Module 5-Breeding for Disease Resistance

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Module 5: Breeding for Disease Resistance

I. Introduction II. Some Concepts of Plant Pathology

a. Bacterial pathogens b. Fungal pathogens c. Nematode pathogens d. Viral pathogens e. Phytoplasm pathogens f. Parasitic plants g. Insect pathogens

III. Genetics of Resistance IV. Breeding for Resistance to Rusts V. Breeding for Resistance to Fusarium Head Blight VI. Breeding for Resistance to Septoria tritici Blotch VII. Breeding for Resistance to Karnal Bunt VIII. Breeding for Resistance to Nematodes

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I. Introduction

Early civilizations were well aware that plants were attacked by diseases. There are references in the Bible to blights, blasts, and mildews (Haggai 2:17, 2 Chronicles 6:28, Amos 4:9). Aristotle wrote about plant diseases in 350 B.C. Theophrastus, the father of botany (372-287 B.C.), in his Historia Plantarum (History of Plants), described rusts on grain crops, in meticulous details. During the Middle Ages in Europe, ergot fungus infected grain and Shakespeare mentions wheat mildew in one of his plays ("He Mildews the White Wheat": King Lear). In India in about the year 500, the situation arose in which plants were thought to be suffering from human ailments. Thus, trees and other plants were thought to suffer from ‘wind’, ‘bile’, ‘phlegm’, ‘jaundice’ and ‘indigestion’. Mathieu Tillet, in 1755, while observing the smuts on wheat, discovered that there were two types of smut: la carie or what the English called "Common Bunt" or "Stinking Smut"; and the second type of smut le charbon or what the English called "loose smut". The distinction between the two types of smut was verified a century later, in 1847, by Louis and Charles Tulasne. To honor Tillet, they named the "Stinking Smut" Tilletia caries. In 1807 Isaac Benedict Prevost was the first to provide definitive evidence that the bunt disease of wheat is caused by a microorganism, smut fungus. He completed detailed experiments on the germination of bunt spores and demonstrated by direct inoculation that they could infect wheat. In 1853 Heinrich deBary published a comprehensive paper that clearly implicated smut and rust fungi as causal organisms of diseases affecting cereal crops. In this and the last century, plant breeders have developed cultivars with genetic resistance to possibly devastating plant pathogens. As this effort has curbed the widespread famine formerly caused by wheat pathogens it can be regarded among the most important contributions to agriculture. However, agriculture is a dynamic trade. Changing agronomic practices and the evolution of new virulent races of pathogens, requires a persistent and continuous effort in disease management. An example of this needed vigilance is occurring now (2007) as a new form of stem rust, has jumped from eastern Africa and is now infecting wheat in Yemen in the Arabian Peninsula. Researchers with the Global Rust Initiative (GRI) and the Agricultural Research Service of the United States Department of Agriculture (USDA-ARS) have confirmed conclusively the existence of the disease in Yemen. There is also

"There is no good theory of disease which does not at once suggest a cure."

Ralph Waldo Emerson

"….when the cause of disease is discovered, consider that the cure is discovered."

Cicero

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evidence that the disease has spread into Sudan but more tests are needed to confirm the finding. Until this discovery, this new strain of stem rust, known as Ug99, had only been seen in Uganda, Kenya and Ethiopia. There is precedence for this, from a virulent strain of another wheat disease, called yellow rust, which emerged in eastern Africa in the late 1980s. Once it appeared in Yemen, it took just four years to reach wheat fields of South Asia. On its way, this new strain of yellow rust caused major wheat losses in Egypt, Syria, Turkey, Iran, Iraq, Afghanistan, and Pakistan, exceeding USD 1 billion in value. There is every reason to believe the new Ug99 strain of stem rust represents a much greater risk to world wheat production. Annual losses of as much as $3 billion U.S. in Africa, the Middle East and south Asia alone are possible.

II. Some Concepts of Plant Pathology Diseases of wheat are numerous and are caused by: bacteria, fungi, nematodes, viruses, phytoplasmas, a single parasitic plant, and insects. For a complete list of known pathogens of wheat, excluding insect pests see:

http://www.apsnet.org/online/common/names/wheat.asp (From the homepage follow “ONLINE RESOURCES” to “COMMON NAMES OF PLANT DISEASES”

to “TABLE OF CONTENTS” and to “#96 WHEAT”.) In order to properly develop a defense against a pathogen, the wheat breeder must know the attacker. The breeder must understand the pathogen’s life cycle (inoculation, infection, proliferation, spread, and latency), its virulence during different environmental conditions and varying stages of wheat growth, along with its epidemiology. The breeder must be able to spot signs of the presence of a pathogen and the breeder must be able to recognize, distinguish, and describe the symptoms that the wheat plant is displaying. Finally, the breeder must understand the economic impacts of a particular pathogen in order to determine the amount of resources that should be directed to resolving the problem.

Inoculation - The introduction of a pathogen to a host. Infection - The pathogen penetrates and establishes a parasitic relationship with the host. Proliferation - The reproduction and growth of the pathogen. Spread - The process by which the pathogen moves within a single host plant or the

process of moving from one infected plant to another. Latency - A time of inactivity of the pathogen (dormancy in advance of proper

environmental conditions). Virulence - The degree of pathogenicity of a given pathogen. Epidemiology - The study of factors influencing the initiation, development, and spread of

infectious disease; the study of disease in populations of plants

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II a) Bacterial pathogens Bacterial plant pathogens are unicellular microorganisms typically 1 to 3 µm in length. They have neither a well defined nucleus, nor a nuclear membrane. Bacteria are spread by insects, air currents, splashing rain, and by mechanical means. Free moisture is usually necessary for infection, and penetration of host tissue occurs through wounds (created by hail, blowing soil particles, insects, or mechanical injury) or leaf openings (stomata and hydathodes). Bacteria reproduce chiefly by binary fission, or cell division yielding two identical daughter cells. Symptoms caused by bacterial diseases may vary greatly, depending on the pathogens involved. A bacterial origin is suggested by water-soaked spots or water-soaked margins around lesions consisting of water-congested green tissue in the early stages of infection. The lesions are greasy and translucent in appearance and may produce exudates. An exudate consists of droplets of bacterial slime emerging from the leaf surface through natural openings (stomata, hydathodes).

Further information on bacterial diseases of wheat can be found in the CIMMYT publication: The Bacterial Diseases of Wheat: Concepts and Methods of Disease Management. Edited by: E. Duveiller, L. Fucikovsky, and K. Rudolph. Available in PDF on line at: http://www.cimmyt.org/

Fig. 1 Example of a bacterial disease cycle

Duveiller et al. 1997

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II b) Fungal pathogens Fungal pathogens of wheat are diverse and constitute the most problematic and costly of the biotic stresses of wheat. Fungi are eukaryotic, non-vascular, saprophytes (taking nourishment from non-living material), or parasites (removing nutrients in a harmful way to a living host). Fungi reproduce by means of spores, both asexually and sexually depending on the species and environmental conditions. Fungal spores spread from plant to plant through wind, water splashing, and by mechanical means. Fungi infect their host when a germ tube growing from a spore enters a plant stomata; or by piercing the cuticle and epidermal cell wall by physical and/or enzymatic means. Within the host, fungi spread by filamentous growth of hyphae forming a mycelium. The lifecycles of the different fungal pathogens can be extremely different from one another; and in order to develop a proper genetic defense against them, a solid understanding of the life cycle of each pertinent fungal pathogen is important.

Further information can be found on a number of fungal pathogens of wheat in the CIMMYT publications: Bunt and Smut Diseases of Wheat: Concepts and Methods of Disease Management. Edited by: R. Wilcoxson, and E. Saari; The Septoria Diseases of Wheat: Concepts and Methods of Disease Management. Edited by: G. Hettel; Septoria and Stagonospora Diseases of Cereals: A Compilation of Global Research. Edited by: M. van Ginkel, A. McNab, J. Krupinsky. Available in PDF on line at: http://www.cimmyt.org/ ; Rust Diseases of Wheat: Concepts and Methods of Disease Management. Edited by: G.P. Hettel.

Fig. 2 – Example of a fungal pathogen’s life cycle

Source: USDA – ARS

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II c) Nematode pathogens Nematodes are a diverse group of worm-like animals. They are found in virtually every environment, both as parasites and as free-living organisms. They are generally minute, but some species can reach several meters in length. Plant parasitic nematodes can be aerial parasites (those feeding on above-ground plant parts) or root and tuber parasites (those feeding on below ground parts). Nematodes can further be classified as migratory endoparasites (mobile, feeding inside the plant), sedentary endoparasites (cease moving once a feeding location inside the plant has been reached), or ectoparasites (feed on the outside of the plant). The nematode life cycle is typically divided into six stages: the egg, four juvenile stages, and the adult. The duration of these stages and of the complete life cycle differs for different species and depends on environmental factors. Further information on nematodes can be found in the CIMMYT publication: Common Diseases of Small Grain Cereals: A Guide to Identification. Edited by: F. Zillinsky. Available on line at Grain Genes (http://wheat.pw.usda.gov/GG2/index.shtml).

Juvenile 3

Juvenile 4

Juvenile 2

male and female nematodes mate and eggs are laid in the soil

Juvenile nematodes develop inside the egg

Second stage Juveniles hatch and are attracted to host root exudates

feeding on host cells and tissue

Fig. 3 Example of a Typical Nematode Disease Cycle.

Source: American Phytopathological Society – Vicky Brewster

Adult

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II d) Viral pathogens Viruses are sub-microscopic particles consisting of genetic material (either DNA or RNA) contained within a protein coat known as the capsid. Viruses are obligate parasites as they can replicate themselves only by the use of another organism’s cellular machinery and intermediate products. Viral infection occurs through wounds, generally being caused by an insect or nematode vector. Viruses can also be spread by fungi, by mechanical means or by being carried on in the seed of infected plants.

Further information on nematodes can be found in the CIMMYT publication: Common Diseases of Small Grain Cereals: A Guide to Identification. Edited by: F. Zillinsky. Available on line at Grain Genes (http://wheat.pw.usda.gov/GG2/index.shtml).

Figure adapted from Nebraska Cooperative Extension

Fig. 4 Example of Viral Disease Cycle

Spring Summer Fall Winter

Mites

Virus

Mites Mites

Virus

Virus

Over-wintering winter wheat crop and other hosts

winter wheat seedlings

hosts other than wheat

volunteer wheat

Infected winter wheat

Arrows indicate virus movement

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II. e) Phytoplasm pathogens Phytoplasmas are similar to bacteria, but unlike bacteria they have no cell wall. They reproduce as bacteria do and are usually found in the water- and food-conducting vessels of an infected plant. Phytoplasmas and spiroplasmas are obligate parasites. Phytoplasmas are transmitted from plant to plant by insects that feed off of phloem tissue Further information on phyoplasms can be found in the CIMMYT publication: Common Diseases of Small Grain Cereals: A Guide to Identification. Edited by: F. Zillinsky. Available on line at Grain Genes (http://wheat.pw.usda.gov/GG2/index.shtml).

Fig. 5 – Example of a Phytoplasm Disease Cycle

Insect vector feeds on vein of infected plant

Insect feeds on annual or perennial plants, Phytoplasmis incubating and is not yet transmitted

Phytoplasm present in salivary glands in large numbers and is injected into plant while insect is feeding

Phytoplasm spreads by phloem throughout the plant

Insect vector feeds on infected plant, picking up the phytoplasm and delivering it to a new plant

Phytoplasmoverwinters in perennial plant

Picture by: S. Mahr; Drawings from: USDA-ARS; Information adapted from: Gianna Sassi Plant Pathology 401 Cornell University

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II f) Parasitic Plants Parasitic plants are flowering plants that gain some or all of their sustenance from another plant. They do this by penetrating host plant vascular tissue through modified roots called haustoria. Striga spp. are obligate root parasites that infect many of the world’s principal grain and legume crops. Although endemic to Africa, Striga spp. occur in over 40 countries worldwide, ranging from Asia to North America. In sub-Saharan Africa, more than two-thirds of the 73 million ha of land used for cereal cultivation is severely infested. Over time, the impact of Striga spp. has increased and it has been described as one of the most serious biological constraints to food production in Africa (Vasey et al. 2005).

Aerial Phase

Underground phase

Host plant

Germination Days 1-2

Attachment to host root Day 2-6

Striga shoot grows utilizing the host root system for water and nutrients Day 6 to weeks 4-6

Weeks 4-7 Striga emerges above ground

Week 9-12 Flowering of Striga

Week 11+ Capsules form on Striga

Week 11+Dissemination of Striga seeds

Fig. 6 Example of a Parasitic Weed Disease Cycle

Adapted from Stringer, 2007

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II g) Insect pathogens The number of insect pests that feed on wheat is numerous. Insects cause injury by feeding, and predispose weakened plants to disease. They produce wounds that serve as infection sites, and often carry pathogens with them from plant to plant. Insects are by far the most numerous and most efficient vectors of viruses and phytoplasmas. Insects with chewing mouth parts remove tissue or cut off young plants. Insect larvae feed on, above and below ground plant parts as well as inhabiting and feeding off of the inside of the plant. Insects can also be problematic, feeding on the grain both pre- and post-harvest.

Further information on insect pathogens can be found in the CIMMYT publication: Common Diseases of Small Grain Cereals: A Guide to Identification. Edited by: F. Zillinsky. Available on line at Grain Genes (http://wheat.pw.usda.gov/GG2/index.shtml).

The pupae overwinter/oversummer within puparia, the hardened skin of the last instar larvae. These puaria, known as the “flaxseed” stage are located just below the surface near the crown of the plant.

Flies deposit eggs on wheat and die in 2 or 3 days

Maggots hatch from the eggs in 3 to 7 days, crawl down the leaves, and feed at the crown or joints along the stem. The maggots develop through three instars over a 25 to 30 day period.

The maggots enter the flaxseed stage for overwinter/oversummer

Flies emerge from the flaxseed stage and disperse throughout the crop

Source: http://www.oznet.ksu.edu/hessianfly/

Fig. 7 – Example of an Insect Pathogen Disease Cycle

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III. Genetics of Resistance Farmers have noticed for centuries that some individual plants in a given species manage to survive disease or epidemics of insects relatively unscathed, while their neighbors succumb to infection or insect predation. In 1905, Sir Roland Biffen of Cambridge, England, wondered whether healthy plants inherited pest resistance, just as they might inherit the tendency to be tall or short. His experiments on two varieties of wheat showed that the ability to resist infection by a rust fungus was indeed inherited in Mendelian fashion, a discovery that intensified attempts by farmers and plant breeders to produce varieties of pest-resistant crop plants. About 50 years later a plant scientist by the name of Harold Flor, while working with flax and the rust fungus Melampsora lini, found that host resistance to a pathogen was not only dependent on the host genetic makeup (resistance genes, R-genes), but also on the genetic makeup of the pathogen (avirulence genes, Avr-genes). With these findings Flor introduced the widely accepted concept of the gene-for-gene theory of disease resistance (often referred to as race-specific or vertical resistance), which predicts that a host will achieve successful disease resistance only if both a host’s R-genes and the pathogen’s corresponding Avr-genes are present. Since that time many R-Avr interactions have been studied and documented. Dozens of R-genes, against many different pathogens have been identified from a variety of plants. These genes encode proteins that can be grouped into superfamilies based on sequence and structure similarity (McDowell and Woffenden 2003); and based on their structure it can be deduced that some of these R-proteins are found in the cell cytoplasm, some are anchored to the cell membrane, and some traverse the cell membrane (Dangl and Jones 2001). There are two models proposed for R-Avr interaction and are depicted in figure 7.

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Plant defense starts with non-self recognition and/or cellular intactness. During host invasion, pathogens release exogenous as well as endogenous elicitors (Vorwerk et al. 2006; Huckelhoven 2007). Elicitors are molecules produced by either the host or the pathogen that in turn induce a response by either the host or the pathogen. Fungal pathogen elicitors include enzymes which break down the cuticle or cell wall polysaccharides. Chitin, the major constituent of fungal cell walls is also known to be an elicitor which triggers a defense response in plants. The bacterial protein, flagellin, is also an elicitor of host defense. Cuticle and cell wall fragments are examples of endogenous elicitors of host defense. As these tissues are under physical or enzymatic attack, released fragments are detected by R-proteins stationed in the cell membrane and a defense response is initiated. Within 15 minutes of pathogen recognition, host cells begin producing new proteins in reaction to the attack (Dangl and Jones 2001). This first response to invasion is known as a basal defense, and includes production of proteins that inhibit pathogen enzymes, structural and chemical remodeling of the host cell wall at penetration sites, and production of antimicrobial agents that will kill the intruder. A second, more extreme, line of defense is known as the hypersensitive response (HR), or programmed cell death (PCD) (Greenberg and Yao 2004; Lam et al. 2001; Day and Graham 2007;

Apoplast Cytoplasm

Host defenses are suppressed

and disease occurs

R

R

Signal transduction

Signal transduction

Defenses activated

Defenses activated

(a)

(b)

(c) Adapted from McDowell and Woffenden 2003

Fig. 7 R-Avr Interaction. A pathogen (grey box) has come into contact with a potential host and is expressing virulence proteins (red). Once inside host cytoplasm, the virulence genes target host defense response proteins (green). (a) The plant cell does not hold an R-protein capable of recognizing any virulence protein, and disease results. (b) Pathogen Avr-protein is recognized by a host R-protein, activating a signal cascade and a defense response. (c) The guard hypothesis: a host defense response protein has been altered by a virulence protein, this is detected by the host R-protein and a signal cascade followed by defense mechanisms being activated soon follows.

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Glazebrook 2005). The hyper sensitive response is a strategy of “scorched earth” where the plant cell introduces toxic molecules into the surroundings; creating a localized environment that is incapable of sustaining life for both the pathogen and the cell itself. The HR is an efficient means of defense against many pathogens, but is ineffective against necrotrophic fungi. Breeders have successfully developed lines resistant to diseases by integrating R-genes into their cultivars for many years; but a durable (sometimes called Horizontal Resistance, Race non-specific resistance, or Qualitative Resistance), long lasting resistance in many cases has been difficult to achieve as pathogens quickly evolve and develop counter resistance genes that circumvent the host cultivars resistance. Breeders often spot this breakdown in resistance and hurriedly integrate a newly found effective R-gene into their populations. In time, the new R-gene loses its effectiveness and the boom-bust and induced co-evolution between crop and pathogen continues. An example of this comes from the United States where in Indiana, 1955; a cultivar was released that held on R-gene conferring resistance to Hessian fly (Meyetiola destructor) attack. Just six years later the Hessian fly population had developed a substantial amount of counter-resistance. By 1964, the Indiana breeders had introduced a second R-gene into their cultivars. This new R-gene provided resistance for eight years before counter-resistance developed. A third R-gene was introduced and released in cultivars in 1971; and again was overcome by the pathogen, this time in a period of ten years (Rausher 2001). Two methods are available to plant breeders in order to increase the durability of their resistant cultivars. The first is known as the High-Dose/Refuge or Multiline strategy (Rausher 2001; Pink 2002). Recall that a host’s resistance to a disease is conferred by the interaction of its R-gene with the pathogen’s Avr-gene. A change due to a mutation of the pathogen’s allele for an Avr-gene will allow the pathogen to circumvent or suppress host defenses and cause disease. Returning to the concepts of population genetics and selection, it is understandable how single R-genes held by the host can be quickly overcome. Suppose p represents a pathogen population’s allele frequency for an Avr-gene that is recognized by a host plant R-gene, while q represents the allele frequency of a new recessive allele formed due to mutation:

1 1 11

1 2 12

2 2 22

: 0.99 0.99 0.005 0.995

: 0.01

: 0.00 0.00 0.005 0.005

A A P p

A A P

A A P q

= → = + === → = + =

and the selection coefficients for each genotype are:

11

12

22

0.90

0.70

0.00

s

s

s

===

.

Following the equation for allele frequencies after selection:

( )( )

12 221 2 2

11 12 22

1

1 2

q ps qsq

p s pqs q s

− + =− + +

,

one can see that the rare mutant allele becomes predominant in just a small number of generations:

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A multiline or refuge will reduce the selection intensity against the A1A1 and A1A2 genotypes by providing an acceptable host for the pathogen; and maintaining (for an extended period) a higher frequency of the Avr-gene, recognizable by the wheat cultivar’s R-gene, within the pathogen population. Suppose 10 to 20 % of the wheat field contains susceptible cultivars to the pathogen A1A1 and A1A2 genotypes; the selection intensities are decreased and the number of generations necessary for the new allele to become predominant increases dramatically:

The multiline strategy requires that some level of disease is acceptable and that the pathogen reproduces by sexual means. Also, multilines may not hold a necessary uniformity that many cropping systems require.

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A second option for the plant breeder in generating a cultivar with durable resistance is know as gene pyramiding. In this strategy several R-genes are deployed in the same cultivar (McDowell and Woffenden 2003; Pink 2002). In theory, pyramiding several “undefeated” R-genes into a single cultivar will provide a more durable resistance as several mutations would need to take place, one at each of the pathogen’s corresponding Avr-loci. With modern molecular biology techniques (See the module: Biotechnology and Wheat Breeding) it is possible to use markers and probes to track the introgression of several R-genes into a single cultivar from various sources during a crossing program. Although many disease resistances often follows the gene-for-gene model some follow a race-non-specific, or qualitative mode of resistance (also known as horizontal resistance) where resistance is controlled by genes with minor to intermediate and additive effects. Combinations of 3 to 5 of these minor genes can result in a high level of resistance (Singh et al. 2000). A third type of resistance is known as partial resistance and most commonly is associated with slow rusting cultivars. Slow rusting as defined by Caldwell (1968) is a type of resistance where disease progresses at a retarded rate, resulting in intermediate to low disease levels against all pathotypes of a pathogen. Partial resistance is a form of incomplete resistance characterized by a reduced rate of epidemic development despite a high susceptible infection type. The components that cause slow rusting of a cultivar are longer latent period, low receptivity or infection frequency, as well as smaller uredial size and reduced duration and quantity of spore production. All of which can affect disease progress in the field. Slow rusting resistance has dominated in CIMMYT’s bread wheat improvement program for more than 25 years. Wheat cultivars susceptible to a disease may in fact show no significant yield reduction (Zuckerman et al. 2006). Protection of yield, or tolerance, in infected plants was defined by Caldwell and Shafer (1958) as the ability of a crop to endure severe epidemics by the pathogen while sustaining only insignificant yield losses as compare with an infected non-tolerant cultivar. The tolerance that some cultivars demonstrate over others is not yet well understood, but it has been proposed that storage of carbohydrates and their mobilization to the sink under stress conditions; or that tolerant plants have the ability to compensate for the loss of photosynthetic leaf area by increasing the photosynthetic capacity of the unaffected leaf area contribute to the tolerance to disease (Zuckerman et al. 1996).

IV. Breeding for Resistance to Rusts By Ravi Singh The three rust diseases, stem (or black), leaf (or brown) and stripe (or yellow) are caused by fungi Puccinia graminis f. sp. tritici, P. triticina and P. striiformis f. sp. tritici, continue to cause losses, often major, in various parts of the world and hence receive much higher attention in breeding. The wheat crop can be protected from rust, or at least the occurrence of epidemics can be reduced, by emphasizing the following three strategies: 1) regional cooperation in monitoring the evolution and migration of new races of rust fungi, 2) enhanced information on the genetic basis of resistance in important wheat cultivars for their deployment, and 3) shift towards breeding and deploying wheat cultivars with durable resistance.

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The South American cultivar ‘Frontana’ is considered the best-known source of durable resistance to leaf rust (Roelfs 1988). The Mexican-Rockefeller Program first used this variety in the 1950s. Later its derivatives, such as ‘Penjamo 62’, ‘Torim 73’, and ‘Kalyan/Bluebird’, showed slow rusting characteristics possibly derived from Frontana. Genetic analysis of Frontana and several CIMMYT wheats possessing excellent slow rusting resistance to leaf rust worldwide has indicated that such adult plant resistance is based on the additive interaction of Lr34 and two or three additional slow-rusting genes (Singh and Rajaram 1992). Leaf rust severity observed in Mexico on most slow-rusting cultivars could be related to the number of minor genes they carry. When susceptible cultivars display 100% leaf rust severity, cultivars with only Lr34 display approximately 40% severity; cultivars with Lr34 and one or two additional minor genes display 10-15% severity; and cultivars with Lr34 and two or three additional genes display 1-5% severity. Leaf rust may increase to unacceptable levels on cultivars carrying only Lr34, or Lr34 and one or two additional genes. However, cultivars with Lr34 and two or three additional genes, referred to as ‘near immune’ (Singh et al. 2000) showed a stable response in all environments tested so far, with final leaf rust ratings lower than 10% (Navabi et al. 2003). The presence of Lr34 can be indicated by the presence of leaf tip necrosis in adult plants, which is closely linked with it (Singh 1992a). Slow-rust resistance to leaf rust is common in spring wheat germplasm and at least 10-12 slow-rusting genes are involved in the adult plant resistance of CIMMYT wheat germplasm (Singh and Rajaram 2002). Lines where Lr34 is absent but still possess high level of slow-rusting resistance are also identified indicating that durable resistance is feasible even in the absence of Lr34. The second slow-rusting resistance gene Lr46 was recently identified in wheat cultivar ‘Pavon 76’ and located in the short arm of chromosome 1BL (Singh et al. 1998; William et al. 2003b). Singh (1992b) and McIntosh (1992) have indicated that the moderate level of durable adult-plant resistance to stripe rust (caused by Puccinia striiformis) of the CIMMYT-derived U.S. wheat cultivar Anza and winter wheats, such as Bezostaja, is controlled in part by the Yr18 gene. This gene is completely linked to the Lr34 gene. The level of resistance it confers by itself is usually not adequate. However, combinations of Yr18 and 3-4 additional slow-rusting genes result in adequate resistance levels in most environments (Singh and Rajaram 1994). Genes Lr34 and Yr18 occur frequently in germplasm developed at CIMMYT and in various countries. Recently identified slow-rusting gene Yr29 is completely linked to slow leaf rusting gene Lr46 (William et al. 2003b). Low disease severity to stripe rust is usually associated with at least some reduction in infection type because stripe rust grows systematically in leaf tissues. This phenomenon results in chlorotic or necrotic stripes and therefore creates difficulties in distinguishing slow rusting resistance from race-specific resistance. Durability and acceptance of adult plant resistance can be expected if the cultivar’s low disease severity is due to the additive interaction of several (4 to 5) partially effective genes (Navabi et al. 2004). Stem or black rust, caused by Puccinia graminis tritici (Pgt), is historically known to cause severe losses to wheat production. However, it has been controlled effectively through the use of genetic resistance in cultivars associated with the green revolution during the 1960s and 1970s. Over 80% of the spring wheat area in developing countries

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is currently sown to cultivars either derived directly from CIMMYT germplasm or from CIMMYT germplasm used as parents. For more than 30 years, a major proportion of the CIMMYT wheat germplasm and germplasm developed by other breeding programs have remained resistant to stem rust. Resistance gene Sr31, located on rye translocation 1B.1R contributed to high level of resistance in several wheat cultivars developed worldwide in recent years. Consequently stem rust disease is often not considered important and in many countries wheat breeding is currently done in the absence of stem rust and research in this area in the last two decades also declined substantially. Detection in 1999 of Pgt race Ug99 in Uganda with broad virulence, including the virulence for Sr31, and its migration to Kenya and Ethiopia has been recognized as a highly significant event and led to the launch of Global Rust Initiative during 2005. All major cultivars currently grown in North Africa, the Middle East and Asia are moderately or highly susceptible to this race. Predominant wind patterns or human errors are likely to introduce this race to above regions and beyond. One of the major challenge to wheat breeding is identifying or developing and diffusing adapted resistant cultivars before this migration occurs. Although some race-specific resistance genes, mostly of alien origin, viz., Sr22, 24, 25, 26, 27, 29, 32, 33, 35, 36, 39, 40, 44, R (1A.1R translocation) and Tmp, can provide effective control; not all can be used in developing cultivars as some of these alien translocations are associated with negative effects on grain yield or quality. Shortening of these alien segments could help their utilization. Improved wheat germplasm that carry Sr24, Sr25, Sr26, SrTmp and SrR genes is already identified and can be used in breeding. Gene Sr24 currently occurs with a relatively high frequency in wheat cultivars and has become ineffective in India and South Africa soon after the release of cultivars that carried this resistance gene. The best strategy therefore is to reconstitute durable adult-plant resistance that once protected the green revolution and subsequent wheat cultivars. If race-specific genes need to be used, they must be deployed in combination to enhance their longevity. Durable stem-rust resistance of some older US, Australian and CIMMYT spring wheats is believed to be due to the deployment of Sr2 in conjunction with other unknown minor, additive genes. McFadden transferred Gene Sr2 to hexaploid wheat in the 1920s from tetraploid emmer wheat cultivar ‘Yaroslav’. The slow rusting gene Sr2 confers by itself only moderate levels of resistance. Its presence can be detected through its complete linkage with the pseudo-black chaff phenotype. A large number of wheat lines were evaluated during 2005 in Kenya under the stem rust epidemic caused by Ug99. Genotypes with pseudo-black chaff phenotype showed varying degrees of disease severity with a maximum severity reaching to about 60% compared with 100% severity for highly susceptible materials. Moreover, the host reaction for these genotypes on the same internode varied from moderately resistant to susceptible. These observations clearly indicated that although slow rusting resistance gene Sr2 continues to confer at least some resistance, the level of resistance was not sufficient when this gene is present alone under high disease pressure in Kenya. Sr2 was detected in several highly resistant old, tall Kenyan cultivars, including ‘Kenya Plume’ (Singh and McIntosh 1986), and CIMMYT-derived semidwarf cultivar ‘Pavon 76’. These cultivars have shown a maximum disease score of 15MR (15% disease severity with moderately resistant reaction). Because Pavon 76 is susceptible to race Ug99 at the seedling stage, its resistance, as speculated earlier (Rajaram et al. 1988), is based on multiple additive genes

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where Sr2 is an important component. Wide testing of improved wheat germplasm also has helped in identifying additional sources of adult-plant resistance. These sources are being used at CIMMYT to incorporate durable stem-rust resistance into high yielding, widely adapted wheat cultivars using the methodology described below. Breeding for durable resistance based on minor additive genes has been challenging and often slow, for several reasons: 1) a sufficient number of minor genes may not be present in a single source genotype, 2) a source genotype may be poorly adapted, 3) there may be confounding effects from the segregation of both major and minor genes in the population, 4) crossing and selection schemes and population sizes are more suitable for selecting major genes, 5) reliable molecular markers for several minor genes are unavailable, and 6) the cost associated with identifying and utilizing multiple markers is high. One suggested approach is to use recurrent selection schemes to accumulate several minor genes in a single genetic background. Such selection schemes have often been more of a scientific interest than actually being applied in breeding. Selection for resistance alone will not generate important popular cultivars, unless it is simultaneously combined with other traits, such as high yield and quality. However, such germplasm carrying combinations of minor genes should be very useful in transferring these genes to modern cultivars. A successful example of breeding for resistance based on minor genes is the resistance to leaf and stripe rusts in wheat, which took about 30 years of continuous effort at CIMMYT. In the early 1970s, S. Rajaram, influenced by the concept of slow-rusting resistance in wheat proposed by R. Caldwell and partial resistance to late blight of potato put forth by J. Niederhauser, made a strategic decision: to initiate selection for slow-rusting resistance to leaf rust in CIMMYT spring wheat germplasm. In the early phase of breeding he maintained plants and lines in segregating populations that would show 20-30% rust severity with compatible infection type. This strategy led to the release of several successful wheat cultivars, such as ‘Pavon 76’ and ‘Nacozari 76’, in Mexico and other countries. These slow-rusting lines were used heavily in the crossing program and resulted in the wide distribution of minor genes within CIMMYT spring wheat germplasm. The genetic basis of such resistance started to become clear in the early 1990s. High-yielding lines that combine four or five additive, minor genes for both leaf and stripe rusts and show near-immune levels of resistance were developed in the 1990s (Singh et al. 2000). Three or four lines carrying different minor genes were crossed (3-way and 4-way crosses), and plants in large segregating populations were selected under artificially created rust epidemics. Races of pathogens that have virulence for race-specific resistance genes present in the parents were used to create the epidemics. The resulting highly resistant lines are now being used in a planned manner to transfer these minor resistance genes to well adapted, “farmers’ choice” cultivars that are currently grown across large areas but have become susceptible to rust races in Mexico. Based on genetic information on the number of additive, minor genes that must be transferred to achieve the desired level of resistance, the crossing and selection scheme described below was developed and applied. This strategy has allowed simultaneous transfer not only of resistance genes but also other minor genes with small effects that increase the yield potential or improve the grain quality of an adapted cultivar.

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To transfer minor gene-based resistance into a susceptible adapted cultivar or any selected genotype, we use a ‘single backcross-selected bulk’ scheme (figure 10), where the cultivar/genotype is crossed with a group of about 8-10 resistance donors; 20 spikes of the F1 plants from each cross are then backcrossed to obtain 400-500 BC1 seeds. Selection is practiced from the BC1 generation onwards for resistance and other agronomic features under high rust pressure. Because additive genes are partially dominant, BC1 plants carrying most of the genes show intermediate resistance and can be selected visually. About 1600 plants per cross are space-grown in the F2, whereas population sizes of about 1000 plants are maintained in the F3-F5 populations. Plants with desirable agronomic features and low to moderate terminal disease severity in early generations (BC1, F2 and F3), and plants with low terminal severity in later generations (F4 and F5) are retained. We use a selected-bulk scheme where one spike from each selected plant is harvested as bulk until the F4 generation, and plants are harvested individually in the F5. Bulking of selected plants poses no restriction on the number of plants that can be selected in each generation, as harvesting and threshing are quick and inexpensive, and the next generation is derived from a sample of the bulked seed. Because high resistance levels require the presence of 4 to 5 additive genes, the level of homozygosity from the F4 generation onwards is usually sufficient to identify plants that combine adequate resistance with good agronomic features. Moreover, selecting plants with low terminal disease severity under high disease pressure means that more additive genes may be present in those plants. Selection for seed characteristics is carried out on seeds obtained from individually harvested F5 plants. Small plots of the F6 lines are then evaluated for agronomic features, homozygosity of resistance, etc., before conducting yield trials. Resistant derivatives of several cultivars and genotypes were recently developed using the above methodology. In each case we could identify derived lines that not only carry high levels of resistance to leaf rust or yellow rust or both, but also show about 5-15% higher yield potential than the original cultivar. We believe this approach to wheat improvement allows us to maintain the characteristics of the original cultivar while improving its yield potential and rust resistance. It should be noted that having minor gene-based resistance in several backgrounds should ease future selection for these resistance genes.

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Fig. 10 Strategy for Breeding Durable Rust Resistance

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V. Breeding for Resistance to Fusarium head blight Fusarium Head Blight (FHB, Fusarium head scab) is one of the most destructive diseases of wheat in areas with warm and humid climatic conditions. This disease poses a grave threat to wheat and barley production and industries throughout the world since it brings about mycotoxin contamination. The mycotoxins, such as deoxynivalenol (DON) and nivalenol (NIV), are produced by Fusarium fungi and cause acute food poisoning in people who eat infected wheat and barley products. The mycotoxins also affect animals fed on diseased grains. Over the years FHB has become widespread, partly because of the climatic changes brought about by global warming and also due to the increase in reduced-tillage practices. Genetic and breeding studies on FHB resistance have been carried out in Japan since the 1960s. CIMMYT also started a breeding program for FHB resistance approximately 20 years ago. Several species of the genus Fusarium cause fusarium head blight (FHB) or scab, which is a main production constraint in environments where humid and semi-humid conditions, caused by frequent rainfalls, coincide with flowering in wheat. The Yangtze River basin of China with about 7 million ha has traditionally been known to be highly prone to the disease epidemics. Disease incidences leading to epidemics are now frequent in various other developing countries, e.g., Argentina, Brazil and Uruguay, where residue retention is a common practice for conservation agriculture. Introduction of maize-wheat crop rotation further increases the disease build-up. Sources of resistance to FHB have been divided into three groups: China and Japan, Argentina and Brazil and Eastern Europe (Singh and Rajaram 2002). More recently additional sources, including some hexaploid synthetic derived wheat lines have also been identified to carry moderate resistance. Earlier genetic analysis indicated that a few additive genes confer resistance in Chinese and Brazilian wheats, and genes present in Chinese sources are different from those in Brazilian sources (Singh et al. 1995, Van Ginkel et al. 1996). Although several genomic regions are now known to contribute quantitative resistance (Buerstsmayr et al. 2002, Anderson et al. 2001), a gene from Chinese cultivar ‘Sumai 3’ in the short arm of chromosome 3B has shown the largest and consistent effect in reducing disease severity and mycotoxin accumulation (Anderson et al. 2001). The Chinese sources are probably the best resistances currently available and must be combined with other sources of resistance. The Chinese cultivars that best combined with CIMMYT materials to transmit scab resistance are ‘Sumai#3’, ‘Ning 7840’, ‘Shanghai#5’, ‘Yangmai#6’, ‘Suzhoe#6’, ‘Wuhan#3’ and ‘Chuanmai 18’. Further progress in enhancing the level of resistance beyond the current level can come from a breeding strategy that would favor the accumulation of multiple minor genes from various sources into a single genotype. This would involve intercrossing parents where resistance is located in different genomic regions, followed by growing large segregating population and using flanking markers linked to resistance locus for selection in early segregating generations. Conventional field screening must wait until the F4 or F5 generations when homozygosity has increased significantly. Progenies that show higher levels of resistance than the parental sources could be used for a targeted transfer to high yielding cultivars that would already by then carry moderate levels of resistance from the ongoing breeding efforts. This strategy is currently practiced at CIMMYT to accumulate resistance genes from different sources.

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In 2006 CIMMYT modified their FHB screening system for greater screening capabilities, accuracy and precision. These changes have included the following:

• Changing the primary screening site from Toluca to El Batan (headquarters), Mexico • Implementing an automated, programmable misting system • Using precision CO2 sprayers for liquid inoculum application

Fine-Misting System Approximately 1.9 hectares were placed under a programmable misting system to provide uniform humidity conditions for favorable FHB conditions. This misting system includes about 1,600 DAN modular micro-sprinklers (Figure 1A) spaced at distances of 3 x 4 meters, and operated automatically by a programmable timer to provide humid conditions 24 hours a day. The system is programmed for misting both day and night, with misting intervals and duration varying depending on the time of day (more frequent misting during the driest periods of the day). CO2 Sprayers CO2 backpack sprayers were purchased from R&D Sprayers with an 8002VS flat fan nozzle for precise application of inoculum (Figure 1B). Liquid inoculum was applied at a concentration of 100,000 macroconidia/ml of a mixture of three isolates of F. graminearum at a pressure of 40psi and a rate of 39ml of inoculum per meter. Wheat plots were first sprayed at anthesis, and barley plots were first sprayed at heading. Inoculum was re-applied at the same rate three days after the first inoculation. Grain spawn inoculum (colonized maize grains) was also spread in the FHB evaluation plots to ensure adequate levels of the pathogen for the generation of the disease. By screening germplasm using the method above, recommendations were made to CIMMYT breeders on germplasm for further use of these materials by their programs and development of new FHB resistant wheat (Table 1).

A B C

Figure 11: CIMMYT Primary FHB Screening Site, El Ba tan, Mexico: A) Micro-sprinkler of misting system, B) CO2 backpack sprayer used for inoculations C) Examples of symptoms observed.

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Table 1 – Accessions That Have Been Recommended for Further Use Based on Recent (2006) FHB Trials in a Disease Nursery at CIMMYT

Cross Selection History SHANGHAI -33GH-0M-0Y-0Y-0SCM-0FGR-0FGR-0FGR

GONDO/TNMU CMSS92M01425S-015M-0Y-0Y-050M-16Y-2M-0Y-2SJ-0Y 0FGR-0FGR

IVAN/6/SABUF/5/BCN/4/RABI//GS/CR A/3/AE.SQUARROSA (190)

CMSS00M00007S-030M-1Y-4SCM-010Y-0FGR

IVAN/6/SABUF/5/BCN/4/RABI//GS/CR A/3/AE.SQUARROSA (190)

CMSS00M00007S-030M-1Y-8SCM-010Y-0FGR

IVAN/6/SABUF/5/BCN/4/RABI//GS/CR A/3/AE.SQUARROSA (190)

CMSS00M00007S-030M-1Y-24SCM-2Y-0FGR

GONDO/FINSI CMSS00Y02909S-030Y-030M-030Y-2M-1M-0Y

TNMU/6/CEP80111/CEP81165/5/MRN G/4/YKT406/3/AG/ASN//ATR

CMBW91Y01692S-13Y-2AL-3AL-010Y-3M-0Y-3PZ-0Y

MAYOOR//TK SN1081/AE.SQUARROSA (222)/4/CS/LE.RA//CS/3/PVN/5/PRINIA

CSSS00B00011S-2Y-10M-2Y-8FGR-1Y-0FGR-0BI

80456/YANGMAI 5//SHA5/WEAVER/3/PRINIA CMSS98M00896T-040Y-0100M-040Y-040M-030Y-53M-1Y-0M

SUM3/3/CS/LE.RA//CS/4/YANGMAI 158 -0FGR

WUH1/VEE#5//CBRD CMSS92M01863S-015M-0Y-050M-0Y-13M-0Y-0FGR-0FGR-0FGR

80456/YANGMAI 5//SHA5/WEAVER CMSS97M01288S-030M-020Y-030M-015Y-29M-2Y-2M-0Y-020SCM

NG8675/CBRD//SHA5/WEAVER CMSS97M01295S-040M-020Y-030M-015Y-40M-1Y-2M-0Y-020SCM

GONDO/CBRD CMSS97M01318S-040M-20Y-010M-010Y-5M-2Y-2M-0Y-020S

BAU/MILAN//CBRD CMSS97M01333S-030M-100Y-010M-010Y-6M-1Y-2M-0Y-020SCM

EMB16/CBRD//CBRD CMSS98M00761T-040Y-0100M-040Y-040M-030Y-15M-2Y-0M

80456/YANGMAI 5/3/PF70354/BOW//DUCULA/4/DULUS

CMSS99Y03242T-040M-040Y-040M-030Y-030M-17Y-2M

YANGMAI 5*2/4/MOR/VEE#5//DUCULA/3/DUCULA CMSS99Y03149F-040M-040Y-040M-030Y-030M-15Y-2M-2M-0Y

SHA4/CHIL/4/CAR422/ANA//TRAP#1/3/STAR CMSS99M02404S-040M-030Y-030M-2Y-1M-2M-0Y

SHA3/SERI//G.C.W1/SERI/3/SHA3/SERI//YANG87-142

CMSS00Y02958S-030Y-030M-030Y-5M-1M-0Y

NG8675/CBRD//MILAN/3/NG8675/CBRD CMSS95M01814T-050Y-0100M-050Y-050M-040Y-030M-3Y-0M-0SCM-0Y-0FGR-0FGR

NING MAI 9558 -0CHN-0SCM-0Y-0SCM-0Y-0FGR-0FGR

TINAMOU CM81812-12Y-06PZ-5Y-5M-0Y-3AL-0Y-2M-010Y-0M-3PZ-0Y

TRAP#1/BOW//TAIGU

DERIVATIVE

CMSS94Y01180S-0300M-0100Y-050Y-050M-41Y-030M-3PZ-

0Y-3M-0Y-0SCM-0Y-0FGR-0FGR

TRAP#1/BOW//TAIGU

DERIVATIVE

CMSS94Y01180S-0300M-0100Y-050Y-050M-41Y-030M-1SJ-

0Y-2M-0Y-0SCM-0Y-0FGR-0FGR

SHA3/CBRD -0SHG-1GH-0FGR-0FGR-0SCM-0FGR-0FGR-0FGR

SUM3/3/CS/LE.RA//CS/4/YANGMAI 158 -0FGR-0FGR-0FGR

YANGMAI 5 -0CHN-0SCM-0Y-0SCM-0FGR-0FGR-0FGR

TRAP#1/BOW//TAIGUDERIVATIVE CMSS94Y01180S-0300M-0100Y-050Y-050M-41Y-030M-3PZ-

0Y-0FGR-0FGR

EMB27/KLORI F37023-901F-904F-902F-903F-901F-453F-901F-900F

BR23/EMB27 -7TSB-0Y-0SCM-0Y-0SCM-0Y-0FGR-0FGR

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VI. Breeding for Resistance to Septoria tritici Blotch Septoria tritici blotch (STB), caused by Mycosphaerella graminicola (anamorph Septoria tritici), is currently the most serious foliar disease of wheat grown under temperate (15–20°C) and humid climates in Europe, South America, North Africa, and Central Asia. Severe infections can cause up to 60% losses. Resistant cultivars reduce the use of costly fungicide treatments some of which have become obsolete as some pathogen isolates have become immune (Arraino and Brown 2006). The population of M. graminicola is highly diverse genetically (McDonald and Linde 2002) and the fungus reproduces sexually several times during the wheat growing season, which increases the risk that the pathogen overcomes host resistance (Zadoks 2003). Likewise, this high evolutionary rate probably explains the widespread failure of strobilurin fungicides after most M. graminicola isolates appeared to spread the mutation G143A in a very few years in the United Kingdom (Lucas 2003). Useful sources of resistance include Bobwhite, Kavkaz-K4500, Corydon, Catbird, and Milan (Singh and Rajaram 2002). Kavkaz-K4500, Veranopolis, and Catbird have isolate-specific resistance. This suggests that several resistance genes can be pyramided in a single cultivar (Chartrain et al. 2004; Chartrain et al. 2005). Isolate specific resistance is near-complete, oligogenic, and follows a gene for gene relationship, whereas quantitative or partial resistance is incomplete, polygenic, and isolate nonspecific. Twelve genes for resistance to STB have been identified (Chartrain et al. 2004). Specific interactions between cultivars and isolates can easily be assessed by using a detached seedling leaf technique to study resistance in wheat (Arraiano et al. 2001). The detached seedling leaf technique has a number of advantages over field and glasshouse trials:

• The test can be carried out on several pathogen isolates at one time. • It is conducted under controlled conditions at any time of year. • Little glasshouse space is required. • The labor required to set up the trail is offset by no labor being needed

for watering, fertilizing, or other maintenance of plants. • Results are highly correlated with results from the field.

The following protocol for the detached seedling technique is abbreviated from Arraiano et al. (2001):

• Seeds of the cultivars to be tested are pre-germinated on wet filter paper in darkness at 25°C for 24 h.

• Seeds moved to the refrigerator at 5°C for 48 h. • Seeds moved back to 25°C for 24 h. • Germinated seeds are grown in potting soil, in a glasshouse until first or

second leaf is expanded (12-15 days following germination). ∗ Inoculum is produced from sporulating cultures of M. graminicola, grown on

potato dextrose agar for 7 days under near-ultraviolet light for 16 h per day at 15°C.

∗ Cultures are flooded with sterile distilled water and scraped to release conidia. ∗ The concentration of conidia suspension is adjusted to107 spores/mL and

Tween 20 is added to 0.15% v/v.

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• Wheat seedlings are evenly sprayed with spore suspension to run off. • Leaves are left to dry for 30 min. • Three 5 cm sections cut from the middle of the primary leaves. ∗ Water agar is prepared (10g/L) containing 100mg/L benzimidazole (used to

retard senescence) ∗ 50 mL dispensed into an appropriate number of 8x12x2 cm clear polystyrene

boxes. ∗ Rectangular sections (3x9 cm) are cut from the center of the agar. • 8 to 10 Seedling leaf sections per box are laid, top surface up, across the

gap(created by cutting out of the rectangular sections) so that the cut ends rest on the agar.

• Strips of agar are laid over the cut edges of the leaves. • The boxes are closed and covered with black plastic or foil. • The boxes are incubated at 20°C for 48 h in darkness. • The boxes, then, are uncovered and left under white phosphorescent light for

12 days at 20°C. • Boxes are then moved to conditions with near-ultraviolet light at 15°C to

promote sporulation (14 days after inoculation) • The percentage of leaf area covered by lesions bearing pycnidia is scored 4 to

5 times at intervals of 2-4 days for 19-28 days after inoculation. (Assessments carried out under a 40x dissecting microscope).

New computer tools may lend themselves useful in scoring of diseases. Image analysis software can be a faster and more reliable method for scoring of diseases than that which can be done with the eye which is more subjective and can tire. For further information on how image analysis software can help quantify disease incidence see Mirik et al. (2006). In field studies at CIMMYT disease is rated using a double digit scale: After anthesis, spot blotch severity is evaluated using the double-digit scale (00-99) developed as a modification of Saari and Prescott’s scale for assessing severity of foliar diseases of wheat (Saari and Prescott, 1975; Eyal at al., 1987). The first digit (D1) indicates disease progress in canopy height from the ground level, while the second digit (D2) refers to severity measured based on diseased leaf area. Both D1 and D2 are scored on a scale of 1 to 9. Since the rate of disease progress in the field can be extremely high in some regions, it is often needed to take repeated scores to properly assess the level of resistance (Dubin et al., 1998; Duveiller et al., 1998). It is recommended to take several individual disease scores per plot at 3- to 7-day intervals over a 3- to 4-week period between anthesis and dough stage depending on seeding date. For each score, percent disease severity is estimated based on the following formula: % severity = ((D1/9) x (D2/9) x 100) The area under the disease progress curve (AUDPC) is calculated using the percent severity estimates corresponding to the three to four ratings shown below:

,

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where, xi = severity on the ith date, ti = ith day, and n = number of dates on which disease is recorded. The AUDPC (%-day) measures, the amount of disease as well as the rate of progress. It may be appropriate to standardize the AUDPC to take into account different epidemics situations and planting dates. In this case AUDPC should be divided by the total number of days in the evaluation period (AUDPC/day) to better compare genotypes or among epidemics. A good example of how to identify sources of race-specific resistance is underway at the CIMMYT station in Toluca. Ten nurseries, each containing the same 30 wheat cultivars replicated twice in each nursery have been planted. Each nursery has been inoculated with a different strain of Mycosphaerella graminicola, and disease has been scored as described above four times over the season. By doing this, the breeder can identify which cultivars hold R-genes for specific pathogen isolates. Cultivars holding resistance to different races of the pathogen can then be put into a double cross scheme in an attempt to introgress a number of R-genes into a single cultivar. A number of cultivars that can be used as a source for resistance are listed in table 2 taken from a study done by Chartrain et al. (2004).

VII. Breeding for Resistance to Karnal Bunt Karnal (Tilletia indica) bunt or partial bunt is the most recently described smut disease of wheat, being first reported around Karnal, India in 1931. Since that time it has been reported in several countries including India, Nepal, Pakistan, Afghanistan, Iran, Iraq, South Africa, Mexico, and U.S.A. Although high disease severity does not cause a significant loss in grain yield, a crop containing just 1-4% of infected kernels is sufficient to render wheat grain unpalatable; and disease incidence of 5% causes distinct deterioration in flour quality. Since the 1940s wheat cultivars have been reported to be resistant to Karnal Bunt under field conditions in India. Resistant cultivars have also been reported in to originate in China, Brazil and from synthetic varieties derived by T. turgidum x A. tauschii (Fuentes- Davila et al. 1994; Multani et al, 1988; Villareal et al. 1996). Studies on the mode of inheritance and allelic relationship among genes conferring Karnal bunt resistance in wheat have identified two partially recessive and four partially dominant genes (Fuentes-Davila et al. 1995). Other studies suggest resistance is oligogenic and additive in nature; requiring two or three genes for resistance and as many as nine genes available from a variety of sources (Sharma et al. 2005; Harjit-Singh et al.1999). Inoculation of resistant lines with different Karnal bunt isolates reveal variable levels of disease incidence; suggesting that available resistance to Karnal bunt is race-specific which may require R-gene pyramiding for durable resistance (Datta et al. 1999). As sufficient resistance appears to rely on just a few genes, a backcross strategy as the one described above for the rusts would be an effective breeding method in developing a locally adapted and resistant cultivar

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Table 2 – Sources of Resistance to STB and Percentage of Leaf Area Covered by Lesions Bearing Pycnidia of 12 Different Mycosphaerella graminicola isolates

Wheat line Origin of line CA30 USA

IPO001 N’LANDS

IPO323 N’LANDS

IPO87019 URUGUAY

IPO88004 ETHIOPIA

IPO89011 N’LANDS

IPO90004 MEXICO

IPO90012 MEXICO

IPO92006 PORTUGAL

IPO94269 N’LANDS

ISR398 ISRAEL

ISR8036 ISRAEL

SENAT Denmark 1 10 0 1 0 6 17 7 3 25 6 2 GENE USA 6 8 0 20 53 1 20 37 39 0 10 0 TE9111 Potugal 17 10 0 0 4 33 50 1 17 12 0 1 MILAN CIMMYT 9 - 0 10 77 1 12 19 25 9 34 29 ISRAEL493 Israel 6 48 7 21 0 12 7 6 44 16 6 30 CHAUCER UK 18 24 71 1 0 23 27 6 5 30 30 2 EQUINOX UK 3 7 65 17 22 36 24 28 61 30 28 10 ARINA Switzerland 45 18 0 37 7 8 8 82 78 44 19 6 TONIC UK 25 37 63 31 8 6 23 84 59 24 14 9 MENTANA Italy 20 24 39 49 42 5 38 47 44 3 29 41 FLAME UK 25 29 3 37 30 59 33 66 64 27 15 2 VERNOPOLIS Brazil 7 59 0 9 0 58 3 3 53 17 18 29 REAPER UK 16 3 64 22 54 41 36 48 69 34 41 9 OLAF USA 29 45 1 30 3 47 0 1 52 15 18 24 RIBAND UK 49 44 71 34 0 38 30 56 59 32 24 13 LONGBOW UK 23 34 57 27 0 38 42 72 54 35 42 24 BULGARIA88 Bulgaria 21 50 5 51 44 37 36 57 65 28 18 37 FRONTANA Brazil 35 38 53 39 71 16 37 67 69 5 35 17 CATBIRD CIMMYT 3/ 4 1 3 13 43 81 89 80 55 0 49 BALDUS Netherlands 71 51 30 44 2 63 54 78 66 55 26 9 SHAFIR Israel 18 64 2 66 95 55 58 87 59 24 44 46 CHINESE SPRING

China 38 44 9 15 96 55 90 99 57 42 16 39

KK (L6.A.4) CIMMYT 68 10 1 0 78 35 78 93 79 13 0 4 COURTOT France 59 88 81 34 76 4 89 100 79 63 66 73

Isolate mean 20 33 55 26 44 26 39 64 53 29 24 14 Grey shaded figures indicate significance for specific interactions of wheat line with M. graminicola isolates – Wheat lines in bold are those with best resistance.

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It has been reported that selection for resistance to Karnal bunt is exceptionally difficult as environmental conditions play a significant role in this pathogen’s virulence and nurseries are both expensive and difficult to maintain (Datta et al. 1999; Sukhwinder-Singh et al. 2003).

VIII. Nematodes It has been estimated that plant-parasitic nematodes account for 10% of worldwide crop loss. Cereal Cyst Nematodes are a complex group of 12 described and several undescribed species with Heterodera avenae being the most economically important. Yield losses from H. avenae vary from 15-20% in Pakistan to 40-92% in Saudi Arabia. The damage threshold is dependent on a number of variables including wheat cultivar, soil type, the pathotype and ecotype of the nematode, and climactic conditions of the geographical region. Potential damage from nematodes is increased where wheat is grown in stressful environments such as poor soil nutrition, temperature stress, water stress, or where there is pressure from other pathogens (Nicol et al. 2003). . Identification of Cereal Cyst and Lesion nematodes is difficult, and has historically been accomplished through comparative morphology. A key to the identification of nematodes can be found at: http://nematode.unl.edu/nemakey.htm and may be useful in identifying a specific nematode pest. More recently laboratory techniques including AFLP markers have been developed to identify and discriminate both cyst and lesion nematodes (Andrés et al. 2001; Mokabli et al. 2001; Orui and Mizukubo 1999; Curran 2002). A handful of Cyst resistance genes (Cre) have been identified in wheat and have been deployed to provide effective resistance in parts of the world. In Europe and North Africa, Cre1, provides good resistance against H. avenae; but has proven ineffective against those races specific to Asiatic and Australian wheat growing regions. Greater regional usability is offered by Cre2 and Cre4 which come from Aegilops spp. Table 2 contains a number of sources that could be used to introgress nematode resistance genes into a breeding population. Host resistance to nematodes is still poorly understood and collaboration between research institutions like CIMMYT and country programs will be necessary to improve and develop more reliable host resistance. Table 3 suggests that both horizontal and vertical resistance to nematodes is available in existing germplasm. If nematode resistance is a part of a program’s breeding objectives, cultivars with proven resistance should be chosen as a donor parent in a backcross strategy with a locally adapted recurrent parent. Stated earlier, nematode pressure depends on many variables and successfully screening and selecting for cultivars holding resistance genes may be difficult as nematode pressure will vary from site to site and from year to year. Establishing a disease nursery for nematode resistance selection would be helpful. In order to keep nematode populations at a maximum an understanding of the problem nematode’s preferred environment is necessary. By maintaining an optimal soil ecosystem for the nematodes, and offering them susceptible hosts (also useful as checks) season after season in the same location; a large population of nematodes will be maintained, offering better disease pressure and an opportunity for more effective selection.

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Table 3. Principal sources of genes used for breed ing resistance to Heterodera avenae in cerealsb

Cereal species Cultivar or line Origin Genetic information Remarksc Used

Wheat

Triticum aestivum Loros, AUS 10894 -, Australia 1 dominant gene, Cre1 (formerly Ccn1) on chromosome 2BL

S, India; pR to several pathotypes NW. Europe, Australia

Katyil Australia Ccn1 S, India Australia

Festiguay Australia CreF on chromosome 7L? pR in cv Molineux Australia

AUS4930 = 'Iraq 48'

Iraq ? R, to several cereal cyst pathotypes and species and Pratylenchus thomei

Australia, France, CIMMYT (under evaluation)

T. durum Psathias - ? S, to some pathotypes -

also pR

7654, 7655, Sansome,

- ? S, to some pathotypes France

Khapli also pR

Wild grass relatives

Aegilops tauschii (T. tauschii)

CPI 110813 Central Asia On chromosome 2DL, Cre4 R, Australia (Ha 13) and several other countries

Australia synthetic hexaploid lines

Ae. tauschii (T. tauschii)

AUS 18913 - 1 dominant gene on chromosome 2DL, Cre3

R, Australia (Ha13) and several other countries

Australia advanced breeding lines

T. variabilie 1 West Asia Gene Rkn-mn1 on chromosome 3U or 3Sv

R, to various pathotypes and Meloidogyne naasi and H. latipons

France, Algeria, Spain, India, Syria

T. longissimum 18 - ? R and pR to several pathotypes France (under evaluation)

T. ovatum 79 Mediterranean basin

? R and pR to several pathotypes France (under evaluation)

T. triunciale (Ae. triuncialis)

TR-353 Spain 1 dominant gene, Cre7 (formerly CreAet)

R, to several pathotypes (French, Swedish, Spanish)

Spain (under evaluation)

T. geniculata (Ae. geniculata)

? Spain, Bulgaria, ? R, to several H. avenae France, CIMMYT (under evaluation)

T. ventricosum (Ae. ventricosa)

VPM 1 Jordan, Tunisia On chromosome 2AS, Cre5 (formerly CreX)

populations and H. latipons R, to French pathotype (Ha12)

France, Australia (under evaluation)

T. ventricosum (Ae. ventricosa)

11, AP-1, H-93-8 Mediterranean basin

On genome Nv, Cre2 R, to Spanish, French and UK (Ha11) pathotypes

Spain (under evaluation)

T. ventricosum (Ae. ventricosa)

11, AP-1, H-93-8, H-93-35

Mediterranean basin

1 dominant gene on chromosome 5Nv, Cre6

R, to Australian pathotype (Ha13), not effective against Spanish (Ha71)

Spain, Australia (under evaluation)

b Information unavailable from reference = -; no published scientific studies conducted =? c R = resistant; pR = partially resistant; S = susceptible

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Questions to Test Understanding: 1. Describe symptoms of bacterial diseases.

2. What are R-genes and Avr-genes?

3. How is a plant defense mechanism triggered and how does it operate?

4. What strategies do breeders use to increase the durability of their resistant

cultivars?

5. What strategy is CIMMYT using to develop Ug99 resistant cultivars?

6. What are the challenges for selecting for resistance to Septoria Tritici

Blotch (STB)?

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