Goals of USDA-ARS Strategic Action Plan Built on understanding stem rust epidemiology • Stem Rust Assessment and Pathology • Detection and Identification • Monitoring and Reporting • Germplasm Enhancement, Gene Discovery, Development of Molecular Markers • Regional Variety Development, Evaluation and Implementation • Disease Management • Communication and Outreach Another way to breed durably resistant wheat varieties to stem rust is to accumulate many minor genes for resistance, making it difficult for the pathogen to overcome all the small effects (CIMMYT approach). Also important can be how resistant varieties are used in the field. Mixtures or blends of varieties can dilute the amount of spores and serve as partial barriers to disease spread. Designation Pedigree Sr Gene Combination ARS04-1267 KS98HW151-6/KS00HW120//NC94-4680 22, 38, 1A.1R ARS05-0005 TX85-264*2/TTCC512 36, 1A.1R ARS05-0403 TX98D1170*2/TTCC365 24, 36, 38 ARS05-0456 Neuse/TX98D2334 36, 38, (+Lr34) ARS05-1034 KS2016/Trego 38, 1A.1R ARS07-0050 WX98D022-U44/TX98D1519 36 (+Lr34) ARS07-0116 AR93035-4-2/PI564341//TX85-264 36, 38, 1A.1R, (+Lr34) Pyramiding Stem Rust Genes Theoretically, durability of a resistance gene is a function of the amount of fitness penalty imposed on pathogen. So, if avirulence genes in the stem rust pathogen affect the pathogen’s fitness differentially, then mutations in them should differentially affect fitness, and resistance genes in wheat corresponding to them should be differentially durable. Those avirulence genes with biggest effect on fitness may correspond to those resistance gene with greatest durability. So, which resistance genes that are pyramided may be at least as important as whether and how many resistance genes are pyramided. Pyramids are especially effective if pyramided genes have different modes of action (low potential for cross-resistance). Breeding for Stem Rust Resistance • Resistance breeding (regardless of type of resistance) is focused also on reducing pathogen fitness (the combined ability of an organism to survive and reproduce). • Pathogen fitness is quantifiable: Reproductive rate; Infection efficiency; Aggressiveness (amount of disease caused - Disease severity and AUDPC); Frequency of a strain relative to other strains can be used as an estimator of fitness. How Resistance is Expressed • Resistance is a relative trait. It is the restriction of development of a pathogen, that may vary in degree from immunity (no development) to only slight impeding or delay of the pathogen (relative to a susceptible reaction). Ways to Minimize Wheat Stem Rust from Thriving • Development and deployment of resistant varieties (reduce rate and amount of pathogen build-up); all-stage resistance and adult-plant resistance; gene pyramids; avoid high-effect single gene resistance. • Diversification of resistance - gene deployment; varietal diversification; blends or mixtures • Reduce or eliminate overwintering and oversummering. • Eliminating susceptible alternate host. • Fungicides to reduce rust population size and protect yield of susceptible varieties. Ways to Minimize Wheat Stem Rust from Surviving • Elimination of Highly Susceptible Varieties • Disease escape • Rust Monitoring - surveillance plots; race identification and surveys • Organized communication between farmers, Extension personnel, crop advisors, and researchers; rapid response • Fungicides – If caught early and limited occurrence Ways to Minimize Wheat Stem Rust from Arriving • Reduce global population size of rust • Regulatory methods – Import/Export restrictions on pathogen cultures and plant material • Traveler Advisories • International Cooperation • Unified Concept of Pathogen Movement and Disease Development • Regardless of the type of organism, successful invaders must negotiate a sequence of events that includes arriving, surviving, and thriving in a new environment. • In order to manage wheat stem rust, we must minimize or eliminate the pathogen from arriving, surviving and thriving. • Factors that Promote Spread of Stem Rust • Susceptible Wheat Varieties • Continuous host in space and time • Conducive Environment • Moisture on plants (dew, 6-8 hours) • Uredospore Germination (2-300C) • Uredia Sporulation (5-400C) • Virulent Rust Races • Large Population Size

Stem rust race analysis and nomenclature Z.A. Pretorius Department of Plant Sciences University of the Free State South Africa Landmark studies Stakman, EC & Piemeisel, FJ. 1917. Biologic forms of Puccinia graminis on cereals and grasses. J. Agric. Res. 10: 429-495. Stakman, EC, Piemeisel, FJ & Levine, MN. 1918. Plasticity of biologic forms of Puccinia graminis. J. Agric. Res. 15: 221-250. Stakman, EC & Levine, MN. 1922. The determination of biologic forms of Puccinia graminis on Triticum species. Minn. Agric. Res. St. Bull. 8. Stakman, EC, Stewart, DM & Loegering, WG. 1962. Identification of physiologic races of Puccinia graminis var. tritici. USDA-ARS E617. Why race analysis? (McIntosh et al., 1995) To determine the range of pathogenic variation in a region To screen for resistance in cultivars To confirm that altered host responses are due to race changes To build a collection of races for use in research and breeding To understand the mechanisms of variation Other labs are doing it Pathogenic variation in a region Pathogenicity Virulence profile (avirulent for Sr5,6,8b / virulent for Sr9e,17,31) Occurrence and distribution Which races occur where and at what frequency Screening for resistance Cultivars Commercial varieties Seedling stage Postulation of Sr genes Adult plant resistance Need to knock out seedling genes Breeding lines Genetic studies Altered host response Confirm that atypical host reponse (e.g. susceptibility) is the result of pathogenic adaptation Altered host response Confirm that atypical host reponse (e.g. susceptibility) is the result of pathogenic adaptation Culture collection Maintain a viable collection of important rust cultures Use in screening and research Host resistance Relationships among isolates Inheritance studies Mechanisms of variation Recombination Completion of the sexual cycle Mechanisms of variation Recombination Completion of the sexual cycle Mutation TTKSK TTKST (mutated for Sr24 virulence) TTKSK TTTSK (mutated for Sr36 virulence) Mechanisms of variation Recombination Completion of the sexual cycle Mutation TTKSK TTKST (mutated for Sr24 virulence) TTKSK TTTSK (mutated for Sr36 virulence) Migration Detection of TTKSF in South Africa Migrated from East Africa Probability of variation events Race analysis terminology The pathogen Biologic form Physiologic race Race Biotype Strain Pathotype Variant The host Differentiating line Differential Tester line NIL Single gene line The process Monitoring Surveying Surveillance Tracking Typing Analysis Characterization Pathogenic variability Terminology Host pathogen interaction Basic compatibility Compatible (= susceptible interaction) Incompatible (= resistant interaction) Host phenotype Reaction / Response Low reaction (= resistant) High reaction (=susceptible) Pathogen phenotype Pathogenicity Low pathogenicity (=avirulence) High pathogenicity (=virulence) Host-pathogen interactions (McIntosh et al., 1995) Gene-for-gene interaction Incompatiblity between host and pathogen involves corresponding genes in each organism Consequence of at least one host resistance gene and one pathogen avirulence gene (LIT = LR:LP) Host-pathogen interactions (Brown & Ogle, 1997) Collecting rust cultures Regular surveys Commercial fields Variety trials Trap nurseries Volunteer plants Considerations Main and off-seasons Co-worker involvement Regular stops All major production areas Neighbouring countries Cost per collection Handling field samples during survey Collect 3-5 leaves or stems Paper or glassine envelopes Fold leaves neatly Avoid wet samples, plastic bags, high temperatures Record site,crop and disease information as accurately as possible Record keeping Excel spreadsheet Accession number (e.g. Pgt_2009_01) Collection date Location GPS coordinates Host Disease severity Date processed Race Reference isolate no. (e.g. Pgt_2009_01/2) Storage position and date Back-up data Processing field samples Store samples in cool, dry place Excess moisture will cause moulds Process within 3 mo. of collection Processing field samples Collect spores with cyclone collector connected to vacuumpump Spray onto seedlings (one pot per collection) using the inter-changeable collector / inoculator and air vacuum / pressure system Flush inoculation booth between isolates Always protect uninoculated plants against background contamination Always use sterile pipette tips for adding oil to each sample Processing field samples Increase Pgt on McNair 701 or Morocco seedlings Plant McNair on weekly basis during survey season Add maleic hydrazide to increase pots (0.3 g / L; 50 mL/pot at emergence; prepare and store MH properly) Inoculation equipment and procedures Incubation specifications Allow oil to evaporate completely Stripe rust: 5-12°C, 24-48 h Mist lightly with distilled H20 before incubation Stem rust: 18-25°C, 12-24 h Expose seedlings to some light during incubation Leaf rust: 10-25°C, 12-16 h Do not “overdew”, i.e. we don’t want runoff Dew chamber options Moist plastic bags  Condensation chamber (several designs) Ultrasonic humidifier  Mist plants and place in closed container Working with rust pathogens Differentiating lines and systems  Composition of sets  Growing differentials (seedlings) Single-pustule vs bulk isolates  Inoculating differential sets  Infection types Temperature specificity  Rating infection type patterns  Avirulence / virulence formulae  Race nomenclature systems  Long sets Inoculation of differential sets  Field samples have been increased on susceptible seedlings  Decide to inoculate bulk spore sample on differential set, or make single pustule isolates Establishing single-pustule isolates  Collect with cyclone collector directly from leaf tips if possible  Alternatively, trim leaves down to one pustule only  Incubate in dew chamber overnight  To germinate contaminant spores  Place in isolation cubicle  Collect spores 2-3 days later  Increase on suscept, or inoculate differential set directly Stem rust differential set  Use international set plus own differentials International set Mini sets  Decide on ~6 critical Sr genes for detecting Ug99 or any other race in your region (e.g. Sr9e,24,31,36,38,C)  Plant only one pot and inoculate with field sample Inoculating differential sets  Pgt single uredinium usually requires one cycle of spore multiplication before inculating sets  Inoculate when majority of seedlings are at the one and a half leaf stage (7-10 days after planting)  Remember fertilization and keep uninoculated plants free from background contamination Infection types (Knott, 1989) Infection types  Where are the cut-off points for resistance and susceptibility?  Resistance: 0 to 2  Susceptibility: 3 to 4 Leaf surface  Leaf and stripe rust: rate pustules on upper surface  Stem rust: lower surface Seed of differential lines  How do we keep lines pure?  Single plant selections  Head descriptions  Agronomic and harvesting practices  DNA fingerprinting  Mass production Growing differentials  Number differentials and always maintain same order  Plant 4-7 genotypes per 9-cm diam pot  Plant 5-10 seeds per differential  Fill pots with potting medium to same level  Water from below if soil is hydrophilic  Add some water from above if soil mix is hydrophobic  Sow seeds in defined clumps Cover with thin and uniform layer of soil or vermiculite; compact slightly Continue with sub-irrigation Fertilize at least twice, starting two days after emergence e.g. Chemicult™, Multifeed ™ Temperature specificity R gene expression is influenced by temperature Some low ITs expressed at high temperatures e.g. Lr13 Some low ITs expressed at low temperatures e.g. Lr18, Lr34, Sr15 Gene-for-gene-for-environment interaction Race nomenclature systems Stakman standard races (Pgt) Avirulence / virulence combinations plus arbitrary pathotype no. North American system Decanary system Avirulence/virulence formulae Isolate 1: Sr5, 9e, 24 / 36, 31 Isolate 2: Sr9e, 24 / 5, 36, 31 Isolate 3: Sr36 / 5, 9e, 24, 31 North American system Jin, Y., Szabo, L., Pretorius, Z.A., Singh, R.P. & Fetch, T. 2008. Detection of virulence to Sr24 within race TTKS of Puccinia graminis f. sp. tritici. Plant Disease 92(6): 923-926. Pathogenicity H (High) = Susceptible IT 3-4 (race is virulent for Sr gene / Sr gene is ineffective) L (Low) = Resistant IT 0-2 (isolate is avirulent for Sr gene / Sr gene is effective) Long sets  Inoculate “long sets”, consisting of all other numbered genes as well important tester lines and cultivars, with bulk samples and individual pathotypes  To detect virulences not revealed by differentials Working with rust pathogens  Storing type cultures  Retrieving cultures from storage  Heat shock  Rehydration  Testing germination Storing type cultures  Increase on selective host  Harvest spores  Dry over silica gel or glycerol in dessicator for 2-3 days  Store in cryovials at -70°C, or in glass vials or aluminium foil sachets in liquid nitrogen Retrieving cultures from storage  Place vial in water at ~45° for 6 min  Open vial, place in beaker filled with wet vermiculite, and cover with cling wrap for 2-3 h prior to inoculation  Not necessary to heat shock Pst Testing germination  Place suspension droplets on water agar  Incubate in dark at ~20°C for 3 h  Determine germination percentage microscopically (stereo, with light from below) Working with rust pathogens  Handling contaminants  Retest if unsure  Sub-culture single pustule isolates  Increase on selective host  Run differentials Resources  Human  Experienced staff  Financial  Staff, surveys, infrastructure, operational costs  Physical Resources  Physical  General laboratory  Greenhouses  Temperature control  Pre-inoculation facility  Pots and potting medium  Irrigation  Nutrition  Water bath  -70 C freezer  Inoculation facility  Inoculation accessories (collectors, inoculators, capsules, oil, Tween, dispensette, pipettes, Mcartney bottles, etc)  Dew chamber (leaf and stem vs stripe rust)  Germ plasm collection and its management  Increase of seed and maintaining purity Digital photography  Avoid direct sunlight  Use boot of car for field pics  Avoid direct flash  Use natural light  Experiment!  Pick contrasting background  Avoid background shadows  Tape leaves meticulously  Use optical zoom Pitfalls  Lack of experience  Lack of attention to detail  Poor technique and quality control  Mixed / wrong seed sources  Lack of key genotypes  Mixed cultures  Contamination with background inoculum  Escapes / failures  Lack of replication / confirmation  Infrastructure failures  Environmental variation  Powdery mildew  Hyperparasites Questions and Answers

Stem rust –Ug99 Stem rust ● Stem rust was and still continues to be one of the most feared diseases of wheat. ● Several references dating back to Biblical times relate to epidemics of cereal rusts and smut inflicted upon the Israelites as punishment for their sins (Chester, 1946). ● Fragments of stem rust-infected wheat from the Bronze Age have been discovered in Israel (Kislev, 1982). ● Numa Pompilius (715-672 BC) described the Roman festival of “Robigalia” that was established to protect cereal crops. ● “Black rust”- caused by Puccinia graminis tritici ● Linear relationship in grain yield losses ● High disease severity-100%losss ● Under control in most regions except East Africa since Green Revolution Detection of Race Ug99 of P. graminis in Uganda during 1999 ● First report of virulence for Sr31 and Sr38 (Pretorius et al. 2000 Plant Dis. 84:203) ● Stem rust did not appear in Uganda during the following years ● Presence in Kenya realized in 2002 although Kenyan data indicate that it may have existed since 1999 ● Detected in Ethiopia in 2003 ● Most leading cultivars and breeding lines became susceptible to Ug99

Cereal Rust Surveillance & Monitoring Systems - GPS and Survey Tools Dave Hodson, AGP Division, FAO Njoro, Kenya Sept 2009 Outline of Session  Introduction – Surveillance & monitoring – GPS & tools  GPS Practical  Using data / information tools Ug99 – A call to action Challenges for a rust monitoring system  Highly mobile, trans-boundary pathogens  Need to monitor huge areas  Irregular outbreaks  Changing nature of pathogen (5 known variants in Ug99 lineage)  Timely field surveys + effective data flows, data analysis & management  Timely & targeted information dissemination  Where to start? Model Systems? FAO Desert Locust network Potential Outline – Rust Monitoring System Survey / Sampling – Tools & Protocols  BGRI protocols manual – Field survey forms – GPS protocols – Sampling protocols – “Quick Sets” – race analysis  R. Park (Uni. Sydney)  K. Nazari (ICARDA)  A. Yahyaoui (ICARDA)  Z. Pretorius (Uni. Free State)  K. Cressman (FAO)  T. Fetch (Ag. Agri-Food Canada)  Yue Jin (USDA-ARS)  D. Hodson (CIMMYT) Field Survey Procedure  At survey location, switch on GPS  Wait until GPS receives satellite signals  Record location with GPS – “Mark a Waypoint”  Fill location (latitude and longitude) and elevation details on survey form  Survey for (stem) rust, fill in survey form  Collect sample (s)  Switch off GPS  Move to next survey location – approx. 20km away from 1st location  Repeat GPS DATA Using GPS – 1,2,3  1. Switch On 2. Receive satellite signals (4 satellites) “Ready to Navigate” 3. Record Location “Mark a Waypoint” Survey Forms  Developed at GRI workshop, Ethiopia Oct 2007  Field tested, refined and simplified  GPS location a key component! Continued Expansion of Survey Network On-line Information Tools  RustMapper (Google Earth/web-browser)  Near real-time: sites, winds, wheat, susceptibility  http://www.cimmyt.org/gis/rustmapper/  http://www.cimmyt.org/gis/rustmapper/RustMapper_Web.html Race Analysis  A bottle-neck  Reliance on North American labs – (low volume, seasonality issues)  Substantial on-going efforts to increase capacity in regions New Information Tools  Forthcoming: Rust SPORE – web information portal – smart pdf maps – pathotype tools  Technical collaboration: University of Aarhus, Denmark – Looking at common platform / tools – Pathotype Tracker? Will Stem Rust Keep Following the “Rules”?  No room for complacency  A need for monitoring & surveillance in “low risk” areas Risk of Accidental Transfer (the “747 route”) Long Distance Dispersal – “Rare Events” Conclusion  Ug99 lineage – a wake up call against complacency  Clear need for global monitoring & surveillance (Including in “low” risk areas)  Elements of a global monitoring system being put in place by an international coalition  An effective network is essential  Simple survey tools (GPS & forms) provide vital foundation information

Cereal Rust Surveillance & Monitoring Systems - Using GPS Data Dave Hodson, AGP Division, FAO Njoro, Kenya Sept 2009 Overview  International Data Handling – Tools – Information Products  National Data Handling – Tools & Methods International Information Tools  RustMapper & RustMapper Web – Google Earth platform – Survey sites, winds, wheat – Updated automatically every 5 days – Needs internet  Smart PDF – Lite, simple – Web browser or Acrobat v9  Others... National: Using GPS Data  Several simple free tools – Data capture, mapping  1. Google Earth – Direct GPS connect & map  2. DNR Garmin – Data download, file conversion  3. DIVA-GIS – Data import, mapping Google Earth - GPS  Mapping / Visualization – Connect GPS via data cable – Switch on GPS – Open Google Earth  Tools  GPS  Garmin  Import Note: Works off-line (uses cached images) DNR Garmin  Data download and file conversion – Free download http://www.dnr.state.mn.us/mis/gis/tools/arcview/extensions/DNRGarmin/DNRGarmin.html – Connect GPS via data cable – First use: Set projection – None (WGS 84, Lat / long) – Download – Waypoint or Track – Save File – SHP (unprojected) [for use in DIVA-GIS or other GIS] or direct to Google Earth DIVA-GIS  Mapping, Analysis, Data Import – Free GIS software (and data) – http://www.diva-gis.org – 1. SHP file (from DNR Garmin) – add layer (s) – 2. Text file with Lat/long (csv, tab deliminated...) [NB: Use simple headers!]  Data  Import points to shapefile  From txt – Create Maps

GENETICS OF RUST RESISTANCE GENETICS OF RUST RESISTANCE  Basic compatibility  What do we mean by resistance?  Resistance and susceptibility are relative terms Seedling Responses to Stem Rust - seedlings Phenotypes  What you see depends upon the genotype of the host plant AND the genotype of the pathogen isolate, race or pathotype  The gene-for-gene relationship Needs  For breeders to use resistance it helps to know something about the resistance phenotype and how it is inherited - how many genes - are they dominant - do they interact - do they work against all races - How much protection do they provide Types of Resistance  Seedling or all-stage resistance – characterized by hypersensitivity  Adult plant, post seedling, APR - Hypersensitive or small ‘susceptible’ pustules – slow rusting Studying Resistance  What is the difference between genetics and breeding? Genetics of Resistance - 1  Types of crosses - R x S or S x R - R x R – tests of allelism  What generations to study - F1, F2, F3,.... - BC, TC F1 or F2  Homozygous lines – DH, SSD, Sub.  Others?? Genetics of Resistance - 2  No. of individuals or lines  No. of individuals within lines  What pathotypes?  Should pathogen cultures be absolutely pure? Interpreting Genetic Data  Where to partition between R & S  Hypothesis making  Testing the hypothesis  Validation of the hypothesis – progeny testing; 3 : 1 becomes 1 : 2 :1 – larger populations – more crosses Independent Segregation at Two Loci With selfing - by phenotype - 15:1 or 9:3:3:1 - by genotype - 15:1 or 1:2:1:2:4:2:1:2:1 With testcrossing – by phenotype - 3:1 or 1:1:1:1 - by genotype - 3:1 or 1:1:1:1 If low infection types conferred by two genes are different then genetic ratios can be subdivided Location on Genes on Chromosomes  Aneuploid analysis - monosomic analysis  Association with markers - placement of markers on chromosomes using deletion stocks - nullisomic-tetrasomics - ditelosomics - deletion stocks Linkage of Two Genes on One Chromosome  Two genes located on one chromosome can be independently inherited. Why?  As two genes become closer together on a single chromosome they tend to be associated or linked in coupling or in repulsion  Crossing-over in meiocytes occurs at the 4-strand stage – the number recombinant gametes will not exceed 50%, i.e. independent segregation Association Mapping - 1  If you know the DNA sequence of a gene, then a cloned sequence of the gene can be used as a perfect or functional marker  If you have a DNA sequence that is close to a gene of interest, that sequence can be used to select the gene with an accuracy that depends on its closeness Association Mapping - 2  If lines are related, a gene marker association is likely to occur between the relatives  If gene marker relationships hold between relatives then it is possible they will hold across lines that are not known to be related  Gene marker associations therefore can be used to screen germplasm collections as a means of gene identification  The accuracy of prediction will depend on the closeness of linkage Qualitative and Quantitative Inheritance  QTL analysis involves the correlation of components of trait variation with DNA markers whose locations in the genome are known or can be determined by using deletion stocks Using Genetic Information in Breeding Programs  Identifying gene combinations may not be possible due to masking effects  It helps to know what genes are combined or are being combined  Predicting population sizes needed to achieve breeding targets  Use in pre-breeding  Availability of special lines for breeding-related research  Molecular breeding Using Genetic Information in Breeding for Resistance  Single major genes should be avoided  Combining major genes (gene pyramiding or gene stacking) – aided by markers  Minor genes or QTLs can be combined together in known combinations – aided by markers  Combinations of major and minor genes made possible by markers Final Question  Is rust control about protecting crops from loss of yield, or about reducing the amount of inoculum to infect future crops?

Crop calender “Green Bridge” Yr27 Pst pt; A New Threat to wheat production in Asia  1998 sever stem rust infections occurred in Uganda with virulence for Sr31+ Sr38  A new race identified and designated as TTKS (Ug99)  2001-03 Subsequently detected in Kenya and Ethiopia  2006 Ug99 was recorded in Sudan (early 2006) and later same year in Yemen  2006 virulence for Sr24 detected in Kenya  2007 Ug99 was confirmed in Iran  2007-08 Ug99+vir Sr24 and vir Sr36 in Kenya  2009 Ug99 was detected for new areas in Yemen and Iran  CWANA 42 m. ha  Subcontinent 36 m. ha Total 78 m. ha  Estimated wheat production 170m. t. Modest 10% crop loss= Approx. losses could reach up to 17 m.t Genetic basis of Resistance Pathogenic variability  Seedling  Race analysis  Adult-plant  Biological trap plots Advanced rust labs for race analysis  Yellow rust  1956-1992 IPO, in Wageningen ▪ 5000 samples from 60 countries Stubbs, 1988  Aarhus University, Denmark  Stem rust  The International Virulence Gene Survey for Puccinia graminis (1969-1971) ▪ Prof. I.A. Watson, The University of Sydney, Distributing seed of 120 lines and cultivars to 19 rust laboratories ▪ Cereal Disease Laboratory, USDA-ARS, University of Minnesota ▪ Cereal Research Center, AAFC, Winnipeg, CANADA Stem rust race analysis regional laboratories  Plant Breeding Institute Cobbitty, Australia  Seed and Plant Improvement Institute  ICARDA  CIMMYT  India  Ethiopia  Turkey  Pakistan Potential labs  Egypt  Kenya  Uzbekistan Pathogenic variation: Physiologic specialization • International system (Stem Rust) Developed by Stakman and Piemeisel (1917), revised in 1962  Used 12 differential host cultivars representing three ploidy levels  12 cultivars were used worldwide until 1950  The race analysis is based on seedling resistant reaction  Resistant reaction: (0, 0;, 1,2, X)  Susceptible reaction 3 and 4  2. Modified potato- Phy. infestans system  It was introduced in Australia and New Zealand  The system uses selected Int. differential cultivars  An ordered set of differential with sequential numbers 3. Coded system (Stem Rust) In the United States (Roelfs et al., 1982):  Three sets of four single gene host line First set: resistances in the international diff. set (Important in differentiating races of sexual population of Pgt in the USA) Other sets: Second and third sets have resistance that become important for breeding program  Tracking Ug99 and Rust Surveillance  Race analysis & seedling assessments of Sr-genes ▪ Seed and Plant Improvement Institute (SPII, Karaj, Iran)  Tracking Ug99 and Rust Surveillance  Race analysis & seedling assessments of Sr-genes ▪ ICARDA, Aleppo, Syria  Tracking Ug99 and Rust Surveillance  Race analysis & seedling assessments of Sr-genes ▪ Turkey. Central Research Institute for Field Crops  Full race analysis  Seedling and adult-plant assessments (local races)  Tracking Ug99 and Rust Surveillance  Race analysis & seedling assessments of Sr-genes ▪ Ethiopia. Ambo Plant Protection Research Center  Full race analysis  Seedling resistance screening  Adult-plant assessments (Ug99)  Tracking Ug99 and Rust Surveillance  Race analysis & seedling assessments of Sr-genes ▪ Egypt  Full race analysis  Seedling and adult-plant assessments (local races)  Tracking Ug99 and Rust Surveillance  Race analysis & seedling assessments of Sr-genes ▪ Pakistan  Full race analysis  Seedling and adult-plant assessments (local races) ▪ Georgia  Full race analysis  Seedling and adult-plant assessments (local races) ▪ Kazakhstan  Full race analysis  Seedling and adult-plant assessments (local races) ▪ Potential lab  Uzbekistan Pathogenic variation Biological Trap Plots  The International Yellow rust Trials Project. The initiative was taken by the Committee for Yellow Rust Research in 1955.  This project was designed to collect information on:  Epidemiology of yellow rust  The physiologic specialization of the yellow rust  The behavior of resistant and susceptible varieties tested under different environmental conditions (Zadoks, 1961) Pathogenic variation, Biological Trap Plots IRN Early 1950’s (USDA) RDTN Early 1970’s (CIMMYT) IDTN Late 1970’s (Ford Foundation, CIMMYT) CIMMYT Cereal Disease Monitoring (1970’s) Compositions of ICARDA Rust Trap Nurseries  IYRTN # 50 entries  Differential cultivars  Avocet Near Isogenic Lines  Known cultivars for adult-plant resistance genes  Commercial cultivars in CWANA region  ISRTN # 65  Monogenic lines for Sr-genes  Different sources of key Sr-genes for Ug99  Commercial cultivars (BW and DW)  Ug99 Trap Nursery # 25  Key Sr-genes, Commercial cultivars Simple approache  Each entry in two rows (1 meter)  Natural infection= No artificial inoculations Iran, Turkey, Ethiopia, …  Modified Cobb’s Scale  Severity (0-100)  Reactions (R, MR, MS, S)  Consistency  Time of scoring?? GPS Stem rust differentials, field differentiations Testing sites of ISRTN and Ug99, 2009  Field- based pathogenicity surveys  Yellow rust ▪ 2nd International Yellow rust Trap Nursery ▪ Yellow rust differential cultivars ▪ Yellow rust Avocet Near Isogenic Lines (NILs) ▪ Known cultivars for APR genes ▪ Commercial cultivars in CWANA  76 location in 28 countries ▪ Virulence for Yr2, Yr6, Yr7, YrA and Yr31 is fixed in all locations. ▪ Yr5 and Yr15 are effective in all locations ▪ Virulence for Yr9 and Yr25 was very common ▪ Virulence on Yr1, Yr8, Yr10. Yr17, Yr18, Yr24, and Yr27 was varied ▪ Frequency and distribution of virulence forYr27 is increasing  Field- based pathogenicity surveys  Stem Rust example RTN08.xlsx ▪ 4th International Stem Rust Trap Nursery & ▪ 2nd International Ug99 Stem Rust Trap Nursery ▪ Stem rust monogenic lines ▪ Known cultivars for APR gene/s ▪ Commercial cultivars in CWANA  In 73 location in 24 countries ▪ Drought conditions in most of the areas ▪ Kenya, Ethiopia, Yemen, Sudan, Iran*, Pakistan*, Georgia ▪ High infection on Sr31 in Kenya, Ethiopia, Yemen and Sudan ▪ High infection on Sr24 only in Kenya. ▪ HI for Sr24, Sr25 in Sudan and Yemen (Winter type/ del length sensitivity) ▪ HI on Sr36, Kenya (Ug99) other locations (local population) Use of Rust Trap Nurseries  The use of trap nurseries would provide:  Assessment of pathogenic variation in where there is no laboratory facilities and expertise  Assessment of race-specific seedling resistance genes  valuable information on field responses of APR genes  Interaction of resistance genes/ resistance sources to environmental conditions in different high yielding background cultivars  Preliminary information on pathogen change  National program will generate annual information from various locations within each country  Annual meeting are organized and information is presented by national program coordinator  Current changes: Seed distributions & multiplications of Sr differentials  Large scale seed multiplication (4 X 4) ▪ Sr -monogenic lines-ICARDA source (45) ▪ Canada, Manitoba, NA differentials (14 + 20) ▪ Yellow rust differentials (17) ▪ Yellow rust Near Isogenic Lines (19) ▪ Leaf rust isogenic lines (39)  Australian Differential sets (2 X 2) ▪ Yr differentials (25) ▪ Yr NILs (19) ▪ Lr- differential set ▪ Sr- differential set  North American Sr- differentials (2 X 2) ▪ Monogenic lines, CANADA (45 lines) ▪ North American System (20) Monogenic lines Sr14, T. Fetch 2009  Capacity building: priorities and requirements?  Mechanism of standardizing protocols  Upgrading facilities: priorities and requirements?  How to re-activate and link regional and international rust networks?  Exchange information?  Financial supports to national, regional & international rust activities? International Collaboration is the key to success

Methodologies to Screen Wheat for Resistance to Rusts Sridhar Bhavani, Ravi Singh and Davinder Singh Greenhouse Evaluation of Seedlings Greenhouse Evaluation of Seedlings  Race-identification  Race-specific genes identification  Inoculation of 8-10 days old seedlings  Infection type characterization 10-14 days after inoculation Planting in the Greenhouse Planting of Segregating Lines in the Greenhouse Increasing Pure Rust Isolates in the Greenhouse Increasing Rust Inoculum in the Greenhouse Collection of Spores in the Field Storage of rust inoculum  Normal refrigeration of vacuumed dried spores  Ultra-freezing at about –50 C  Freezing in liquid nitrogen Storage of Rust Inoculum Greenhouse Evaluation in Seedlings Greenhouse Screening of Adult-plants  Can identify both race-specific and slow rusting type of resistance  Inoculation of newly emerged flag leaves or stem  Characterization of infection type or slow rusting components Mechanisms (Components) of Slow Rusting Resistance  Longer latent period  Reduced infection frequency  Smaller uredinia or smaller infection area  Reduced spore production Result in: Slow disease progress in the field Field Screening  Creating proper disease pressure Natural vs. artificial epidemics  Spreader rows Universally susceptible cultivars Differentially susceptible cultivars Methods of Rust Inoculation  Injecting by syringe Spore-Water-Tween 20 suspension  Spraying Spore-Water-Tween 20 suspension Spore-Light weight nonphytotoxic mineral oil suspension  Dusting Spore-Talcum powder or Wheat flour mixture Injecting with syringe Spraying with ultra-low volume sprayer Spraying with hand held atomizer Dusting with a cloth bag Dusting with hand held duster Dew formation: necessity for success Planting Spreader Rows for a Uniform Rust Pressure

Major Genes For Stem Rust Resistance Harbans Bariana Australian Cereal Rust Control Program – Since 1975 Diversity – Regional Acknowledgements Past and present staff and students of the Rust Group, the University of Sydney Plant Breeding Institute Major genes – Why? Current global diversity Identification of new sources of resistance Role of markers Commitment to the cause of breeding for resistance Major Gene Resistance The Driver for Durability Low or no inoculum Grow resistant cultivars Monitor pathogen populations and withdraw susceptible cultivars quickly from commercial cultivation, for example Sr36 in Australia Control over-summering Stem Rust Resistance – Australia 1930s - Sr2, Sr6, Sr17, Sr30 1940s - Sr11, Sr9b, Sr12 1950s - Sr11, Sr9b, Sr17 1960s - Sr2, Sr5, Sr8a, Sr9b, Sr9g, Sr11, Sr12, Sr17,Sr26 Sr30 Current – Sr2, Sr8b, Sr13, Sr15, Sr9e+Sr36, Sr22, Sr24, Sr26, Sr30, Sr33, Sr36, Sr38, Sr45 (These are genes that effective against predominant pathotypes in combinations) Diversity – Regional • Northern region Sr2 + Sr30, Sr2+Sr38 +Sr30, Sr38+Sr36, Sr38+Sr26, Sr24+Sr36 (Durum – Sr9e, Sr8b, Sr13) • Southern region Sr30, Sr24, Sr15, Sr38 (Durum – Sr13) • Western region Sr2+Sr30, Sr30, Sr24, Sr13 and Sr8a+Sr15 Stem Rust Resistance – Global International Nurseries - Sr2, Sr8a, Sr9g, Sr17, Sr23, Sr30, Sr31, Sr25, Sr24, Sr33, Sr36, Sr45, SrTmp, uncharacterised, APR European cultivars - Sr5, Sr8a, Sr8b, Sr9g, Sr12, Sr30, Sr31, Sr36 and Sr38, Sr29 (possibly present in some), APR Durum nurseries - Sr9e, Sr8b, Sr13 Synthetics – Sr9e, Sr8b, Sr13, Sr33, Sr45, Sr46, Uncharacterised Stem Rust Resistance – New Sources Watkin’s collection Two putatively new seedling resistance genes and some potentially new APR genes (non Sr2) Swiss cultivar Arina APR gene on chr. 5B Synthetics plus international nurseries Some sources may have either gene combinations or new sources of resistance Stem Rust Resistance – Markers Seedling Markers are vailable for Sr22, Sr24, Sr31, Sr32, Sr33, Sr36, Sr26, Sr39, Sr40, Sr45, Sr46, SrR Adult Plant Resistance Sr2 In winter wheat Forno Lr34 provides moderate level of protection and marker is available Thatcher-derivatives with Lr34 provide high levels of resistance Current and Near Future Targets – Major genes Pyramiding of Sr2, Sr13, Sr22, Sr26, Sr24, Sr33, Sr36, Sr39, Sr40, Sr45 and Sr46 in different combinations for stem rust resistance Care should be taken not to loose stripe rust and leaf rust resistance Choice of parents should include Lr34/Yr18 and Lr46/Yr29 and major genes that effective predominant pathotypes in the target regions Breeding Methodologies Parent building/ Pre-breeding – Backcross for major genes and single backcross followed by recurrent selection Breeding – depends on sources of resistance and/or agronomic quality of parents Major genes should be used in combinations Marker assisted selection should be applied to develop gene combinations for multiple diseases As more markers for APR genes become available, combinations of major and minor genes can be created Care should be taken not to loose stripe rust and leaf rust resistance Choice of parents should include Lr34/Yr18 and Lr46/Yr29 and major genes that effective predominant pathotypes in the target regions Never forget that breeding for rust resistance is a journey not a destination

Standardization of Stem Rust Note Taking and Evaluation of Germplasm Training – KARI-Njoro September 26-October 7, 2009 Breeding for Stem Rust Resistance in Durum Wheat CIMMYT Durum Germplasm worldwide Historical impact and current responsibility CIMMYT’s Breeding Approach Centralized program, with “eyes” worldwide CIMMYT’s Breeding Approach General selection scheme CIMMYT’s Breeding Approach Shuttle Breeding, key to wide adaptation Continued Progress in Yield Potential Worldwide – Analysis of 22 years of International trials Limitations of activities in Mexico Need to work elsewhere through collaboration with NARS Breeding for Stem Rust Resistance Durum-specific situation and challenges Breeding for Stem Rust Resistance Ethiopia as primary selection site Breeding for Stem Rust Resistance Not much resistance to start with… but enough to work Breeding for Stem Rust Resistance Three-approach strategy… Breeding for Stem Rust Resistance Inter-crossing/crossing available elite lines Breeding for Stem Rust Resistance Inter-crossing/crossing available elite lines Breeding for Stem Rust Resistance Crossing Ethiopian sources of resistance Breeding for Stem Rust Resistance Crossing Ethiopian/landrace sources of resistance Breeding for Stem Rust Resistance Pyramiding resistance genes from bread wheat with markers Molecular Markers Tags attached to a gene or trait of interest Molecular Markers How they are used at CIMMYT Molecular Markers How many are used at CIMMYT? Breeding for Stem Rust Resistance Which marked gene to use in durum wheat? Breeding for Stem Rust Resistance Currently used marked genes Breeding for Stem Rust Resistance Pyramiding resistance genes from bread wheat with markers Breeding for Stem Rust Resistance Pyramiding resistance genes from bread wheat with markers  Septoria Tritici (on DW only) ■ Collaboration with INRAT-Tunisia ■ Hessian Fly ■ Collaboration with INRAM-Morocco ■ Stem Rust Collaboration with EARO (Debre Zeit) and KARI (Njoro  Old, pre-Ug99, problem in Ethiopia  Durum area in Ethiopia has fallen dramatically, partly due to stem rust  Complex virulences, beyond Ug99 and its variants, mostly durum-specific, largely not virulent on bread wheat  Most of CIMMYT germplasm was susceptible although some cultivars were selected by Ethiopian NARS Very few reliable sources of resistance  Inter-cross/cross few sources of resistance in Elite material from CIMMYT or other programs  Cross to Ethiopian sources of resistance and some tall land-race types  “Pyramid” – more than one – major effective genes from bread wheat using molecular marker assisted transfer  Sr13, 14, 22, 25, 26, 27, 28, 29, 33, 35, 39, 40, 43, 44, 45, Tmp, 1A.1R, Sha7 and a few more undesignated genes  Immediate value: Sr22, 26, 35 and Sha7; and to a lesser extent Sr13, 14, 25, 1A.1R and Tmp for use in combinations  Immediate value + Markers available:  Sr 22  Sr 25  Sr 26  Gene: Sr 22  Marker: cfa2123, (Khan et al., 2005)  Chromosome: 7A  Source: Tall, late, BW stock  Type: CO-DOMINANT  Reliability: Good  Gene: Sr 25/Lr 19  Original marker: wmc221, (lsaac et al., 2004)  Durum marker: Dreisigacker, CIMMYT  Chromosome: 7D (7A, for durum)  Source: various bread + durum wheat  Type: CO-DOMINANT  Reliability: Excellent  Added benefit: yellow color  Current Status: ■ Marker-assisted transfer to durum completed for Sr22 and Sr25 ■ Monogenic F6 lines positive for marker to be sent to Debre Zeit for validation (we know we have the marker! But do we have the gene? And does it provide adequate protection?) ■ Pyramiding of Sr22 + Sr25 in already resistant backgrounds (3rd unknown gene) initiated

Goals of USDA-ARS Strategic Action Plan Built on understanding stem rust epidemiology • Stem Rust Assessment and Pathology • Detection and Identification • Monitoring and Reporting • Germplasm Enhancement, Gene Discovery, Development of Molecular Markers • Regional Variety Development, Evaluation and Implementation • Disease Management • Communication and Outreach Another way to breed durably resistant wheat varieties to stem rust is to accumulate many minor genes for resistance, making it difficult for the pathogen to overcome all the small effects (CIMMYT approach). Also important can be how resistant varieties are used in the field. Mixtures or blends of varieties can dilute the amount of spores and serve as partial barriers to disease spread. Designation Pedigree Sr Gene Combination ARS04-1267 KS98HW151-6/KS00HW120//NC94-4680 22, 38, 1A.1R ARS05-0005 TX85-264*2/TTCC512 36, 1A.1R ARS05-0403 TX98D1170*2/TTCC365 24, 36, 38 ARS05-0456 Neuse/TX98D2334 36, 38, (+Lr34) ARS05-1034 KS2016/Trego 38, 1A.1R ARS07-0050 WX98D022-U44/TX98D1519 36 (+Lr34) ARS07-0116 AR93035-4-2/PI564341//TX85-264 36, 38, 1A.1R, (+Lr34) Pyramiding Stem Rust Genes Theoretically, durability of a resistance gene is a function of the amount of fitness penalty imposed on pathogen. So, if avirulence genes in the stem rust pathogen affect the pathogen’s fitness differentially, then mutations in them should differentially affect fitness, and resistance genes in wheat corresponding to them should be differentially durable. Those avirulence genes with biggest effect on fitness may correspond to those resistance gene with greatest durability. So, which resistance genes that are pyramided may be at least as important as whether and how many resistance genes are pyramided. Pyramids are especially effective if pyramided genes have different modes of action (low potential for cross-resistance). Breeding for Stem Rust Resistance • Resistance breeding (regardless of type of resistance) is focused also on reducing pathogen fitness (the combined ability of an organism to survive and reproduce). • Pathogen fitness is quantifiable: Reproductive rate; Infection efficiency; Aggressiveness (amount of disease caused - Disease severity and AUDPC); Frequency of a strain relative to other strains can be used as an estimator of fitness. How Resistance is Expressed • Resistance is a relative trait. It is the restriction of development of a pathogen, that may vary in degree from immunity (no development) to only slight impeding or delay of the pathogen (relative to a susceptible reaction). Ways to Minimize Wheat Stem Rust from Thriving • Development and deployment of resistant varieties (reduce rate and amount of pathogen build-up); all-stage resistance and adult-plant resistance; gene pyramids; avoid high-effect single gene resistance. • Diversification of resistance - gene deployment; varietal diversification; blends or mixtures • Reduce or eliminate overwintering and oversummering. • Eliminating susceptible alternate host. • Fungicides to reduce rust population size and protect yield of susceptible varieties. Ways to Minimize Wheat Stem Rust from Surviving • Elimination of Highly Susceptible Varieties • Disease escape • Rust Monitoring - surveillance plots; race identification and surveys • Organized communication between farmers, Extension personnel, crop advisors, and researchers; rapid response • Fungicides – If caught early and limited occurrence Ways to Minimize Wheat Stem Rust from Arriving • Reduce global population size of rust • Regulatory methods – Import/Export restrictions on pathogen cultures and plant material • Traveler Advisories • International Cooperation • Unified Concept of Pathogen Movement and Disease Development • Regardless of the type of organism, successful invaders must negotiate a sequence of events that includes arriving, surviving, and thriving in a new environment. • In order to manage wheat stem rust, we must minimize or eliminate the pathogen from arriving, surviving and thriving. • Factors that Promote Spread of Stem Rust • Susceptible Wheat Varieties • Continuous host in space and time • Conducive Environment • Moisture on plants (dew, 6-8 hours) • Uredospore Germination (2-300C) • Uredia Sporulation (5-400C) • Virulent Rust Races • Large Population Size