Disease resistance breeding

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1 SEMINAR ON “BREEDING FOR DISEASE RESISTANCE” By R.L. Bhakre Seminar Incharge Dr. K.B. Wanjari Head Department of Agricultural Botany Post Graduate Institute, Dr. PDKV, Akola.

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2 Abnormal condition in the plant produced by an organism. “Physiological disorder or structural abnormality that is deleterious to the plant or to any of its part or products, or that reduces their economic value.” (Stakman and Harrar,1957) “Harmful deviation from normal functioning of physiological processes.”(Anon,1950) What is disease?

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Agrios, G.N. 1998 Different Pathogens Causing diseases

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4 Losses due to diseases Disease reduce biomass (dry matter) & hence Yield. This may happen in either of the following ways: killing of plants (e.g. vascular wilts, various soil borne fungi), general stunting caused by metabolic distruption, nutrient drain or root damage (e.g.many viruses), killing of branches (e.g. some fungal diebacks), damage to leaf tissues ( e.g. many rusts, mildews, blights, leaf spots), damage to reproductive organs including fruits & seeds. Often the loss due to diseases may range from a few to 20 or 30 %; in case of severe infection, the total crop may be lost (Jayaraj,2002).

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5 MAJOR FAMINES OR FOOD LOSSES The wheatless days (1917’s) in USA, due to stem rust epidemics; Irish famine (1840’s) due to potato leaf blight epidemics; Devastation of all Victoria-derived oats (mid 1940’s) in USA due to fungus causing Victoria blight disease; Bengal famine (1943’s) due to brown spot (Drechslera oryzae) disease of rice; Downy mildew epidemic (1973’s) in pearl millet (c.o. Sclerospora graminicola) in India ; and Bacterial blight (X.campestris pv.oryzae) (1980’s) severe out-break in Panjab (A.P.K.Reddy 1980).

Important fungal diseases of major crops in India : 

6 Important fungal diseases of major crops in India DasGupta et al (2003)

Important viral diseases of crops in India : 

7 Important viral diseases of crops in India

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8 History of breeding for disease resistance Theophrastus (3RD Century):cultivated variety differed in their ability to avoid diseases. Benedict Prevost: diseases are produced by pathogen. He showed that wheat bunt was produced by a fungus. Andrew Knight (Mid 19TH Century):crop varieties differed for disease resistance. Biffen(1905):resistance to yellow rust in wheat was governed by a recessive gene segregating in the ratio 3:1 in F2. Erikson (1894):Pathogens ,although morphologically similar,differed from each other in their ability to attack different related host species. Barrus (1911) :different isolates of a microorganism differed in there ability to attack different varieties of the same host species; this finding is the basis for physiological races &/or pathotypes. Flor (1956):Gene-for-Gene Hypothesis.

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9 Agrios, G.N. 1998 Disease Development

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10 Stages of disease development (especially fungal diseases): Contact: landing of pathogen on host tissue Infection: pathogen gains entry in to the host tissue Establishment: pathogen proliferates & spreads within host tissue Development: spore production/multiplication by pathogen & symptoms developed

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11 DISEASE REACTIONS Various reactions of hosts to the pathogen may be grouped into; Susceptible: disease development is profuse & is presumably not checked by host genotype.eg.Agra Local susceptible to wheat rust. r=1 Immune: host does not show symptoms of a disease (100% freedom from infection), r=0 Resistance :less disease than susceptible, r>0 & r<1 Tolerant: a tolerant cultivar is one which endures disease attack (R.M.Caldwell et al. 1958 ) & looks susceptible one (Browning & Frey 1969).

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12 Vertical & horizontal resistance Van der Plank (1963,1968) Vertical Resistance Specific resistance of host to particular race of a pathogen. Also called oligogenic,race spacific,pathotype specific or specific resistance. Controlled by one or few major genes. When controlled by single major gene it is known as monogenic resistance. Each gene has large & easily identifiable effect on resistance. Less stable as can be overcome by appearance of one virulent gene. VR involves hypersensitive reaction to the pathogen. Less effect of environment. VR governed by two or more major gene is more durable than controlled by single major gene. Very effective against few races but totally ineffective against others. Transfer from one host to other is simple.

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13 Horizontal Resistance Resistance of host to all the races of pathogen. Reproduction rate of pathogen is never zero,but is less than one,i.e.r>0 but <1. Also called as race non specific,pathotype non specific, polygenic, partial or general resistance. Controlled by polygenes. Provides protection from all races of a pathogen. It has continuous variation & hence,impossible to classify plants into different clear-cut classes. More durable than VR since it involves sevsral features (polygenes) of host plant. More stable since it require new genes for virulent that is less probable. Influenced by environmental factors. Some degree of effectiveness against all races though may not be equally effective against all. Transfer of HR from one host to another is more difficult than VR.

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14 Mechanism of disease resistance Mechanical Hypersensitivity Nutritional Mechanical : Certain mechanical &/or anatomical features of host may prevent infection. e.g.closed flowering habit of wheat & barley prevents infection by spores of ovary infecting fungi (Macor 1960). Several leaf characters of rice cultivars viz. rolled, narrow & dark green were associated with resistance to bacterial leaf blight (X.campestris pv. oryzae) resistance (C.B.Singh & Y.P.Rao 1971).

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15 Hypersensitivity: Immediately after infection several host cells surrounding the point of infection die leading to death of pathogen or prevents spore production. Found in biotrophic or obligate parasite (e.g. C.O. of rust ,smut etc.). Hypersensitive cell produce chemical/ phenolic compounds (phytoalexins, etc) which are fungitoxic & autotoxic (Deverall 1976) In large no. of cases, immune reaction is due to hypersensitive reaction of host. Nutrional: The reduction in growth & in spore production is generally supposed to be due to an unfavourable physiological conditions within the host. Most likely, a resistant host does not fulfill the nutritional requirements of pathogen & hence limits its growth & reproduction.

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16 Gene-for-gene concept: Postulated by Flor in 1956 based on his work on Linseed rust (c.o. Melampsora lini) For each resistance gene (R gene) in the host, there is corresponding avirulence genes (avr gene) in the pathogen(Flor, 1956).

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17 Genetics of disease resistance in some crop plants D = dominant gene, d = recessive gene, I = inhibitor gene, m = modifier gene

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18 Molecular basis for gene-for gene-relationship On the basis of molecular interactions involved in producing resistant/susceptible responses in the host, the gene-for-gene relationship may be classified into two general groups: Incompatible reaction Compatible reaction

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Incompatibility reaction Found in biotrophic pathogens (obligate parasites) (e.g. rusts & smuts) and is associated with hypersensitive response of the host Only one of the four combinations would lead to the resistant response since the products of R & A would recognize & interact with each other. The product of alleles a & r are unable to recognize each other, & there is no interaction between them hence reaction of host becomes susceptible. Allele A of the virulence gene specifies avirulence. Allele a of the virulence gene governs virulence.

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Compatible reaction Found in heterotrophic pathogens (facultative parasites) e.g. Helmithosprium leaf blight (of sugarcane & maize),Victoria blight of oats etc. The allele for resistance of the host ( R) produces a modified /specific substances ,which is unable to recognize & interact with the product (toxin) of alleles for virulence (a) in the pathogen. Similarly, the allele for avirulence in pathogen (A) specified modified product (generally a toxin), which is not able to interact with the product of the alleles of the susceptibility (r) in the host & due to this reason only one of the fours combinations would lead to susceptibility and rest lead to resistant. Allele A of the virulence gene specifies avirulence. Allele a of the virulence gene governs virulence.

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21 Sources of disease resistance: A known variety Germplasm collection Related species Mutations Somaclonal variation Unrelated organisms

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22 A known variety /cultivated varieties : best sources of resistance since they possess good agronomic characters besides disease resistance. e.g. Cotton variety MCU 5 VT tolerant to Verticillium wilt was isolated from commercial variety MCU 5 of G. hirsutum. Germplasm collection : Potential sources of resistance in all the cultivated crops. e.g. In cotton several germplasm lines resistant to bacterial blight & Fusarium wilt have been identified based on screening of large no. of germplasms in India. Generally germplasm lines have poor agronomic characters.

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23 Related species/ wild species : Often resistance to a disease may not be present in the varieties of the concerned crop species. In such cases, it would be necessary to transfer resistance genes from related species through interspecifc hybridization. Despite many problems (viz. cross incompatibility, hybrid inviability, hybrid sterility and linkages of several undesirable traits with desirable ones) in such gene transfers, it has been successfully and extensively used in many cases.

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24 Some of the wild /related species (genera) of different crops used to transfer or possessing genes for disease resistance.

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25 Screening technique Screening for disease resistance is carried out under field and glasshouse conditions. The glasshouse tests are conducted under controlled conditions &, therefore, glasshouse screening is considered more reliable than field tests. Screening procedure for various types of diseases: Soil borne diseases For soil borne diseases like root rots, colar rots, wilts, etc. the screening is done in disease sick plots. Disease sick plots are developed in three ways : by mixing soil from other sick plots by adding remains of diseased plants, and by adding inoculum developed in the laboratory. The soil from disease sick plots can be used in pots for conducting tests in the glass house.

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26 Air borne diseases For air borne diseases viz, rusts, smuts, mildews, blights, leaf spots, etc., The screening is done either by dusting the spores or by spraying spore suspension on healthy plants. Planting of highly susceptible varieties after few rows of test material is also used to develop the inoculums. Seed borne diseases In case of seed borne disease like smuts and bunts, either dry spores are dusted on the seeds or the seeds are soaked in the spore suspension. Insect transmitted diseases the insect from susceptible varieties are collected and released on healthy plants or juice of diseased plants is rubbed onto healthy plants after causing mechanical injury in healthy plants.

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27 Methods of breeding for disease resistance Commonly used breeding methods are: Selection Introduction Mutation Hybridization MAS Tissue Culture Methods: Somaclonal Variation Somatic Hybridization (Protoplast fusion) Meristem-tip culture (for virus free planting material) Genetic engineering (Transgenics) Tradional Methods Biotechnological Tools

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28 SELECTION Cheapest and quickest method The resistant plants may be multiplied, screened & released as a variety. Kufri Red, potato from Darjeeling Red Round. Pusa sawani, bhindi (A.esculentus) from Bihar is resistant to yellow mosaic under field condition. MCU1, Cotton (G. hirsutum) from CO-4 for resistance to black arm. INTRODUCTION The resistant variety may be introduced & after testing, if found suitable, can be released in the disease prone area. Easy & quick  Introduction also serve as source of resistance in breeding programmes.

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29 Limitation Introduced varieties may not perform well in new area. May become susceptible to concerned disease in the new environment, May be susceptible to the concerned diseases to other diseases common in the new area, e.g. Kenya wheats (T. aestivum) introduced in India were rust resistant but they were highly susceptible to loose smut. Achievments Ridley wheat introduced from Australia has been useful as a rust resistant variety. Early varieties of groundnut (A. hybpagaea) introduced from USA have been resistant to leaf spot (tikka) disease (C.O. Cercospora orachidicola). Kalyan sona & sonalika varieties originated from segregating material introduced from Mexico, & were rust resistant.

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30 Mutations Induced mutations can be utilized by Direct release as a variety (resistant mutant) & by utilization in hybridization programme. e.g. Co 8153, a sugarcane variety developed from variety Co 775 with the use of gamma-ray irradiation was resistant to smut. Hybridization used for (i) transfer of disease resistance from an agronomically undesirable variety to a susceptible but otherwise desirable variety (by backcross method). (ii) combining disease resistance & some other desirable characters of one variety with the superior characteristics of another variety(by pedigree method)

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31 Pedigree method It consists of selecting individual F2 plants for desirable features, including resistance to diseases. Progenies of these selections are reselected in each succeeding generation until homozygosity is obtained. Artificial epiphytotic conditions are created in early segregating generations for the selection for disease resistance. Advantages: This method is quite suited for breeding for horizontal (polygenic) resistance. Occasionlly, transgressive segregation of quantitative characters including disease resistance is encountered.  Achievements: KalyanSona, Sonalika, Malviya 12, Malviya 37, Malviya 206, Malviya 234, & many wheat varieties. Laxmi (American Cotton variety) resistant to red leaf blight was developed through the cross between susceptible parent Gadag1 & the resistant parent CoimbatoreCombodia2.

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32 Backcross methods When resistance is dominant, F1 is back crossed to the susceptible parent. The progeny of first backcross generation is tested for resistance. The resistant plants are again backcrossed to the susceptible (recurrent) parent. After several generations of backcrossing (5-6), plants with characters almost identical to the original susceptible variety are obtained with an added advantage of resistance in them. When resistance is recessive, the progeny of each back cross generation is selfed. At the end of backcross programme, the progeny are selfed & resistant plants are selected. Progenies derived from different resistant plants that are identical in agronomic characteristics are usually bulked to produce the new disease resistant variety. The new variety is almost identical to the recurrent parent, except for disease resistance.

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33 Advantages: 1) When resistant variety is unadapted & agronomically undesirable, backcross method is an obvious choice. 2) Useful to transfer one or few major genes (vertical resistance). 3) Extensive yield trials are usually not required before its release for commercial cultivation. 4) Multiple resistance breeding is possible. Disadvantage: No advancement in yield potential is possible. Achievements: Transfer was the first commercial wheat variety in which rust resistance was transferred from a related species. Rust resistance was transferred to Kalyansona from several diverse sources e.g., Robin, K1, Bluebird, Tobari, Frecor, HS 19 etc.which resulted in release of three multiline varieties of wheat, namely, KSML 3, MLKS 11, & KML 7406. [Source: Dasgupta (1991);Singh (2000)]

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34 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, & to select for disease resistance QTL. In this technique, linkages are sought between DNA markers & agronomically important trait viz., resistance to pathogens etc. Instead of selecting for a trait the breeder can select for a marker that can be detected very easily in this selection scheme.

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35 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: RFLP (Restriction fragment length polymorphism ) RAPD (Random amplified polymorphic DNA markers) SSRT (Simple sequence repeats or microsatellites AFLP (Amplified fragment length polymorphism) STS (Sequence Tagged Sites) SCAR (Sequence Characterised Amplified Region)

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36 Some examples of molecular markers associated with resistance traits in crop plants

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37 SOMACLONAL VARIATION "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 no. of generations & the subsequent regeneration of plants." (Larkin & Scowcroft, 1981). Disease resistant somaclonal variants can be obtained in the following two ways: 1) Screening: Plants regenerated from cultured cells or their progeny are subjected to disease test & resistant plants are isolated. 2) Cell Selection: cultured cells are selected for resistance to the toxin or culture filterate produced by the pathogen & plants are regenerated from the selected cells. Cell selection strategy is most likely to be successful in cases where the toxin is involved in disease development. Resistance was first reported in sugarcane for eye spot disease (Helminthosporium sacchari)

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38 A] A list of disease resistant crop plants obtained by screening of somaclones at the plant level without in vitro selection.

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39 B] Disease resistant crop plants obtained by in vitro selection.

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40 Somatic cell hybridization Involves physical union or fusion of protoplast from two parents. A technique of fused protoplast from two contrasting genotypes for production of hybrids or cybrids which contain various mixture of nuclear & / or cytoplasmic genomes respectively. Because of the inherent totipotey of the isolated cell it is possible to regenerate a whole plant out of the fused protoplasts. Somatic hybrids have been produced between several species such as Arabidopsis thaliang & Brassica compestri, potato & tomato, Nicotiana and Lycopersicon, etc. (Khush & Brar, 1988).

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41 Genetic traits transferred via protoplast fusion. (Chawla,1998)

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42 MERISTEM TIP CULTURE ( for virus free planting material) Cultivation of axillary or apical shoot meristems, particularly of shoot apical meristem is known as meristem culture. Limmaset & Conuet (1972) observed that in virus infected plants, the virus concentration decreases towards the growing point. Morel and Martin (1952) developed the technique of meristem culture for in vivo virus eradication of Dahlia. 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. e.g. Banana free from cucumber mosaic virus, cauliflower free from cauliflower mosaic virus, pea from seed borne mosaic virus, potatoes free from parasrinkle virus X, S Y, a leaf roll & spindle tuber virus & sugarcane from mosaic (Siddiq and Sharma 1995). Also used to develop virus free plants of dahlia, carnation, strawberry, chrysanthemum, archids, etc. (Khush, 1995).

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43 Transgenics for disease resistance Transfer of genes between plant species has played an important role in crop improvement for many decades. Genes expected to confer disease resistance are isolated, cloned & transferred into the crop in question. Useful traits viz.,resistance to diseases, insects & pest have been transferred to crop varieties from noncultivated plants. The overall process of genetic transformation involves introduction, integration & expression of foreign gene in the recipient host plant. Plant that carry additional stably integrated, & 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 and vectorless approach (Horsch et al., 1984.) A virus resistant transgenic variety of squash is in commercial cultivation in USA.

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44 Virus Resistant Transgenic Plants

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Transgenic Virus Resistance Papaya plant Virus Susceptible Papaya Plant

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46 B] Transgenic Plants (Fungal and Bacterial Diseases)

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47 Disease Epidemics An epidemic is a severe outbreak of disease beginning from a low level of infection. Epidemics are common in case of air borne fungi. An epidemic progresses from low infection level. e.g. An epidemic of potato leaf blight may be initiated by a single infector plant / sq. km. Causes of Epidemics: Narrow genetic base of cultivated crops Large acreages under single varieties Introduced pathogens Failure of VR genes (eg. Vertifolia potato variety) Inadvertent breeding for susceptibility.

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48 Measures for Prevention of Epidemics: (Management of Disease Resistance): Kush (1995) propose four approaches to prolong the useful life of a variety with VR Deployment of genes over time Development of genes over space Pyramiding of vertical genes, and Multiline varieties

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49 Deployment of genes over time Strong genes for VR may be employed or may be used to check epidemics in alternate years so that the virulent pathotype against any one of them doesn’t evolve & doesn’t survive even if it did evolve. based on the principle of crop rotation to control certain soil borne pathogens. Deployment of genes over space Strong genes for VR may be employed or may be used to check epidemics in different geographical regions; these regions would run perpendicular to the direction of movement of the pathogen. Thus each strong gene would act as a sieve for the pathotype virulent on the succeeding one(s).

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50 Gene Pyramiding (Watson & Singh, 1953): Incorporation of diverse major genes for resistance to a pathogen / insect in one & the same variety. It is believed that, greater the no. of major genes for resistance in a variety greater would be its longevity. Limitations : May lead to the evolution of “super races” If the super race in produced, which may overcome the combined resistance, then several resistance genes may be simultaneously rendered ineffective & may lead to quick exhaustion of available genes for resistance (Kush, 1995).

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51 Multiline Variety: One of the breeding approaches for a long-range control of diseases in SPCs is the development of multiline varieties. “A multiline variety is a population of plants that is agronomically uniform heterogenous for genes that condition reaction to a disease” This concept was first given by Jensen (1952) for use in oats Similar approach was suggested by Borlaug(1959) in Wheat for controlling stem rust.

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52 Characteristics of a good multiline: It has genotypic diversity with respect to vertical genes. Each vertical genotype must be strong enough to ensure that reduction in the exodemic is maximum. The multiline should have normal resistance to other important diseases of concerned crop & area. The multiline should have phenotypic uniformity with respect to agronomic characters like plant height, maturity & seed type. The multiline should have yield advantage to the extent possible.

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Combines VR and HR Exploit multiple alleles Exploit linked genes Extend life of resistant genes Dynamic system Exploit genetic diveresity Costly Agronomically conservative Breeding grounds for new races Possibility of super race Advantages Limitations

Achievements of Multilines in India : 

Achievements of Multilines in India

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55 Merits of breeding for disease losses caused by disease crops are minimized with use of resistant varieties. reduction in the cost of production resulting in increasing cost benefit ratio. Reduces environmental pollution & heath hazards by reducing use of pesticides. They are non toxic to man, farm animals & wild life (do not contain pesticidal resistance). Genetic resistance is only solution of some of the diseases such as wilts, rusts, smuts, nematodes and bacterial blights.

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56 Demerits of breeding for disease It is long term process which takes 10-15 years to develop agronomically acceptable variety. In some cases, breeding for resistant to one disease leads to the susceptibility to another pests. Breeding for disease resistance is an expensive method (requires adequate finance for long period).

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57 Breeding achievements for resistance breeding for major field crops

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58

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59 Note:-1) Abbreviations used before varieties : T - Tollerent, MT - Moderately tollerent, R - Resistant, MR - Moderately resistant. S - Susceptible 2) Varieties coated as T/MT/MR/R for biotic or abiotic stresses is observed only at the time of their release.

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16-60 THANK YOU The hazardous effect of fungicides, bactericides and insecticides, or their degradation products, on the environment and human health strongly necessitates the search for new harmless means of disease control…….and i.e. Development of resistant varieties.