Placement of South African stripe rust in a global context and development of diagnostic tools for genotyping field samples
BGRI 2015 Plenary Abstract Hester van Schalkwyk
Department of Plant Sciences, University of the Free State, South Africa
Stripe (yellow) rust, caused by the fungus Puccinia striiformis f. sp. tritici (PST), is a major global wheat disease. New PST strains that show higher infection rates and rapid adaptation to less favourable environmental conditions have been observed over the last 15 years. It has also continued to spread to areas where it was not previously recorded. In South Africa, stripe rust was first detected in 1996. In subsequent years three more PST races were observed, with what seemed to be a step-wise virulence gain. A better understanding of the South African PST pathotypes and how they fit in the global context is needed. We aimed to address this by sequencing the genomes of four historical PST isolates displaying the four distinct virulence profiles. This allowed us to characterise the genetic diversity between these stripe rust races and develop diagnostic markers to easily genotype current detections. We also placed the South African PST isolates in context with global PST isolates where sequence data was available. This analysis illustrates that the South African PST races are more closely related to PST from other African countries when compared to isolates from Africa, Europe and Asia. Through pairwise comparison of isolates, we identified 27 candidate effector genes showing specific polymorphisms between the four isolates that could be related to their distinct virulence profiles. We are currently undertaking gene expression profiling of these candidates to determine if these effectors are specifically upregulated during infection–a key characteristic of effector genes. This study has shed new light on the potential origin and adaptation of stripe rust in South Africa and provides tools for rapid genotypic classification of infections in the field.
The Green Revolution involved the deployment of reduced height (Rht) genes to generate shorter wheat varieties with increased grain yields. It also contributed to a reduction in genetic diversity in the modern gene pool. Therefore, the pre- Green Revolution tall wheat landraces may provide a reservoir of genetic variation for economic traits such as rust resistance. Considering the breakdown of a suite of rust resistance genes through the emergence of currently predominant pathotypes (e.g. Ug99 and high temperature adapted isolates of the stripe rust pathogen) after three decades of Green Revolution, the discovery, characterisation and deployment of diverse sources of resistance remains a high priority. We have screened the Watkins wheat landrace collection and discovered, characterised and formally named a suite of new rust resistance genes including Yr47, Yr51, Yr57, Yr63 and Sr49. In addition, genotypes carrying potentially new genes for resistance to three rust pathogens are currently being investigated by students from seven nations representing three continents (Australia, Asia and Africa). Yr47, Yr51, Yr57 and Lr52 have been backcrossed into modern cultivars including the widely adapted cultivar PBW343 (Atilla) using markers developed in our research program. Development of triple rust resistant derivatives in modern wheat backgrounds is in progress. Stocks carrying Yr47, Yr51, Yr57 and Lr52 have been mutated to facilitate cloning of these loci for their eventual use in development of multi-gene cassettes for transformation.
Wheat stem rust is one of the major wheat yield limiting factors in Ethiopia. A stem rust epidemic occurred in the wheat belts of Arsi and Bale zones in the 2013-2014 crop season caused by Pgt race TKTTF that is virulent to the widely grown Ug99-resistant variety Digelu. This epidemic highlighted the need for wheat varieties with resistance to multiple Pgt races. This study was therefore, carried out to evaluate the reaction of the major Ethiopian varieties and advanced breeding lines against the dominant Pgt races in Ethiopia. Races TKTTF, TTKSK, TRTTF and JRCQC were isolated from field samples and multiplied on the susceptible cultivar McNair starting in May 2014. Four wheat stem rust nurseries, each inoculated with a single Pgt race, were established at Kulumsa and monitored from July through October, 2014. Each nursery included 34 entries in two replicates and 137 entries in a single replicate, augmented with six sets of five repeating checks. An additional nursery established at Debre Zeit, containing 551 entries in an augmented design, was evaluated with the epidemic Pgt race TKTTF. These entries included the most relevant Ethiopian bread and durum wheat breeding lines and cultivars, and 34 seedling-susceptible lines to evaluate the race-specificity of adult plant resistance. Stem rust severities for the four races ranged from trace to 80 %. Out of all entries evaluated, 10 were resistant to all four Pgt races, while 11 entries were effective to three of the four races. At Debre Zeit, 31.4% of the entries were resistant to Pgt race TKTTF. This study showed that rapid isolation and increase of Pgt races in Ethiopia is possible to facilitate field screening of breeding lines to select for candidate cultivars with resistance to multiple virulent races of Pgt.
Using ‘speed breeding’ to harness rust resistance: faster, cheaper and easier
BGRI 2015 Plenary Abstract Lee Hickey
The University of Queensland, Queensland Alliance for Agriculture and Food Innovation, Australia
Watch presentation (YouTube)
A new method for rapid generation advance, called ‘speed breeding’, has considerable advantages over DH technology for spring wheat because it provides increased recombination during line development and enables selection in early generations for some traits. The system has been refined over the past 8 years at The University of Queensland, utilizing controlled temperature regimes and 24-hour light to accelerate plant growth and development. The low-cost management system enables up to 6 plant generations of wheat annually – just like Arabidopsis. Currently, three of the six wheat breeding companies in Australia are exploiting speed breeding, and elite lines developed using the technology are in the final stages of yield evaluation. Recently, we developed methods adapted for use in the speed breeding system, which permit year-round high-throughput screening for adult plant resistance (APR) to rust pathogens that attack wheat. In this presentation, we describe the protocols, explain how phenotypes are related to field-based measures and highlight how the system can even handle diverse germplasm, such as winter types and landraces. Our ‘triple rust’ screening methodology enables selection for APR to all three rust pathogens and crossing of selected plants within a single plant generation. We applied the technique to rapidly introgress rust resistance into several Australian cereal cultivars. The technology is also a useful tool to accelerate rust research efforts. RIL populations designed for mapping novel APR genes can be developed within 12–18 months. Experiments to understand gene function in terms of temperature stability and onset of resistance can be performed year-round and if combined with sequencing technologies, such as RNAseq, transcripts involved in rust defence can be rapidly identified and harnessed via the speed breeding system. We will also reveal our current activities aiming to integrate the system with other plant breeding technologies to maximise genetic gain for wheat.
In response to the threat posed by Ug99 (race TTKSK) and a global expert panel assessment, the Borlaug Global Rust Initiative (BGRI) was formed in 2005. This represented one of the most comprehensive global programs to address an emerging crop pathogen threat. For the last decade, surveillance and monitoring has been a key component of the BGRI. Progress in rust surveillance and monitoring over the last ten years is critically reviewed, with a focus on stem rust. The transition from a data poor environment regarding stem rust to a fully functional, comprehensive crop pathogen surveillance system is a notable success. Key components and status of the current system are described, including; the surveillance network, the data management and information platforms, and pathogen tracking. The application of the existing surveillance and monitoring system and the current status of important stem rust races are described. The role that new technologies are playing in the monitoring and tracking of stem rust is highlighted. Recent stem rust epidemics in East Africa provide stark warning of threat that the disease poses and the clear need to continuously monitor evolving stem rust populations. Shortcomings of the existing system are examined and future directions for the surveillance and monitoring system are outlined.
Population structure of Puccinia striiformis f.sp. tritici at the southern part of Pakistani Himalayan region
BGRI 2015 Plenary Abstract M.R. Khan
The University of Agriculture, Peshawar, Pakistan
The Himalayan region of Pakistan has been shown to be the centre of diversity of wheat yellow rust pathogen Puccinia striiformis f.sp. tritici (Pst) with a probable role of sexual reproduction in the population temporal maintenance. However, the populations of southern part of Pakistani Himalayan region remains unexplored, where wheat yellow rust is an important disease on rainfed wheat. The current study was thus carried out to assess the disease status and population structure of Pst prevalent in the southern part of Pakistani Himalayan region, mainly the districts of Kohat, Karak, Bannu, Lakki-Marwat and DI-Khan. A high disease pressure was observed during wheat season in 2013 in the region, where the level of severity ranged from 5% to 100% depending upon the variety tested. Microsatellite genotyping of 102 isolates with 18 SSR markers revealed a high diversity ranging from 0.86 (for DI Khan) to 1.00 (for Karak). The recombination signature was lower compared to the Himalayan populations. Analyses of the population subdivision revealed no clear evidence of spatial structure, with the maximum FST value of only 0.081. The overall diversity was higher in the region as compared to European clonal population, though it was still lower than the recombinant Himalayan populations, which could be attributed to their distance from Berberis spp. plantation zone.
The cereal Mla locus is a rich source of effective resistance genes: cloning the Sr50 gene from rye
BGRI 2015 Plenary Abstract Rohit Mago
CSIRO Plant Industry, Australia
The stem rust resistance genes Sr31 and Sr50 in wheat were both derived from translocations of the short arm of chromosome 1 from rye and conferred resistance to all field isolates of Puccinia graminis f. sp tritici (Pgt) for many years, preventing their distinction as different resistance specificities. We now show that Sr50 confers resistance against the Ug99 strain that overcomes Sr31, whereas a mutant Pgt strain virulent towards Sr50 is avirulent towards Sr31. Because lack of recombination between wheat and rye chromosome arms precludes genetic mapping and so map-based cloning of Sr50, we used a combination deletion mutagenesis and large DNA fragment cloning in bacterial artificial chromosome (BAC) vectors to define this resistance locus. Sequence analysis of a BAC contig spanning the smallest deletion detected with DNA markers at the Sr50 locus identified six coiled coil nucleotide binding site leucine-rich repeat (CC-NB-LRR) genes and a chymotrypsin inhibitor gene closely related to genes at the orthologous barley powdery mildew resistance locus, Mla. Sequencing of these genes from two EMS-induced mutants that had lost no DNA markers revealed mutation in one of the CC-NB-LRR orthologs of Mla. Transgenic complementation tests in stem rust susceptible wheat proved this gene to be Sr50. A survey of a set of rye accessions identified several carrying the gene but occurring in different Mla gene haplotypes based on DNA gel blot patterns and copy number of Mla orthologs. Several different powdery mildew and rust resistance genes including TmMla1 from T. monococcum, 23 Mla alleles from barley and stem rust resistance genes Sr33 from Aegilops tauschii and Sr50 from rye are all members of the Mla clade of cereal R genes. The gene Sr50,was initially thought to be allelic to Sr31, however, appearance of Ug99 showed that this is a different gene and is rye ortholog of barley Mla powdery mildew resistance gene. The cloning of Sr50 gives us an opportunity to screen the rye germplasm for presence of Sr50 and allows us to now do functional analysis of the various domains and understand the mechanism of resistance. The cloning also helps to add very effective resistance to gene cassette. Sr50 is effective against all the stem rust isolates around the globe
Use of large-scale computational resources has permitted the first quantitative study of airborne migration routes of fungal spores between numerous key epidemiological hot-spots of wheat stem rust in Africa, the Middle East and the Indian subcontinent. By coupling a state-of-the-art Lagrangian particle dispersion model (NAME) with mechanistic epidemiological models, we simulate turbulent atmospheric transport of large ensembles of fungal spores from source sites. The models use highly resolved global meteorological datasets from the UK Meteorological Office. We consider release of P. graminis uredinospores from numerous source locations over an 11 year period (2003-2014) and simulate atmospheric trajectories over a 10 km2 spatial sampling grid to elucidate spore deposition rates at national, regional, and continental spatial scales. Our systematic exploration permits the first quantitative perspective and ranking of likely airborne transmission routes of wheat stem rust. We identify migration trends within and between the “Rift valley epidemiological zone”, the Middle East, the Indian Subcontinent, as well as South Africa. Our results indicate (I) consistent seasonal dispersal patterns, (II) likely airborne transmission of stem rust from the Middle East to North-East Africa, and (III) suggest that there is considerable risk of spread of Ug99 or other virulent races from Eastern Yemen to the Indian subcontinent. Model results indicate that over the 11 year study period, viable spore deposition occurred between Eastern Yemen and Pakistan on average 22 days per year during overlapping wheat growing seasons. The validity of the modelling framework has been successfully tested by comparison with survey data from the 2013 epidemic outbreak in Ethiopia, and was recently used as a risk assessment tool to provide rapid response advice in different East-African countries. Known stem rust race distributions are also supportive of the model outputs. The research we have been doing allows a quantitative perspective on likely airborne transmission routes of Ug99 or other virulent races of wheat rust. By that we hope to provide new insights and recommendations for future risk assessment, survey and control strategies and also to contribute to fundamental understanding of epidemiological spread on regional and continental scales. The work we would like to present is the result of a joint effort of Dr Laura Burgin and Dr Matt Hort from the UK Meteorological office, Dr Dave Hodson from CIMMYT, and Dr James Cox, Matthew Hitchings and me from the Epidemiology and Modelling group of Prof Gilligan in Cambridge.
To inform breeders and growers of important changes in virulence and to facilitate development and deployment of resistant cultivars, isolates of wheat rust fungi are routinely evaluated on seedlings of a set of differential wheat lines containing different resistant genes. However, the methods used to evaluate and report virulence changes in most regions of the world seem inadequate for accomplishing these goals and could be improved by adherence to three principles. Firstly, for each region, the resistance genes in the set of differentials should match the effective genes in contemporary cultivars and breeding lines. Most differential sets contain several resistance genes that have been ineffective for decades and do not contain genes found in cultivars and breeding lines. Given the importance of genes for race-specific adult-plant resistance, these should be included in differential sets. Secondly, intermediate reactions on differential lines that had been highly resistant are important warnings of gradual increases in virulence. Naming races requires isolates to be either virulent or avirulent on each line in a fixed set of differentials and is a hindrance to identifying gradual changes in virulence on currently effective genes. Utilizing virulence formulae with a designation for intermediate virulence (e.g. parentheses around the gene or differential) seems to be a simple solution for both documenting partial virulence and for easily changing differentials to match genes in cultivars and breeding lines. Thirdly, the method for evaluating virulence against a particular differential should predict the result of that host-pathogen interaction in the field. Growth stage and environmental conditions are important for expression of some resistance genes, and all currently effective genes are not likely to be expressed under the same conditions. Following these principles will make virulence surveys more predictive of important changes in the field and thereby contribute to more effective management of rust diseases.
Since 1998, when Pgt race TTKSK (Ug99) was first identified in Uganda, seven variants in the Ug99 race group have been reported in nine countries in eastern and southern Africa. Five of these variants (TTKSK, TTKST, TTTSK, PTKSK, and PTKST) have been observed in Kenya. Increased surveillance efforts in recent years have enabled detection of new virulence combinations that threaten wheat production. Three new variants in the Ug99 race group were identified from samples collected in 2013 and 2014 in Kenya. A new race, TTHST that is identical to TTKST but avirulent on Sr30 (IT 2-), was identified from a sample collected in the Central Rift Valley Region in 2013. In 2014, two new races, TTKTK and TTKTT, were identified from a total of nine samples (six collected from cv. Robin, and one from each of Eagle10, NJRBW II, and barley) in multiple regions. These two races are of special concern as both are virulent on SrTmp, a gene that is effective against all previously known races in the Ug99 group. Resistance gene SrTmp is postulated to be the source of TTKSK resistance in cv. Robin (released in 2011 in Kenya, also postulated to have Sr2) and cv. Digalu (released in 2005 in Ethiopia). The presence of new races with virulence on SrTmp may explain the high levels of stem rust severity observed in wheat cultivar Robin in Kenya in the past two years. Genotypic relationships between these new races and known races in the Ug99 race group are being characterized using SNP markers. Cultivars and elite breeding lines from Kenya, CIMMYT, and the US are being evaluated for seedling reactions to race TTKTT. With the detection of these new races, there are a total of eight variants in the Ug99 race group in Kenya.