The Stubbs Pst Culture Collection: Recovery, avirulence/virulence phenotyping and past population structure at a global scale
Department of Agroecology, Aarhus University, Denmark
The "Stubbs Collection", began in 1956 by the late Dutch plant pathologist R.W. Stubbs, refers to a unique historic collection of urediniospore samples of Puccinia striiformis that had been stored in liquid nitrogen for decades. Since 2010 the collection has been maintained by the Global Rust Reference Center (GRRC) in Denmark. Part of the collection is now being in a study of past pathogen diversity. A subset of samples collected between 1958 and 1991 from 35 countries was investigated to assess recovery rate, race identity, and previously undetected virulences. A new method for recovery using an airbrush sprayer and NovecTM 7100 fluid as dispersal agent in inoculating host plants was highly successful, resulting in a 96% recovery from 231 isolates. Phenotyping on the World and European differential host sets and additional wheat genotypes revealed 181 apparently uniform isolates, of which race identities were confirmed for 102. Race identities were updated for additional isolates based on improved resolution due to updated and more informative differential lines. Additional virulences corresponding to Yr17, Yr25, and Yr27 were added, as these were not assayed earlier. The past population structure was investigated by genotyping 212 isolates using 19 multilocus microsatellites. Seven distinct populations were detected, including clonal populations and recombinant populations. These results were compared with recent studies and demonstrated an overall consistent population subdivision at the global scale with clear migration events between populations. The outcome of the study facilitates conclusions about long-term temporal dynamics and overall migration patterns within and among world-wide populations of Pst.
Wheat stem rust effectors: Genomics and functional assay
CSIRO Agriculture, Canberra, Australia
Puccinia graminis f. sp. tritici (Pgt) is one of the most destructive pathogens of wheat. Fungal secreted proteins termed effectors play an important role in modulating the host cellular environment and suppressing the plant defense response to enable fungal growth. They also become targets of plant resistance (R) proteins. We have taken a genomics approach to initially identify candidate effectors. We have built a draft genome for a founder Australian Pgt isolate of pathotype (pt.) 21-0 (collected in 1954) by next generation DNA sequencing. A combination of reference-based assembly using the genome of the previously sequenced North American Pgt isolate CDL 75-36-700-3 (p7a) and de novo assembly resulted in a 92 Mbp reference genome for Pgt isolate 21-0. This draft genome was subsequently used to build a pan-genome based on five Australian Pgt isolates. Transcriptomes from germinated urediniospores and haustoria were separately assembled for pt. 21-0 and comparison of gene expression profiles showed differential expression in ~10% of the genes in germinated urediniospores as well as haustoria. A total of 1,924 secreted proteins were predicted from the 21-0 transcriptome, of which 586 were classified as haustorial secreted proteins (HSPs). We are currently exploring effector gene expression during infection of wheat to reduce this candidate list based on a common expression profile identified for Avr genes in the flax rust fungus. Comparison of 21-0 with two presumed clonal field derivatives (collected in 1982 and 1984) that had evolved virulence on four additional resistance genes (Sr5, Sr11, Sr27, SrSatu) identified mutations in 13 HSP effector candidates. These candidate effectors are being assessed for recognition in wheat accessions with the corresponding R genes using a bacterial type three secretion delivery system based on an engineered Pseudomonas fluorescence strain (Upadhyaya NM et al. Mol Plant Microbe Interact 27:255-264).
Introgression of genes for high grain protein content (Gpc-B1) and Lr24 into leading cultivars by marker assisted backcross breeding
Department of Genetics and Plant Breeding, Institute of Agricultural Sciences, Banaras Hindu University, India
A wheat genotype PBW343+Gpc-B1+LR24 containing the high grain protein content (GPC) gene Gpc-B1 linked to marker Xucw108 was used as the donor parent to transfer Gpc-B1 and Lr24 into Eastern Gangetic Plains (EGP) cv. HUW234 and HUW468 that were released in 1986 and 1999, respectively. The backcrossing program involved the following steps: (i) foreground selection, (ii) marker selection, and (iii) recovery of recipient parent genome. Grain protein contents were recorded for all selected plants from the BC2F2:3 generation. The dominant marker Xucw108 was used for foreground selection, and heterozygous plants were identified through progeny testing. For RPG recovery, both genotypic and phenotypic selection was used. Introgression of the high GPC gene into the recipient background without yield loss was completed in 5 years, starting from 2009-10 (F1) and completed in 2013-14 (BC2F5). A conventional selection program would take the same time to reach BC2F5 but ensuring the transfer of GPC would not not be possible. Ten selected single plants from the BC2F3:4 generation had comparable yields of the parents with 26% higher GPC than the recurrent parent HUW 234. Eight selected plants had comparable yields and 34% higher GPC than HUW 468. Multi-row progenies (BC2F4 and BC2F5) of each selected plant were evaluated in yield traits with the donor and recipient parents as controls during 2012-13 and 2013-14. Two lines based on each recurrent parent were identified with significantly higher GPC with no yield penalty. The study reinforced the belief that MAS in combination with phenotypic selection could be a useful strategy to develop high GPC genotypes without sacrificing grain yield. These lines will be submitted to national trial where MAS derived lines require only two years of testing compared to four years for conventionally bred lines.
Targeting stem rust resistance genes Sr32 and Sr1644 for cloning by mutagenesis and sequence capture
John Innes Centre, UK
Resistance offers the best means of control of the cereal rusts, but must be strategically deployed so as to avoid exposure of single major genes, which have faltered so many times in the past. The pyramiding of multiple effective resistance genes is a strategy that has proven effective in a number of wheat production areas around the world. However, the process of incorporating multiple resistance genes into a single cultivar using standard breeding techniques is time consuming, laborious, and hampered by the problem of linkage drag. If a suite of effective resistance genes could be efficiently cloned and transferred into wheat as a cassette, it would accelerate the development of durably resistant varieties without the problem of linkage drag. Toward this end, we have developed a resistance gene cloning technology based on resistance gene enrichment sequencing (RenSeq) of EMS-derived mutant R gene alleles. As a proof of concept test, we successfully ‘re’-cloned the already characterized gene Sr33 and are now targeting the cloning of eight other effective resistance genes. For the identification of susceptible mutants for the cloning of Sr32 from Aegilops speltoides, we screened 1,109 M2 families with race TPMKC — as a surrogate for race TTKSK. Five susceptible M2 mutants were confirmed by progeny testing. These mutants were also susceptible to race TTKSK. For the population involving Sr1644 from Ae. sharonensis, 1,649 M2 families were screened, yielding 33 M2 families that appeared to segregate for susceptibility. Thirteen of 33 families were confirmed as bona fide susceptible mutants by progeny tests in the M3 generation. Identification of susceptible EMS mutants of Sr32 and Sr1644 suggests that the underlying resistance in these lines is conferred by single genes. We will report on progress to clone and characterize these genes using R gene exome capture and sequencing technology (RenSeq).
Resistance to wheat stem rust in selected accessions of Iranian wheat landraces
Tarbiat Modares University of Tehran, Iran
View mojerlou.pdf (199.61 KB)
Stem rust is a potentially destructive fungal disease of wheat worldwide. In 1998 Pgt pathotype TTKSK virulent to Sr31 was detected in Uganda. The same pathotype was confirmed in Lorestan and Hamedan provinces of Iran in 2007. We used a derivative of race TTKSK to phenotype 62 Iranian wheat landraces (resistant to stripe rust in a previous study) at the seedling stage to this new pathotype (TTSSK). Twenty eight accessions were evaluated for the presence of resistance genes Sr2, Sr22, Sr24, Sr25, Sr26, Sr35, Sr36 and Srweb using SSR markers. None carried Sr2, Sr24 or Sr26, but the presence of Sr22, Sr25, Sr35 and Sr36 was indicated. Some susceptible landraces predicted to carry Sr2 by marker analysis require further investigation. To evaluate defense gene expression in compatible and incompatible stem rust interactions we sampled resistant and susceptible cultivars at 0, 12, 18, 24, 72 hours post-inoculation (hpi). ?-1,3 glucanase expression was studied using qGLU-S and qGLUU-AS primers and a real-time PCR step-one ABI machine, with ?-tubulin and EF1-? genes used as internal controls. In incompatible interactions defense gene expression was increased at 24 hpi, but in compatible interactions the highest level of expression occurred at 12 hpi and was significantly decreased at 18 hpi. The results revealed that expression of defense genes such as ?-1,3 glucanase was earlier in compatible than in incompatible interactions but the expression level was less in incompatible interactions. On the other hand, in susceptible genotypes the expression of defense genes increased immediately after inoculation and declined sharply after infection. In contrast defense gene expression in resistant genotypes began to increase after establishment of the pathogen.
Performance of CIMMYT germplasm in Ethiopia: Key materials for variety development
CIMMYT wheat germplasm flow to Ethiopia started in the late 1960s. Over 90 bread wheat varieties were released over the decades. Of these, about 77% had CIMMYT origins or were derived from CIMMYT materials. Wheat is a traditional rainfed crop grown by 5 million small-scale farmers on 1.6 ha more or less. Yields have increased from 1.0 t/ha in the 1960s to 2.54 t/ha in 2014 mainly due to high yielding semi-dwarf bread wheat varieties and modern agronomic practices. Using such technologies, better farmers often get 5-6 t/ha. The rusts are the most important production constraints. For example, the 2010 yellow rust epidemic debilitated the mega varieties Kubsa and Galama in the highlands. In 2013/14, stem rust caused up to 100% yield losses in the widely adopted bread wheat variety Digalu in Arsi and Bale. This epidemic was caused by Pgt race TKTTF, which is virulent to the gene SrTmp that is present in Digalu, but is avirulent to Sr31, which is overcome by race Ug99 (TTKSK) and derivatives. To avert the increasing threat of rusts, CIMMYT developed a shuttle breeding program where germplasm moves back and forth between Mexico and Kenya and has increased nursery testing sites (Holetta, Kulumsa, Debre Zeit, Sinana, Adet, and Melkassa) in Ethiopia from two to six. The germplasm passes through rigorous tests against major diseases during both the main- and off-seasons. To obtain high yielding rust resistant germplasm, many hundreds of genotypes were introduced and tested over the last two years. In 2014/15, 266 (25%) lines with multiple disease resistances and high yield were promoted to national trials. CIMMYT continues to be an important source of germplasm. Fast tracked variety testing and release, accelerated seed multiplication, demonstration and popularization of new varieties with high yield, multiple disease resistance, and acceptable quality will continue.
Evaluation of wild wheat introgression lines for rust resistance and yield
Kazakh Research Institute of Agriculture and Plant Growing
Wild species are sources and donors of many valuable traits for wheat improvement. We studied winter wheat introgression lines for productivity traits, disease resistance, and protein, globulin, gliadin and glutenin contents as well as grain mineral concentrations. Laboratory and field studies allowed selection in populations segregating for resistance to yellow rust and leaf rust. Lines 1718, 1721-9, 1721-4, 1675 and 1727 had the highest yields (6.2 t/ha) and stable leaf rust and stem rust resistances, but were still variable in response to stripe rust (30-80 S). Lines 1718 (Bezostaya 1 x Ae. cylindrica, genomes CCDD) and 1721 (Bezostaya 1 x T. militinae2 - 6, ABG) were resistant to stripe rust in trials at yield levels of 3.7-7.6 t/ha and from 5.7 to 8.2 t/ha, respectively. Line 1675 (Zhetisu x T. kiharae, ABGD) was resistant to all three rusts. Line 1676 (Steklovidnaya 24 x T. timopheevi, ABG) was resistant to LR and SR at a yield level of 8.3 t/ha, and 1671 (Zhetisu x T. militinae, ABG) was resistant to YR and SR at a yield level of 7.5 t/ha. Protein contents of the lines ranged from 13.6 to 18.4%, and grain mineral contents were above average.
Surveillance and Pgt race analysis in Iran, 2014
Seed and Plant Improvement Institute (SPII), Iran
Stem (black) rust is a potentially important disease in northern, western and southern Iran. A new Pgt race with virulence to gene Sr31 appeared in Iran in 2007. Similar races have spread in Africa and some CWANA countries. In 2014 stem rust was widespread in western, northern, northwestern and central Iran, but at low severities. Thirty-nine stem rust samples were collected for race analysis. After purification and increase each isolate was inoculated to a set of 20 North American differentials in the greenhouse. Infection types were recorded 12-14 days after inoculation using the scale described by McIntosh et al. (1995, Wheat Rusts: An Atlas of Resistance Genes, CSIRO, East Melbourne, Australia). Races TKSTC (59%), TKTTC (20%), TTTTC, KTTSK (virulent on plants with Sr31), TTSTC, PTTTF and TTTTF were detected. Race TKSTC was common in western, northwestern and central Iran. Except for avirulence to Sr17 this race is similar to the race (TKTT) that caused a stem rust epidemic in Ethiopia in 2013.
Rust reactions of lines in a wheat crossing block developed by the Bahri Dagdas International Agricultural Research Institute in 2014
The Central Research Institute for Field Crops, Turkey
Rusts and drought are the principal yield-limiting factors for wheat production in the Central Anatolian region of Turkey. The aim of the study was to determine resistance sources in a crossing block of drought tolerant lines. Seedling tests involving all three rusts were carried out at CRIFC, Yenimahalle, in 2014. Inoculations were made with local Pgt (avirulent on differentials with Sr24, Sr26, Sr27 and Sr31), Pt (avirulent on differentials with Lr9, Lr19, Lr24 and Lr28) and a local Pst population. Reactions were scored 14 days post-inoculation on 0-4 (LR and SR) or 0-9 (YR) scales. Seventeen (19%) genotypes were resistant to stripe rust, 11 (12%) were resistant to leaf rust, and 17 (19%) were resistant to stripe rust.
Comparative analysis of rust resistant and susceptible wheat varieties in Pakistan
International Maize and Wheat Improvement Center (CIMMYT) Pakistan Office
To reduce losses caused by rusts, regular and timely replacement of susceptible varieties with new high yielding, rust resistant varieties must occur. Data from a farmer survey carried out across Pakistan (Punjab, Sindh, KPK and Baluchistan) in 2014 enabled an analysis of the uptake of rust resistant variety NARC 2011. The empirical results indicated that the major sources of information that farmers obtained about NARC 2011 were research stations (83%), seed companies (7%) and fellow farmers (5%). Although production inputs were applied equally to both rust resistant NARC 2011 and rust susceptible wheat varieties the average yield of NARC 2011 (5,063 kg/ha) was superior to high yielding but rust susceptible varieties (4,446 kg/ha). Quality attributes of NARC 2011, including taste, color, dough kneading and chapatti making properties, were preferred by >70% of farmers). Seed availability and accessibility of NARC 2011 were major issues. Farmer awareness of rusts, especially the threat of exotic Pgt race Ug99, needs to be improved.