), mutant screens are underway and pathogen-induced genes are being sought (Argueso and Delaney, unpublished; Rairdan et al., 2000) Slide 21: Plant exhibiting SAR and ISR NR – not recorded Sticher et al. 1997 Disease control methods : Disease control methods Chemical control, Use of Resistant cultivars, Biological control methods Integrated approach Slide 23: IPM Strategies Biological control: protecting, enhancing, and releasing pests' natural enemies, e.g., insects, nematodes, snails, or slugs; Cultural practices: such as ecological landscaping to reduce field size and distance to habitats of natural enemies, erection of barriers, crop rotation, cover cropping, increased reliance on mechanical weed control, improved crop residue management, better water management, and improved pest monitoring; Chemical: with less reliance on synthetics in favor of biopesticides or biochemical pesticides (pheromones, insect growth regulators, and hormones—naturally occurring chemicals that modify pest behavior and reproduction); and Genetic: use of naturally resistant varieties, new varieties bred for resistance, or transgenic varieties, as well as release of sterile pests to prevent reproduction. Slide 24: Biological control of post-harvest disease of oranges Oranges treated with yeast (right) remained healthy, whereas oranges not treated with yeast developed extentsive decay following inoculation with penicillium. Agrios G.N. 1998 Biotechnological approach in disease management : Biotechnological approach in disease management Marker assisted plants breeding Different markers and application in disease resistance Achievements Tissue culture methods Somaclonal variation Somatic hybridization Genetic engineering (Transgenics) Meristem – Tip culture (for virus free planting material) Slide 26: MARKER AIDED SELECTION Marker aided selection (MAS)is potentially useful for breeding for disease resistance in at least four ways: As a substitute for a disease screen, To accelerate the return to the genotype of the recurrent parent during backcrossing, To reduce linkage drag of linked deleterious genes, To select for disease resistance linkage drag of linked delecterious genes, and to select for disease resistance QTL. In this technique, linkages are sought between DNA markers and agronomically important trait such as resistance to pathogens insects and nematodes etc. Instead of selecting for a trait the breeder can select for a marker that can be detected very easily in this selection scheme. Slide 27: MOLECULAR MARKER A molecular marker is a DNA sequence that is readily detected and whole inheritance can easily be monitored . Molecular marker are used to identify and tag desired gene Different markers use for disease resistance are as follows RFLP (Restriction fragment length polymorphism ) RAPD (Random amplified polymorphic DNA markers) SSRT (Simple sequence repeats or microsatellites AFLP (Amplified fragment length polymorphism) Slide 28: Table 1: Some examples of molecular markers associated with resistance traits in crop plants Slide 29: TISSUE CULTURE METHOD 1. SOMACLONAL VARIATION Definition: "The variability generated by the use of a tissue culture cycle." "A tissue culture cycle is a process that involves the establishment of a dedifferentiated cell or tissue culture under defined conditions, proliferation for a number of generations and the subsequent regeneration of plants." (Larkin and Scowcroft, 1981). Table 2 : A] A list of disease resistant crop plants obtained by screening of somaclones at the plant level without in vitro selection. Slide 30: B] Disease resistant crop plants obtained by in vitro selection. Disadvantages associate with somaclonal variation Uncontrollable and unpredictable nature o variation and that most of the variation of no apparent use. The variation is cultivar dependent The variation obtaine is not always stable and heritable. The changes occcur at variable frequencies. Slide 31: SOMATIC HYBRIDIZATION Definition : A technique of fusing protoplasts from two contrasting genotypes for production of hybrids or cybrids which contain various mixtures of nuclear and/ or cytoplasmic genomes, respectively. (Chwala S.H. 1998) Protoplast Isolation and Fusion: The protoplast, also known as naked plant cell refers to all the components of a plant cell excluding the cell wall. Hanstein introduced the term protoplast in 1880 to designate the living matter enclosed by plant cell membrane. The isolation of protoplasts from plant cells was first achieved by microsurgery on plasmolyzed cells by mechanical method (Klerckar, 1892). The true test of protoplast viability is the ability of protoplasts to undergo continued mitotic divisions and regenerate plants. The concentration of protoplasts in a given preparation can be determined by the use of hemocytometer Slide 32: Somatic hybridization for gene transfer of Disease resistance: The family Solanaceae and Brassiceae contains the most commonly used species for somatic hybridization. Both interspecific and intergeneric hybrids have been obtained. Many disease resistance genes viz. potato leaf roll virus, leaf blight, Verticillium, phytophthora have been transferred to Solonum tuberosum from other species where normal crossings would not be possible due to taxonomic or other barriers. Resistance to black-leg disease (Phoma lingam) has been found in B. nigra, B. jncea and B. carinata and after production of symmetric as well as asymmetric somatic hybrids between these gene donors and B. napus, resistant hybrids have been developed (Sjodin and Glimelius, 1989). Attempts were made to introduce tolerance in Brassica napus against Alternaria brassicae from Sinapsis alba (Primard et al, 1988) and beet cyst nematode from Raphanus sativus (Lelivelt et al. 1993). Resistance has been introduced in tomato against various diseases like TMV, spotted wilt virus, insect pests and also cold tolerance. Kobayeshi et al (196) reported the incorporation of disease resistance genes from wild species Solanum ochranthus a woody vine like tomato relative to L. esculentum. Hansen and Earle (1995) reported that black rot disease caused by Xanthomonas compesris is a a serious disease in cauliflower. The somatic hybrids were produced by protoplast fusion of B. oleracea with B. napus. Some of the examples of incorporation of resistance genes via protoplast fusion technique have been listed in Table. Slide 34: Problems and Limitations of Somatic Hybridization Application of protoplast methodology requires efficient plant regeneration from protoplasts. Protoplasts from any two species can be fused. However, production of somatic hybrid plants has been limited to a few species. The lack of an efficient selection method for fused product is sometimes a major problem. The end-products after somatic hybridization are often unbalanced (sterile, misformed, and unstable) and are therefore not viable, especially if the fusion partners are taxonomically far apart. Regeneration products after somatic hybridization are often variable due to somaclonal variation, chromosome elimination, translocation, organelle segregation etc. It is never certain that a particular characteristic will be expressed after somatic hybridization. To achieve successful integration into a breeding program, somatic hybrids must be capable of sexual reproduction. All diverse intergeneric somatic hybrids reported are sterile and therefore have limited value for new variety development. It may be necessary to use back-fusion or embryo culture to produce gene combinations that are sufficiently stable to permit incorporation into a breeding program. To transfer useful genes from a wild species to a cultivated crop, it is necessary to achieve intergeneric recombination or chromosome substitution between parental genomes (Chawla,2002). Slide 35: Transgenics for disease resistance Transfer of genes between plant species has played an important role in crop improvement for many decades. Useful traits such as resistance to diseases, insects and pest have been transferred to crop varieties from noncultivated plants. The overall process of genetic transformation involves introduction, integration and expression of foreign gene in the recipient host plant. Plant that carry additional stably integrated, and expressed foreign genes transferred (transgenes) from other genetic sources are reffered to as transgenic plants. The capacity to introduce and express diverse foreign genes in plants was first described in tobacco by Agrobacterium mediated Horsch et al., 1984. And vectorless approach (Paszhkowski et al. 1984.) Slide 36: GENETIC ENGINEERING Genes expected to confer disease resistance are isolated, cloned and transferred into the crop in question. In case of viral pathogens, several transgenes have been evaluated, viz., virus coat protein gene, DNA copy of viral satellite RNA, defective viral genome, antisense constructs of critical viral genes, and ribozymes. Viral coat protein gene approach seems to be the most successful (Singh, 1998) A virus resistant transgenic variety of squash is in commercial cultivation in USA. In case of bacterial and fungal pathogens, resistance has been sought by expression of the following transgenes: 1) genes enoding insensitive target enzymes, 2) genes specifying toxin inactivation, 3) expression of antibacterial peptides, 4) expression of bacterial lysozymes, 5) genes specifying artificially programmed cell death, 6) expression of heterologous phytoalexins, 7) genes encoding ribsome inactivating proteins, 8) expression of heterologous thionins, 9) ectopic expression of pathogenesis related proteins and 10) ectopic expression of chitinases. In almost all the approaches, transgenic plants showed increased resistance to the concerned diseases (Singh,2000). Slide 37: Table 4: A] Virus Resistant Transgenic Plants B] Transgenic Plants (Fungal and Bacterial Diseases) CP- Coat protein, PVX – potato virus X, PVY - Potato virus Y, TMV- Tobacco mosaic virus, PLRV – potato leaf roll virus, PSB MV- Pea seed born mosaic virus, RVMV- Rice yellow mosaic virus Slide 38: MERISTEM TIP CULTURE Defination : Cultivation of axillary or apical shoot meristems, particularly of shoot apical meristem is known as meristem culture. (Chawala H.S. 1998) Morel and Martin (1952) developed the technique of meristem culture for in vivo virus eradication of Dahlia. Meristem culture involves the development of an already existing shoot meristem and subsequently, the regeneration of adventitious roots from the developed shoots. It usually does not involve the regeneration of new shoot meristem When the objective is to free the stock from a virus, it is essential that the apical meristem should be excised along with a minimum of the surrounding tissue but when objective s vegetative propogation, the size of shoot tip used for culture is not important. Generally, explants taken for actively growing plants at the beginning of growing season are the most suitable. In practice shoot tip of upto 1 micro meter are used when the objective in virus elimination. Slide 39: Advantages associated with meristem tip culture For production of disease free plants Meristem tip culture in generally followed where aim is to produce disease free plants. It has been demonstrated that the shoot apices of virus infected plants are frequently devoid of viral particles or contains very low viral concentrations. Though chemotherapeutic and physical agents have been used for production of virus free plants but with limited success. In vitro culture has becomes only effective technique to obtain virus free plants in potato, banana. Chrysanthemum, gladiolus, Pelargonium etc. from stock systemically infected not only with virus but with various other pathogens. Meristems are genetically stable and can be regenerated into pathogen free plants meristems have been identified as excellent material of germplasm preservation of crop species with seed borne viruses also. Disadvantages Facilities required are costly Special skills are required to carry out the work Errors in maintenance of identify, introduction of an unknown pathogen, or appearance of an unobserved mutant may be multiplied to very high levels in short time. Slide 40: CONCLUSION Modern agricultural biotechnology is one of the most promising developments in modern science. Used in collaboration with traditional or conventional breeding methods, it can raise crop productivity, increase resistance to pests and diseases, develop tolerance to adverse weather conditions, improve the nutritional value of some foods, and enhance the durability of products during harvesting or shipping. With reasonable biosafety regulations, this can be done with little or no risk to human health and the environment. THANK YOU You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.