11th International Cereal Rusts and Powdery Mildews Conference
John Innes
Centre,
Recommended abstract citation format:
Jafary H, Niks RE, 2004. Mapping of quantitative genes in barley determining the resistance to the heterologous wheat leaf rust fungus (Puccinia triticina). Proceedings of the 11th International Cereal Rusts and Powdery Mildews Conference, Norwich, England, 22-27 August 2004, abstract 1.4, Cereal Rusts and Powdery Mildews Bulletin [www.crpmb.org/icrpmc11/abstracts.htm].
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ABSTRACTS
Part 1 : Lectures
1.1 In search of durable resistance
Michele C. Heath
michele.heath{a}utoronto.ca
More than 20 years ago, Roy Johnson
coined the term “durable resistance” to describe disease resistance proven to
be effective for long periods of time. Not surprisingly, producing durable
resistance has been the goal of much plant pathological
If it is assumed that inducible defenses elicited during most examples of
nonhost resistance are the result of “pattern recognition” analogous to the
recognition of pathogen molecules (elicitors) during the innate immune response
in animals, then successful a pathogen must have adapted to “overcome” such
defenses in its host species. Understanding how a pathogen can overcome
constitutive and inducible plant defenses in its host plant is important for
developing ways of “putting back” durable resistance into these
pathosystems. The study of dikaryon infection structures suggest that initial
rust fungal development requires a sequence of plant signals as well as the
ability of the fungus to inhibit prehaustorial defenses and to prevent an HR
after haustorium formation. Studies of monokaryon infection suggests that
defenses associated with plant cell wall penetration are disrupted in the host
species by a localized reduction in adhesion between the plant cell wall and
the plasma membrane, and that protein synthesis and specific gene expression
are suppressed in a susceptible cultivar prior to the fungus entering the cell
lumen. Data suggest that although active defenses against rust fungi during
nonhost resistance are part of the same battery of defenses used by plants
against other pathogens, the mode of overcoming them may not be the same as
that used by othe
The recent rapid accumulation of information from host and nonhost plant-pathogen interactions involving biotrophic fungi means that we are rapidly reaching the point where protocols for engineering of disease resistance that is not easily overcome can be envisaged. However, the practicality of producing durable resistance in an agricultural setting still present large difficulties.
1.2 The ancient cell death suppressor BAX Inhibitor-1 induces susceptibility of barley to appropriate and inappropriate powdery mildew fungi
Ralph Hueckelhoven, Ruth Eichmann, Holger Schultheiss,
Ralph.Hueckelhoven{a}agrar.uni-giessen.de
Penetration into barley epidermal cells and subsequent haustoria formation is the key step in establishment of compatibility of barley powdery mildew fungus (Blumeria graminis f.sp. hordei, Bgh) and susceptible host plants. Vice versa, broad spectrum resistance, such as mediated by the recessive mutant mlo-gene, is expressed as penetration resistance with cell wall-associated defence. Interestingly, both successful penetration and apoplastic defence are accompanied by accumulation of reactive oxygen intermediates, O2•- and H2O2, respectively (1). Cell death, reactive oxygen intermediate accumulation and penetration resistance are linked in barley via regulation by the MLO protein. Therefore, we isolated the ancient cell death suppressor BAX Inhibitor-1 from barley (2, 3) and studied its role in penetration resistance to Bgh. Single-cell overexpression of wild type BAX inhibitor-1 strongly supported susceptibility of barley to Bgh (2, 3). A functional GFP:BI-1 fusion protein was detected in the ER and nuclear envelope by confocal laser scanning microscopy. Astonishingly, overexpression of BAX inhibitor-1 was sufficient to break both mlo-mediated resistance to Bgh and nonhost resistance to B. graminis f.sp. tritici (3, 4). Since BI-1 is able to suppress cell death in animals and plants as well as penetration resistance in barley, there appear to be conserved overlapping pathways that regulate cell death and defence responses, possibly in all higher eukaryotes (2).
(1) Hückelhoven and Kogel, 2003 Planta 216, 891–902
(2) Hückelhoven (in press) Apoptosis
(3) Hückelhoven et al., 2003 Proceedings of the National Academy of Science USA 100, 5555-5560
(4) Eichmann et al. (in press) Molecular Plant-Microbe Interaction
1.3 Comparison of gene expression profiles in barley epidermis in response to Blumeria graminis f.sp. hordei and Blumeria graminis f.sp. tritici
Wubei Dong, Dimitar Douchkov and Patrick Schweizer
Institute of Plant genetics and Crop Plant Research (IPK), Gatersleben, Germany
wubei.dong{a}ipk-gatersleben.de
Plants often respond in similar ways to host and nonhost pathogens (Thordal-Christensen, 2003). Up to now, most of the identified resistance mechanisms are shared by host and nonhost. Considering the clear difference between host and nonhost resistance with respect to durability, the question about the key components of nonhost resistance is appealing. We compared nonhost and host expression profiles in barley epidermis in response to Blumeria graminis f.sp. tritici (Bgt) and Blumeria graminis f.sp. hordei (Bgh) by using a barley 10k cDNA array. At 6h, 12h, 24h and 36h after inoculation, epidermis was stripped and RNA isolated. cDNA probes were labeled with 33P and hybridized to the array membranes. Clustering analysis indicated that the host and nonhost induced expression patterns are similar. Bgt and Bgh responses at earlier stages show clearer differences, mostly at 12 hours after inoculation. At later time points, the expression patterns are more similar. 276 up-regulated genes were shared by host and nonhost responses in two independent inoculation and array experiments, whereas 244 genes were nonhost specifically induced and 101 genes host specifically induced. Our results suggest that quantitative differences in the expression of pathogen regulated genes might be a critical factor in nonhost resistance. Currently we are verifying our array results by a functional screening system based on RNAi.
Thordal-Christensen H, 2003. Fresh insights into processes of nonhost resistance. Current Opinion in Plant Biology 6, 351-7.
1.4 Mapping of quantitative genes in barley determining the resistance to the heterologous wheat leaf rust fungus (Puccinia triticina)
H Jafary, Rients E Niks
Laboratory of Plant Breeding, Wageningen University, Wageningen, The Netherlands
Hossein.Jafary{a}wur.nl
Very little is known on the genetic
basis of non-host resistance of crops to specialised pathogens. In order to
investigate the inheritance of this resistance, we chose barley, since in this
species some accessions in the seedling stage are still somewhat susceptible to
heterologous rust species like the wheat leaf rust, Puccinia triticina,
implying that barley is nearly a non-host to this rust species. By accumulation
of genes for susceptibility we developed a barley
1.5 Suppression of various forms of rust and powdery mildew resistance of wheat and barley
Balázs Barna
Plant Protection Institute of the Hungarian Academy of Sciences, Budapest, Hungary
bbar{a}nki.hu
Heat pre-treatment of wheat seedlings significantly increased the number of pustules or lesions of stem rust on susceptible or hypersensitive resistant cultivars respectively. The reaction type did not change on either susceptible or resistant plants; however, benzimidazole pre-treatment abolished the effect of heat predisposition.
Heat shock pre-treatment of near isogenic barley lines expressing various resistance genes against powdery mildew (Blumeria graminis f. sp. hordei) decreased resistance to the pathogenic fungus.
Near isogenic lines, with and without Mla, Mlg or mlo resistance genes, of barley cvs. Pallas and Ingrid were immersed in hot water (48-49 oC) for 25 seconds one day before inoculation with Bgh race A6. Heat predisposition significantly increased the number of powdery mildew colonies on susceptible leaves, the number and size of lesions with limited sporulation on Mla lines and the number of visible lesions on Mlg lines. Heat treatment did not affect visible symptom on mlo lines.
In addition, heat pre-treatment increased susceptibility in all barley lines with or without a resistance gene by improving the development of the fungus (secondary germ tube, haustoria formation) and suppressing the resistance responses of plants (papilla formation, epidermal HR and epidermal H2O2 generation). In the mesophyll, however, elevated HR and H2O2 accumulation were observed, probably due to slight damage by heat stress.
Since, in both wheat/rust and barley /powdery mildew interactions, the susceptibility of originally susceptible plants was increased, it seems that a basal resistance, which is suppressed by heat pre-treatment, exists in all plants.
Changes in membrane leakage, antioxidants and possible heat shock proteins will be discussed in relation to the decrease in resistance.
1.6 Analysis of the mechanism of RAC/ROP GTPase activity in susceptibility of barley to the powdery mildew fungus
Holger Schultheiss, Krystina Opalski,
Institute of Phytopathology und Applied Zoology, University Giessen, Germany
holger.schultheiss{a}agrar.uni-giessen.de
Penetration of a barley epidermal cell is the crucial step in pathogenesis of the barley powdery mildew fungus Blumeria graminis f.sp. hordei (Bgh). To prevent penetration by Bgh, attacked cells have to focus the defence machinery to the site of fungal invasion. A prerequisite for cellular polarisation is the rearrangement of the cytoskeleton. Pharmacological inhibition of cytoskeleton rearrangement leads to enhanced penetration of powdery mildew fungi into barley epidermal cells.
Small monomeric G-Proteins of the RAC/ROP family are known to be involved in the regulation of actin structure. Using a candidate RT-PCR approach we identified six cDNAs coding for small RAC/ROP GTPases in barley. The transient knock-down of HvRACB using the sequence-specific dsRNA interference technique enhanced the penetration resistance of barley to Bgh (Schultheiss et al., 2002). Vice versa, single-cell overexpression of constitutive active (CA) HvRACB reduced background resistance. Histochemical staining of actin filaments in transformed cells revealed an influence of CA RACB on the host cytoskeleton. Additionally, overexpression of CA RAC3, CA ROP4 and CA ROP6 enhanced the accessibility of barley to Bgh by an unknown mechanism. The specificity of this effect is given by the fact that CA RACD and CA RAC1 did not influence the barley - Bgh interaction (Schultheiss et al., 2003).
Analysis of GFP:HvRAC/ROP transformed cells revealed varying strengths of plasma membrane-association of barley RAC/ROPs. A close link between localisation and function of barley RAC/ROP proteins was obvious because delocalised CA RAC/ROP mutants failed to induce accessibility to Bgh.
Together, some of the RAC/ROP proteins appear to be specific barley susceptibility factors involved in processes supporting parasitic entry of Bgh into epidermal host cells.
Schultheiss H, Dechert C, Kogel K-H, Hückelhoven R, 2002. A small GTP-binding host protein is required for entry of powdery mildew fungus into epidermal cells of barley. Plant Physiology 128, 1447-1454.
Schultheiss H, Dechert C, Kogel K-H, Hückelhoven R, 2003. Functional analysis of barley RAC/ROP G-protein family members in susceptibility to the powdery mildew fungus. Plant Journal 336, 589-601.
1.7 Powdery mildew, cereal cells and sustainable crop production
Tim Carver, Elena Prats, Alan Gay, Luis Mur, Barry Thomas and Hitoshi Kunoh
IGER, Aberystwyth, UK
tim.carver{a}bbsrc.ac.uk
Sustainable cereal cropping needs reliable disease control and various strategies exploiting host resistance have been proposed. Specific (R gene) resistance is unreliable because cereal mildews evolve virulence rapidly. Combining R genes within a cv. might decrease the risk, but, more simply, epidemics can be suppressed by growing cv. mixtures/multilines containing different R genes. An alternative is to breed for durable resistance. Apart from the (exceptional?) case of barley mlo resistance, durable resistance is likely to be under polygenic control and hard to manipulate. Nevertheless, independent resistance mechanisms can impede pathogenesis at different stages. Even if each mechanism has a minor effect, their combination may confer effective resistance. This conclusion arises from studies that generally use a single inoculation and precisely controlled conditions. But this is an over-simplistic approach to understanding natural complexities. For instance, in fields where leaves are challenged repeatedly, outcome of an initial attack drastically affects subsequent cellular responses through locally induced resistance or susceptibility. New evidence shows such effects can be instigated within 30 minutes of inoculation, probably being mediated via conidial extracellular material. Very recently we have also found dramatic effects of mildew attack on stomatal behaviour. In all barley lines examined, stomatal conductance was reduced during attempted fungal penetration in the light. In suscepts, stomata subsequently shut during dark periods but persistently failed to open fully in light, whereas in mlo barley normal stomatal response was largely restored by 36 hours. In contrast, where R gene resistance caused epidermal cell HR, nearby stomata locked permanently open, even in darkness, thus these apparently disease-free plants were severely compromised. Such complexities must be considered in designing strategies for exploiting resistance in sustainable production.
1.8 The role of the actin cytoskeleton in pathogen defense in barley
Marco Miklis, Uwe Zierold, Patrick Schweizer,
Max-Planck Institute for Plant Breeding, Koln, Germany
panstrug{a}mpiz-koeln.mpg.de
Barley Mlo encodes a protein with seven transmembrane domains that modulates basal defence to the powdery mildew fungus (Blumeria graminis f. sp. hordei, Bgh). Recessive mutations (mlo) in Mlo-mediate resistance to all Bgh isolates. Although calmodulin has been recently identified as MLO-interacting protein, the exact molecular mechanisms by which Mlo modulates plant defence remains to be elucidated. Re-mutagenesis of mlo-resistant plants revealed two genes (Ror1 and Ror2) that are necessary for full mlo resistance. To identify additional components, required for Mlo-mediated susceptibility or mlo resistance, we established a high-throughput approach in which we apply a combination of a single cell transient expression assay and dsRNAi-technology. Single cDNA clones of an epidermal cDNA-library of Bgh infected Hordeum vulgare leaves are introduced into a dsRNAi-vector. Both Mlo and mlo leaves were biolistically transfected with these constructs and subsequently, after pathogen challenge, investigated for an alteration of basal resistance or broad spectrum resistance, respectively.
Here we present the identification of a dsRNAi-construct compromising resistance in both genetic backgrounds (Mlo and mlo) of Hordeum vulgare leaves and further examination of the functional role of the gene putatively knocked-down.
1.9 Involvement of nitric oxide in papilla-based resistance and the hypersensitive response of barley attacked by blumeria graminis
Elena Prats, LAJ Mur and TLW Carver
Institute of Grassland and Enviromental Research, Aberystwyth,UK
elena.prats{a}bbsrc.ac.uk
Nitric oxide is emerging as a major signal in plant-pathogen interactions. In model plant-pathogen systems NO acts synergistically with reactive oxygen intermediates to orchestrate the hypersensitive response (HR). However, there is need for analyses of its role in economically important crop/pathogen interactions. The NO-specific stain, 4,5-diaminofluorescein-2-diacetate (DAF-2DA), was used to image and quantify NO production from 6 to 24 hours after the interaction of Blumeria graminis with barley (Hordeum vulgare) cv. Pallas (susceptible) and isoline P01 that carries allele Mla1 conditioning a rapid hypersensitive response (HR). Localised fluorescence after staining indicated that in both barley lines NO was abruptly generated around 10 h after inoculation (h.a.i.) beneath appressorial germ tubes at sites of papilla formation. In P01 a second burst of NO production that spread throughout attacked cells was initiated around 12 h.a.i. and this preceded whole-cell autofluorescence indicative of death due to HR. That fluorescence due to DAF-2DA staining was truly indicative of NO was supported by application of the NO scavenger, 1H-imidazol-1-yloxy-2-(4-carboxyphenyl)-4,5-dihydro-4, 4, 5, 5-tetramethyl-3-oxide (C-PTIO) which suppressed the fluorescence. In addition, C-PTIO application increased penetration frequencies in both barley lines, indicating a role for NO in papilla-based resistance. Furthermore, C-PTIO application slightly delayed cell death in P01 while, conversely, application of an NO donor, sodium nitroprusside, slightly accelerated cell death in P01 and increased cell death frequency in Pallas. The data show NO generation to be one of the earliest responses of barley epidermal cell defence against B. graminis attack and suggest its importance as a signal and regulatory factor in both the initiation and development of effective papillae, and in the HR.
1.10 Barley MLA protein abundance is controlled by RAR1 and is rate-limiting for efficient resistance to powdery mildew
Stéphane Bieri, Stefan Mauch, Qian-Hua Shen, Francesca Ceron, Ken Shirasu, Paul Schulze-Lefert
Institute of Plant Biology, University of Zürich, Switzerland
sbieri{a}botinst.unizh.ch
The complex barley Mla locus is highly polymorphic and harbors approximately 30 known race-specific resistance (R) genes to the powdery mildew fungus (Blumeria graminis f sp hordei). Nine molecularly isolated Mla powdery mildew R genes each encode CC-NB-LRR type proteins with an additional C-terminus (CT) lacking known structure/function motifs. The high sequence conservation (>90% identity) of the gene products and an identical gene structure suggests that many genetically characterized powdery mildew R genes at Mla are variants of a single gene at the complex locus. Previously, we have initiated a functional analysis by reciprocal domain swaps between MLA R proteins that confer different recognition specificities and exhibit differential requirement on RAR1/SGT1, two conserved proteins with putative co-chaperone-like functions. This enabled us to assign a role for the LRR-CT unit in recognition specificity and revealed that RAR1/SGT1 dependence can be uncoupled from recognition specificity. Transgenic barley lines expressing functional epitope-tagged MLA variants were used in biochemical experiments, indicating the existence of membrane-associated and cytoplasmic MLA pools in non-challenged plants. We have investigated MLA abundance in Rar1 wild type and rar1 mutant backgrounds. We will present evidence indicating a general role of RAR1 in controlling MLA steady state levels in non-challenged plants. Our data suggests that MLA protein abundance is a direct determinant for the efficiency of the resistance response.
1.11 Barley leaf rust affect barley’s susceptibility to powdery mildew
KL Olesen and MF Lyngkjær
Risø National Laboratory, Roskilde, Denmark
Karen.lambaek.olesen{a}risoe.dk
Powdery mildew attack on a barley epidermal cell induces responses in both the epidermis and the underlying mesophyll tissue. Depending on the outcome of the powdery mildew attack, the responses may affect both susceptibility and resistance of the surrounding epidermal cells. However, we do not know if a pathogen attack in the mesophyll tissue from e.g. barley leaf rust, affects mildew susceptibility in the overlying epidermal cells. This was examined in the present study. Barley leaves were first inoculated with either a compatible or incompatible barley leaf rust isolate. When the rust had reached the mesophyll, the leaves were challenge inoculated with a compatible powdery mildew isolate. In both situations the epidermal cells showed reduced susceptibility towards powdery mildew, indicating that rust infection attempts in the mesophyll leads to induced resistance in the epidermis. However, the induced resistance was also observed well before the rust fungi infected the mesophyll, i.e. immediately after an epidermal cell was crossed by a rust hypha. Therefore the reduced susceptibility was more likely caused by growth of rust hyphae across the epidermal surface, than by rust attack in the mesophyll. Our finding supported this, because every time a rust hypha passed across an anticlinal cell wall, a short lateral branch was induced and below this, the epidermal cell accumulated papilla like material including autofluorescent compounds. Furthermore, the epidermal nucleus often moved towards the crossing rust hypha, indicating recognition of the hypha by the epidermal cell. To eliminate the surface effect of the rust, the abaxial side of the leaf was inoculated with rust and incubated for 5 days allowing the infection to progress all the way through the mesophyll cell layers. Then the adaxial side were challenge inoculated with mildew. This eliminated the reduced mildew susceptibility in the epidermal cells. Furthermore, epidermal cells on the adaxial leaf surface containing a rust haustorium (formed by rust growing from the abaxial surface) show increased mildew susceptibility. These epidermal cells, containing haustoria of both pathogens showed no signs of cell death and were able to support growth of both fungi at the same time. In conclusion, growth of rust hyphae across epidermal cells induces resistance to powdery mildew. Contrary, rust infection and haustorium formation in an epidermal cell (infected by rust from the inside of the leaf) strongly suppress resistance responses to powdery mildew. However, it is still unclear if responses to a pathogen in one tissue directly affect susceptibility to pathogens in other tissues in barley.
1.12 Resistance to leaf rust in wheat conferred by slow rusting gene Lr46
Julio Huerta-Espino1 and Ravi P Singh2
1INIFAP-CEVAMEX, Chapingo, Mexico. 2International Maize and Wheat Improvement Center (CIMMYT), Mexico
J.Huerta{a}cgiar.org
Leaf rust, caused by Puccinia triticina, is an important disease of wheat (Triticum aestivum) worldwide. Genes Lr34 and Lr46 are the only designated genes that confer slow rusting resistance. Chromosome substitution lines ‘Lalb(Pvn1B)’ and ‘Lalb(Parula 7D)’ carry genes Lr46 and Lr34 in chromosomes 1BL and 7DS, respectively in the susceptible background ‘Lalbahadur’. We used these lines to determine the effect of Lr46 on three components of slow rusting in adult plants in two replicated greenhouse trials. Fully extended flag leaves were uniformly inoculated with Mexican P. triticina race MCJ/SP and after incubation plants were placed in a greenhouse maintained at 15-200C. The mean latent periods of substitution lines with Lr46 and Lr34 were 117% and 126% and mean uredinium size 72% and 33% respectively, compared to that of the susceptible check. Lalbahadur, its chromosome 1B monosomic line and four Lalb(Pvn1B) substitution lines were grown in field trials for two seasons in northwestern Mexico under fungicide protected and non-protected conditions. Leaf rust epidemic was initiated by inoculating the spreader rows with race MCJ/SP. The area under the disease progress curves of susceptible Lalbahadur and its monosomic line were similar and those of substitution lines were significantly reduced to 36-43%. Losses of 48% and 46% in grain yields for the susceptible checks were significantly higher than the losses of 19-25% for the four Lr46 carrying substitution lines. Presence of Lr46 also reduced losses in grain weight, test weight, biomass, etc. From our studies we draw two conclusions: 1) both Lr46 and Lr34 have pleiotropic effects on the components of slow rusting, and 2) although Lr46 does not confer adequate protection when present alone, its presence slows down the leaf rust progress in the field and thus reduces the losses in grain yield significantly. Gene Lr46 must be combined with other slow rusting genes to achieve satisfactory control.
1.13 Modifers of disease resistance
Lesley A Boyd, James Melichar, Ruth MacCormack
John Innes Centre, Norwich, UK
lesley.boyd{a}bbsrc.ac.uk
Many mutation-derived alleles have found their way into commercial crop varieties (www-infocris.iaea.org). For cereals, the mlo allele of barley, conferring resistance to the fungal pathogen causing powdery mildew, is a well documented example (Jorgensen, 1992). The ability of a mutation-derived allele to enhance resistance would indicate either a modifier effect of the gene on the resistance process, or an alteration in a gene product that prevents the establishment of a compatible association between the plant and the pathogen. A number of mutants exhibiting enhanced resistance to the biotrophic fungal pathogens causing yellow rust, brown rust and powdery mildew have been isolated from wheat (Boyd et al., 2002). The mutant phenotypes are developmentally regulated in that resistance is expressed at specific, plant growth stages (Boyd and Minchin, 2001). Preliminary genetic analysis suggests that in many of the mutants the enhanced resistance is due to a single mutation of partial effect (Smith et al., 2004). Mapping populations are being analysed using DNA markers to identify the mutant loci responsible for the enhanced resistance to each fungal pathogen. Histological examination of yellow rust development and arrest of the pathogen in two of the mutants suggests that enhanced resistance is not due to an early, hydrogen peroxide mediated event.
Boyd LA, Minchin PN, 2001. Wheat mutants showing altered adult plant disease resistance. Euphytica 122, 361-68.
Boyd LA, Smith PH, Wilson AH, Minchin PN, 2002. Mutations in wheat showing altered field resistance to yellow and brown rust. Genome 45, 1035-40.
Jorgensen JH, 1992. Discovery, characterisation and exploitation of Mlo powdery mildew resistance in barley. Euphytica 63, 141-52.
Smith PH, Howie JA, Worland AJ, Stratford R, Boyd LA, 2004. Mutations in wheat exhibiting growth stage specific resistance to biotrophic fungal pathogens. Molecular Plant-Microbe Interactions (in press).
1.14 Flor revisited: Systems biology in barley-powdery mildew interactions
Rico A Caldo1, Dan Nettleton2, Dennis Halterman1,3 and Roger P Wise1,3
1Department of Plant Pathology and Center for Plant Responses to Environmental Stresses, 2Department of Statistics, 3Corn Insects and Crop Genetics Research, USDA-ARS, Iowa State University, Ames, USA
rpwise{a}iastate.edu
Active plant defense to microbial attack is highly dependent upon recognition events involving associated gene products in the host and the pathogen. Both perception of general and specific pathogen-associated molecules result in signal transduction cascades ultimately leading to disease resistance. General elicitors, which include proteins, glycoproteins, peptides, carbohydrates and lipids, signal the presence of the pathogen and are able to trigger defense responses in a non-cultivar specific manner. In contrast, specific effectors, encoded by pathogen avirulence genes, trigger cultivar-specific responses resulting in hyperactivation of basal defense, which is often accompanied by hypersensitive cell death. This specific recognition in plant-pathogen interactions conforms to the gene-for-gene hypothesis and is determined by direct or indirect interaction of host resistance (R) proteins and cognate pathogen-derived avirulence (AVR) effectors.
To ascertain the global framework of host gene expression during biotrophic pathogen invasion, we analyzed in parallel the mRNA abundance of 22,792 host genes throughout 36 (genotype x pathogen x time) interactions between barley (Hordeum vulgare) and Blumeria graminis f. sp. hordei (Bgh), the causal agent of powdery mildew disease. A split-split-plot design was used to investigate near-isogenic barley lines with introgressed Mla6, Mla13, and Mla1 CC-NBS-LRR resistance alleles challenged with Bgh isolates 5874 (AvrMla6, AvrMla1) and K1 (AvrMla13, AvrMla1). A linear mixed model analysis was employed to identify genes with significant differential expression (p-value<0.0001) in incompatible and compatible barley-Bgh interactions across six time points after pathogen challenge. Twenty-two host genes, of which nine were of unknown function, exhibited highly similar patterns of up-regulation among all incompatible and compatible interactions up to 16 hours after inoculation (hai), coinciding with germination of Bgh conidiospores and formation of appressoria. In contrast, significant divergent expression was observed after 16 hai, during membrane-to-membrane contact between fungal haustoria and host epidermal cells, with notable suppression of the steady state levels of most transcripts in compatible interactions. These findings provide a link between the recognition of general and specific pathogen-associated molecules in the expression of plant defense responses and its implications on the evolution of host-specific resistance from the recognition and prevention of the pathogen’s suppression of plant basal defense.
Research supported by USDA Initiative for Future Agriculture and Food Systems (IFAFS) grant no. 2001-52100-11346, USDA National Research Initiative (NRI) grant no. 02-35300-12619, and the USDA-CSREES North American Barley Genome Project.
1.15 Gene transcript profiling of individual Blumeria graminis attacked barley epidermal cells
Torben Gjetting1, Peter Hagedorn1, Patrick Schweizer2, Michael F. Lyngkjær1
1Risø National Laboratory, Roskilde, Denmark. 2Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany
m.lyngkjaer{a}risoe.dk
Resistance or susceptibility in barley to the powdery mildew fungus (Blumeria graminis f.sp. hordei, Bgh) is determined at the single cell level. Even in ‘compatible’ interaction, attacked barley epidermal cells tries to prevent fungal penetration by reinforcing their cell wall. However, this defence is only partially efficient and a number of fungal penetration attempts will succeed and the attacked barley epidermal cell will be infected. The molecular basis of the interaction between barley and powdery mildew has been studied intensively at the leaf and tissue level. However, the mixture of infected and uninfected epidermal cells on a leaf makes it very difficult to relate Bgh-induced gene expression to resistance or susceptibility. To overcome this problem we have extracted contents from single barley epidermal cells after inoculation with Bgh, using glass micro-capillaries and micromanipulation and are for the first time able to separate induced gene expression in resistant and infected cells. Here expression data for two situations are presented: 1) the attacked barley cell resisted fungal penetration and 2) the attacked barley cell was infected by the fungus and contained a fungal haustorium. The contents of mRNA in the micro-extract was purified and processed into cDNA and amplified by PCR. The cDNA pools were used as template in gene specific PCR of selected genes, as radioactively labelled samples in dot-blot/array hybridisation (Gjetting et al., 2004). Detailed expression analysis was performed on micro-arrays spotted with 10 K barley genes and showed very different gene expression profiles for resistant and infected cells, ca. 2800 genes were significantly induced or suppressed in one of the situations. Two examples are GLP4 – encoding a germin-like protein that accumulated specifically in resistant cells, while GRP94 – encoding a molecular chaperone, accumulated in infected cells.
Gjetting T, Carver TLW, Skøtt L, Lyngkjær ML, 2004. Gene expression profiling of individual barley epidermal cells attacked by powdery mildew. Molecular Plant-Microbe Interactions 17, 729-738.
1.16 Positional cloning of powdery mildew resistance genes at the Pm3 locus of hexaploid wheat and characterization of a Pm3 haplotype
Nabila Yahiaoui, Payorm Srichumpa, Susanne Brunner and Beat Keller
Institute of Plant Biology, University of Zürich, Switzerland
nabila{a}botinst.unizh.ch
In wheat, race-specific resistance to the fungal pathogen powdery mildew (Blumeria graminis f. sp. tritici) is controlled by Pm genes. There are 10 alleles conferring resistance at the Pm3 locus (Pm3a to j) on chromosome 1AS of hexaploid bread wheat (Triticum aestivum L.). For the map-based cloning of the Pm3b gene from the large genome of hexaploid wheat, we have used a combined analysis of genomes from wheat species with different ploidy levels. Genetic mapping of Pm3b was performed in hexaploid wheat. The diploid Triticum monococcum and the tetraploid Triticum turgidum ssp. durum provided models for the A genome of hexaploid wheat and allowed us to establish a physical contig spanning the Pm3 locus. Although haplotypes at the Pm3 locus differed markedly between the three species, a large resistance gene-like gene family was consistently found at the Pm3 locus. Partial sequence conservation between resistant line Chul and T. monococcum combined with mutational analysis allowed the identification of a Pm3b candidate gene. The candidate gene, a member of the CC-NBS-LRR type of disease resistance genes conferred race-specific resistance to wheat powdery mildew in a transient single cell transformation assay. Haplotype studies in lines carrying known Pm3 specificities (Pm3a to j) indicated a good conservation of the haplotype in the Pm3 region. This led to the rapid identification of additional alleles at the Pm3 locus. The successful cloning of the Pm3 genes provides molecular tools to study disease resistance gene specificity determination and disease resistance gene evolution in cultivated and wild wheat species.
1.17 Isolation and characterization of the leaf rust resistance gene Lr10 from hexaploid wheat
Beat Keller, Catherine Feuillet, Silvia Travella, Nils Stein and Laurence Albar
University of Zurich, Switzerland
bkeller{a}botinst.unizh.ch
Subgenome map-based cloning was performed to isolate the Lr10 resistance gene on chromosome 1AS in hexaploid wheat. A T. monococcum BAC contig spanning more than 450 kb of the region orthologous to the Lr10 locus in hexaploid wheat was established and full-length sequencing of 211 kb spanning the gene identified two resistance gene analogs which cosegregate with Lr10 in more than 3000 F2 plants. The orthologs of both genes (T10rga1 and T10rga2-1A) were isolated from the resistant hexaploid wheat variety Thatcher Lr10. Haplotype studies at the Lr10 locus showed that recombination is totally suppressed between the two genes and that further genetic analysis cannot help to identify Lr10 from the two candidates. Evidence for the identity of the gene was obtained through mutational and transformation analysis: Single point mutations were identified in the T10rga1 gene in three independent EMS mutants of Thatcher Lr10. In addition, T10rga1 conferred resistance to leaf rust in transgenic wheat plants demonstrating that it is the Lr10 gene. Interestingly, leaf rust resistance was dramatically increased in transgenic plants compared to wild type. Our data demonstrate that map-based cloning is feasible in hexaploid wheat using adequate genetic, evolutionary and genomic tools.
1.18 The structure and possible origin of the barley mlo-11 mildew resistance allele
Pietro Piffanelli, Luke Ramsay,
Robbie Waugh, Abdellah Benabdelmouna, Angélique D´Hont, Karin Hinze, Jørgen
Helms Jørgensen, Paul Schulze Lefert, and Ralph Panstruga
panstrug{a}mpiz-koeln.mpg.de
Barley plants
carrying loss-of-function alleles (mlo) of the Mlo locus are
resistant against all known isolates of the widespread powdery mildew fungus.
The only mlo resistance allele recovered to date from a natural habitat,
mlo-11, was originally retrieved from Ethiopian landraces and nowadays
controls mildew resistance in the majority of cultivated European spring barley
elite varieties. Haplotype analysis revealed that the mlo-11 allele is
likely to have arisen only once after barley domestication. Resistance in mlo-11
plants is linked to a complex tandem repeat array inserted upstream of the
wild-type gene. The repeat units consist of a truncated Mlo gene
comprising 3.5 kb of 5’ regulatory plus 1.1 kb of coding sequence. The repeat
array generates aberrant transcripts that impair accumulation of Mlo
wild-type transcript as well as protein. We exploited meiotic instability of mlo-11
resistance and recovered susceptible revertants in which restoration of Mlo
function was accompanied by excision of the repeat array. We infer cis-dependent
perturbation of transcription machinery assembly by transcriptional
interference in mlo-11 plants as a likely mechanism leading to disease
resistance.
1.19 Diversity, dispersal and evolution of Puccinia striiformis f.sp. tritici
Mogens S Hovmøller and Annemarie F Justesen
Danish Institute of Agricultural Sciences, Research Centre Flakkebjerg, Slagelse, Denmark
mogens.hovmoller{a}agrsci.dk
Genetic diversity is often low in populations of Puccinia striiformis f.sp. tritici although it may vary considerably between region, year and host variety. We have studied diversity in samples of P. striiformis from north-west Europe, north-east Africa and Central Asia, measured by AFLP variation and virulence traits assayed by extended sets of differential varieties. The sampling in NW-Europe covered the UK, France, Germany and Denmark in a period of 15 years, whereas samples from NE-Africa and Central Asia were obtained in 2002 and 2003. In all cases, they were collected from a wide range of locations and varieties in field trials and from farmers’ fields in these regions.
Diversity for AFLP was generally very low in the NW-European P. striiformis population, whereas diversity between populations from different continents was much larger. However, we also observed clonal lineages originating from very distant areas, which had almost identical AFLP patterns, suggesting that spores of the yellow rust fungus may potentially move across very large distances within a relatively short period of time. In the NW-European material, we observed some cases with diversity for virulence pathotype within clonal lineages defined by AFLP polymorphism based on up to 256 +2 primer combinations, which suggest that mutation for gain or loss of specific virulence/avirulence traits may occur fairly frequently. There was no indication for any kind of recombination within the time and area represented by the European samples.
The population dynamics of P. striiformis have a large impact on the expected control of yellow rust by host resistance, leading to either a decrease or an increase in the ability of specific sources of resistance to control yellow rust in specific areas. A striking example has recently been observed in Denmark: yellow rust was absent under natural conditions in 1996, indicating an extinction of the P. striiformis population in Denmark at that time; the subsequent re-colonization of a new population from external sources resulted in a complete change in susceptibility for many varieties. For instance, some of the most widely grown varieties between 1997 and 2003 were fully resistant under field conditions, although the underlying source of resistance was largely ineffective for yellow rust control in the early 1990s. This points to the need for ongoing monitoring of virulence dynamics in different wheat growing regions and for encouragement of breeders to select sources of resistance which are less vulnerable to the inevitable and continual dispersal and evolution of P. striiformis.
1.20 Clonality of wheat yellow rust population in France and high diversity in China
Jérôme Enjalbert, X Duan, A Wan, M Leconte, C de Vallavieille-Pope
INRA Grignon, France
enjalber{a}grignon.inra.fr
Puccinia striiformis f.sp. tritici (PST) has long story of breaking down cultivar resistance genes used by breeders. In Europe, a clonal behaviour of the species has been demonstrated using molecular markers, associated with a long distance dispersion capacity. Despite these spore flows, French PST populations present a strong geographic structure for virulence genes, the northern and southern population possessing different pathotypes. Using microsatellite and AFLP markers we described the neutral diversity underlying this differentiation on highly selected genes by analysing the genetic structure of 356 French isolates belonging to 10 pathotypes collected over a 12-year period. Both on microsatellite and AFLP polymorphisms, specific alleles were described for the South-specific pathotype. This strong genetic distance reveals that northern French population belongs to the North-Western European population, whereas the southern population is related to a more extended Mediterranean population, the two sub-populations resulting from the divergence of two clonal lineages. Such steady polymorphism should be explained on the basis local adaptation of the two clonal sub-populations.
Using the same markers, we analysed a sample of 163 isolates collected in 2001 in the Gansu province, a highland region considered to be the over-summering area of PST in China. As previously suggested, genetic analyses do not support the hypothesis of a strictly clonal reproductive mode. Joint analysis of Chinese and European genotypes revealed that Mediterranean isolates have intermediate genotypes between Chinese and North-Western European ones. In comparison to European PST population, the Chinese AFLP diversity is approximately ten fold. This high diversity confirms the importance of Gansu province for the over-summering of yellow rust. The genetic divergence between Europe and China is to be related to the presence of recombination and the diffusion of wheat in Eurasia.
1.21 Analysis of molecular and pathogenic variability of Puccinia striiformis f.sp. tritici pathotype (104E137A-) in Australia
K Nazari1, FJ Keiper2, and CR Wellings1
1The University of Sydney, Plant Breeding Institute, Camden, Australia. 2Molecular Plant Breeding Cooperative Research Centre, South Australian Research and Development Institute, Adelaide, Australia
kumarse{a}camden.usyd.edu.au
Pathogenic variability has been reported in the Puccinia striiformis f.sp. tritici population following the initial introduction of pathotype 104E137A- into Australia in 1979. AFLP and classical pathogenicity analyses were performed to study genetic variability in 10 accessions of pathotype 104E137A- collected from 1979 to 1989, using single spore isolates. A total of 596 scorable fragments were detected using 9 primer combinations (35 to 86 loci per primer combination); 292 of these were polymorphic. Cluster analysis revealed that the majority of the accessions were genetically similar (82% similarity), however the accession representative of the first pathotype collected in 1979 was clearly differentiated (38% dissimilarity between this cluster and the other isolates). This finding was supported by the number of polymorphic loci detected for this accession (244), mean number of pairwise differences between the accessions (118.267±59.48), and the significant genetic differentiation detected between the accessions (FST=0.476). AMOVA analysis revealed that 47.63% of the observed genetic variation was partitioned among the 10 accessions, with 52.37% detected within accessions (among single spore isolates). Comparisons were made between molecular genetic variability and pathogenic assessments using an extended set of differential varieties.
1.22 Has the European barley powdery mildew population reached Beijing? Not yet!
Antonin Dreiseitl1, Junmei Wang2, Jianming Yang2 and Qiuquan Shen2
1Agricultural Research Institute Kromeriz Ltd., Czech Republic, 2National Barley Improvement Centre, Zhejiang Academy of Agricultural Sciences, Hangzhou, China
dreiseitl{a}vukrom.cz
The virulence of 236 single colony isolates of Blumeria graminis f. sp. hordei collected from four Chinese locations was studied in 2003. For this, 20 differential varieties of barley possessing various genes for resistance to powdery mildew were used. The determined virulence frequencies were compared with analogical results of the Czech (Central-European) population from 2002. Both populations significantly differ from each other. In the Chinese population, virulences to resistance genes possessed by seven differential varieties were detected. No virulence to the resistance genes Mlg, Mlp, Mlat and to any used resistance genes of the Mla locus (except Mla8) was found. There were differences also among Chinese sub-populations but all of them exhibited common (“Chinese”) characteristics. Distinction between the Chinese and European isolates and their low complexity (at least until now) does not correspond with the hypothesis of Limpert et al. (2002) about the virulence accumulation of airborne nomadic pathogens in the direction of prevailing winds, i.e. from west to east ("from Paris to Beijing").
Limpert E, Bartoš P, Buchenauer H, Graber WK, Müller K, Šebesta J and Fuchs JG, 2002. Airborne nomadic pathogens: does virulence accumulate along the way from Paris to Beijing? Proceedings 6th EFPP 2002, Prague, Plant Protection Science 38 (Special Issue 1), 60–64.
1.23 Epidemic development of yellow rust in a growing wheat crop: parameters for epidemic development
Karsten D. Bjerre1, Lisa Munk1, Mogens S. Hovmøller2, and Hanne Østergård3
1KVL, Denmark. 2DIAS, Flakkebjerg, Denmark. 3Risø National Laboratory, Denmark
kdb{a}kvl.dk
Epidemics of yellow rust (Puccinia striiformis) in winter wheat were observed in a field experiment (randomized incomplete block design) with plot sizes of 8 by 8 metres. Spores were dispersed from infected seedlings in small pots, which were located centrally in each plot. Disease assessments were done in 40 subplot units in varying distance from the initial spore sources of each plot. By this, synchronized epidemics were observed in subplot units characterized by identical crop development stage but with different loads of initial inoculum due to varying distances from the source. Observations were done in year 2001 at May 22 (GS 32), June 7 (GS 50), June 20 (GS 61), and July 4 (GS 75), corresponding to four yellow rust generations. Yellow rust epidemic development was analysed by disease progress between adjacent assessment dates (three periods). A discrete time step logistic growth model extended with effects of spore influx was used in analysis of disease severity changes during subsequent periods. The model is: yt+1(yt) = r*(yt+m) /(1+(r-1)*(yt+m)/K), where yt is the disease severity at time point t, r is the finite epidemic rate, K is the maximum disease severity, and m is the disease effect of spore influx. Period specific model parameters were estimated and treatment (variety * isolate) effects were evaluated. During the first and the last period, the growth model assumption of a constant leaf resource was not met due to flag leaf emergence and/or leaf senescence. An approximate correction for canopy changes was done by estimating disease severities on all leaves present throughout a period. Parameter estimates based on such normalized severities were closer to estimates from the middle period (where the canopy did not change) than estimates based on original observations. The discrete time growth model was well suited for estimation of period specific epidemic parameters.
Pielou E, 1977. Mathematical Ecology. US, New York: Wiley.
1.24 Modelling various manifestations of disease spread at different scales
Samuel Soubeyrand, Ivan Sache
INRA, Grignon, France
samuel.soubeyrand{a}grignon.inra.fr
Consider monocyclic experiments carried out to observe spread of an airborne plant disease. A lot of such experiments, whose procedures and aims are various, have been performed. These experiments particularly contrast each other by their spatial scales, from few decimeters to hundreds of meters.
Depending on the spatial scale, both sampling practices and disease manifestations vary. Sampling practices are especially concerned with sampling units (nature, quantity, distribution in the space) and disease measure (intensity, incidence, severity); they depend on the scale mainly because of cost and time considerations. Disease manifestations are, for example, decreasing concentration of symptoms with distance, anisotropy of spread, variations in host receptivity and host sensibility; they are the answers to the question "What can be seen in that experiment?"
We propose a modelling framework used to describe and compare what can be seen at different scales. It is based on two characteristics: the infectious potential and the infection law. It allows us to build models adapted to various disease measures and various disease manifestations. Parameters of these models can be estimated from measures of disease spread.
To exemplify our approach, we present two experiments concerned with spread of wheat rusts, and the two corresponding models. The first experiment deals with short scale and lesion counts on individual leaves. The second experiment deals with growers' field scale and incidence on square meter quadrats. Moreover, the second experiment differs from the first one by its anisotropic spread. These various features are taken into consideration in the two models we propose.
1.25 Molecular genetics of the barley stem rust disease: the barley perspective
Arnis Druka, N Rostoks, R Brueggeman, J Nirmala, T Drader, T Cavaleer, L Zhang, D Kudrna, B Steffenson and A Kleinhofs
SCRI, Dundee, UK
adruka{a}scri.sari.ac.uk
Stem rust disease in barley is caused by biotropic fungus Puccinia graminis f. sp. tritici. Remarkable feature of the incompatible interaction between barley and the stem rust fungus is its durability. For over 50 years, a single barley gene, Rpg1 has been successfully and extensively utilized in agriculture as only resistance gene to the stem rust. To understand molecular basis of this durability research in A. Kleinhof’s lab has focused on isolation and characterization of the Rpg1 and other barley genes involved in the incompatible interaction between barley and the stem rust fungus.
The Rpg1 gene was recently cloned. Comprehensive information on allelic variation, structure, tissue and subcellular localization and enzymatic activity of Rpg1 has accumulated. Potential Rpg1 suppressor genes were identified and isogenic transgenic barley lines were constructed. Independent, complex genetic locus encoding barley stem rust resistance genes, rpg4 and RpgQ were genetically characterized and several candidate genes were identified. The rpg4 locus confers resistance to the relatively recently emerged Rpg1 resistance breaking Puccinia graminis pathotype, Pgt QCC and the RpgQ locus confers resistance to the rye stem rust pathogen Puccinia graminis f. sp. secalis causing serious infections also on barley.
The possible molecular and cellular mechanism of the barley resistance to the stem rust disease will be discussed in the context of the present knowledge on the barley resistance genes and by integrating information available about cellular events accompanying fungal infection process.
The potential of applying some of the recently developed high throughput technologies to study stem rust disease will also be addressed.
1.26 Fine mapping of durable, broad-spectrum rust resistance genes in wheat (Triticum aestivum)
Wolfgang Spielmeyer1,2, Raja Kota1,2, Evans Lagudah1
1CSIRO Plant Industry, Canberra, Australia. 2Graingene, Griffith, Australia – a joint venture between AWB Limited, CSIRO, GRDC and Syngenta Seeds
wolfgang.spielmeyer{a}csiro.au
In wheat, durable rust resistance genes have provided protection against infection by Puccinia sp. worldwide for many decades. These genes are effective during the adult plant growth stage and have been described as ‘slow rusting’. The quantitative resistance response to a broad-spectrum of pathotypes is not accompanied by a hypersensitive reaction. We have started with the fine mapping and positional cloning of some of these genes including genes conferring durable leaf and stripe rust resistance (Lr34/Y18) and stem rust resistance (Sr2). Most progress has been made with the Sr2 gene, which was positioned within the distal 25% portion of the short arm of chromosome 3B of wheat. A high-resolution map was constructed using wheat ESTs that were previously positioned by the NSF deletion mapping project to the distal region of 3BS. A contiguous rice sequence that corresponds to the syntenic region in wheat was used to develop additional markers which resulted in the fine mapping of the resistance gene to a small genetic interval. The trait that causes pigmentation of the stem (pseudo black chaff) and is associated with resistance, was also mapped to the same genetic interval. We have begun to use a chromosome 3B specific BAC library of ‘Chinese Spring’ to isolate large DNA segments that span the target interval of a susceptible haplotype before deciding on the best approach to isolate a candidate gene for Sr2. With Lr34/Yr18 we are using an advanced backcross derived family that allows the mapping of the rust resistance genes as major genes to identify tightly linked flanking markers in preparation for the development of a high resolution mapping family.
1.27 A novel approach for assigning leaf rust resistance genes to chromosomes in wheat
Colin Hiebert, Julian Thomas, and Brent McCallum
Agriculture and Agri-Food Canada, Cereal Research Centre, Winnipeg, Canada
chiebert{a}agr.gc.ca
A new method has been used to place the unassigned leaf rust resistance (Lr) gene LrW (temporary designation) to wheat chromosome 5B. This gene has received the official name Lr52. Most Lr genes have been assigned to chromosome using monosomic analysis. Our technique used haploid-derived aneuploids to determine the chromosome carrying Lr52. Haploids (n=3x=21) were produced from plants carrying a single source of resistance, Lr52, and were pollinated with the leaf rust susceptible cultivar ‘AC Foremost’. Nearly all of the hybrids produced were resistant. However five progeny from a population of 417 were susceptible to leaf rust because of the failed transmission of most of the chromosome carrying Lr52 due to the irregular meiosis of haploid wheat. Microsatellite markers (SSR) showed that chromosome 5B from the resistant haploid parent was deficient in all susceptible hybrids. The chromosome location of Lr52 was confirmed by the demonstration of linkage to SSR markers on the short arm of chromosome 5B. No other Lr genes are on the short arm of chromosome 5B, therefore Lr52 is a new gene. A number of other genes are being analyzed using this same technique.
1.28 Breeding cereals for rust resistance in Australia
Robert F Park
The University of Sydney, Plant Breeding Institute, Camden, Australia
robertp{a}camden.usyd.edu.au
Rust diseases have caused significant losses to Australian cereal crops, and continue to pose a serious threat to production. Because cereal crop yields are generally low, genetic resistance remains the most economical means of rust control. Resistant cultivars also contribute significantly to reducing oversummer rust survival on self-sown cereals. A policy of releasing only rust resistant wheat cultivars in northern New South Wales and Queensland has resulted in industry-wide protection from the rust diseases for the past 40 years. The Australian Cereal Rust Control Program conducts annual pathogenicity surveys for all cereal rust pathogens in Australia, undertakes genetic research aimed at identifying and characterising new sources of resistance and provides a germplasm screening and enhancement service to all cereal breeding groups. These three activities are inter-dependent and closely integrated. Some recent changes in the wheat rust pathogens in Australia, including the detection of virulence for four seedling resistance genes (Yr17, Lr24, Lr37 and Sr38) and the introduction of a new pathotype of the wheat stripe rust pathogen, have provided new and significant challenges for rust resistance breeding. Several examples are given to illustrate the way in which rust isolates are used to provide information on rust resistance in cereals that can be used in breeding for resistance.
1.29 The impact of wheat stripe rust in South Africa
Zacharias A Pretorius
University Free State, South Africa
pretorza.sci{a}mail.uovs.ac.za
Since the detection of wheat stripe rust in South Africa in August 1996, it has affected the wheat industry in many ways. At the time of detection no screening or anticipatory breeding for resistance to Puccinia striiformis f. sp. tritici existed and any form of host resistance was unintentional from a South African perspective. A widespread epidemic, intensified by cultivar susceptibility, favourable weather conditions and farmer ignorance, developed in the spring wheat regions during 1996. This epidemic provided the foothold for stripe rust in South Africa, resulting in epidemics in the central and western Free State in 1997, the eastern Free State in 1998, and all summer rainfall wheat growing areas in 2002. Direct costs confronting farmers are chemical seed treatment, foliar sprays and yield and quality losses. On farm and experimental data have shown that stripe rust can reduce yield by more than 50% and hectolitre mass by 14%. The 1998 epidemic on ca. 42,000 ha winter wheat in the eastern Free State resulted in losses of ZAR 12 million. Indirect losses due to stripe rust include the initial rejection of approximately 50% of breeding lines in leading programmes, the replacement of adapted parental genotypes, a reduced frequency in releasing new cultivars, the phasing out of susceptible cultivars, additional resources for field screening, race surveys and germplasm development, and the inhibitory effect of stripe rust epidemics on leaf and stem rust development in disease nurseries. A further implication was the recommendation of stripe rust-resistant but stem rust-susceptible cultivars in certain areas, leading to a recurrence of P. graminis f. sp. tritici in the Western Cape. Stripe rust oversummers on volunteer wheat and grasses in production areas as well as in the highlands of Lesotho. It is clear that the disease has become endemic in South Africa and that resistance breeding will continue.
1.30 Assessment of adult plant resistance to stripe rust of wheat
Zacharias A Pretorius, L Pienaar, CM Bender
University of the Free State, Bloemfontein, South Africa
pretorza.sci{a}mail.uovs.ac.za
Reliable flag leaf ratings of wheat (Triticum aestivum) to stripe rust, caused by Puccinia striiformis f. sp. tritici, are often difficult to achieve under greenhouse conditions. In addition to specific temperature and moisture requirements, the germination and penetration frequency of this pathogen has been recognized as lower than other foliar-infecting cereal rust fungi. In greenhouse studies flag leaves of mature wheat plants do not respond well to prolonged periods of saturation at <10° C and leaf damage often adds to poor quality stripe rust data. To determine whether adult plant resistance can be detected in primary leaves, seedlings of selected wheat varieties were exposed to various combinations of temperature and light cycles before or after inoculation. Although considerable variation in primary leaf infection types to pathotype 6E16A- was observed, no environment appeared conducive to the expression of rust phenotypes that correlated with adult plant responses observed in the field. A system was then optimized for screening adult spring wheats by growing them in seedling cones in a greenhouse. Results were most accurate and consistent when plants were inoculated between heading and flowering. The system was validated by comparing field and greenhouse data for the 23rd and 24th CIMMYT elite spring wheat trials, as well as for segregating populations of Baviaans x Avocet S and Sunmist x Avocet S crosses. Despite some underestimation of resistance, the greenhouse system was found reliable for pure lines. Segregating ratios were mostly similar between the greenhouse and field environments, but minor genes in Avocet S influenced the reaction types in greenhouse-grown plants.
1.31 Development of a doubled haploid mapping population and linkage map for studying rust resistance in wheat
R Prins1, VP Ramburan1,2, ZA Pretorius3, LA Boyd4, WHP Boshoff5, PH Smith4, JH Louw2
1ARC-Small Grain Institute, Matieland, South Africa. 2University of Stellenbosch, Matieland, South Africa. 3University of the Free State, Bloemfontein, South Africa. 4John Innes Centre, Norwich UK. 5ARC-Small Grain Institute, Bethlehem, South Africa
pretorza.sci{a}mail.uovs.ac.za
A doubled haploid population of 150 wheat lines was constructed from the F1 of a cross between the cultivars Kariega (stripe rust resistant, stem rust susceptible) and Avocet S (stripe rust susceptible, stem rust resistant) using the wheat-maize technique. Segregation and linkage analysis of 214 DNA markers, two storage protein markers, leaf tip necrosis (Ltn) and the stem rust resistance gene Sr26 yielded 31 linkage groups of which 28 were chromosome-anchored. These linkage groups covered the entire genome of 21 chromosomes. SSR and AFLP markers were advantageous for chromosome anchoring and consolidation of the various linkage groups, respectively. A significant feature of the linkage map is a relatively low level of polymorphism for markers on the D genome, constituting approximately 19% of all markers mapped. The Kariega X Avocet S doubled haploid population and linkage maps have emerged as valuable resources for genetic studies of qualitative and quantitative traits of economic importance in bread wheat. In this regard two major QTLs for stripe rust resistance have been identified in Kariega, as well as confirmation of the Thinopyrum elongatum translocation to the long arm of chromosome 6A in Avocet S.
1.32 Identification of slow rusting resistance to leaf rust in durum wheat
Sybil A Herrera-Foessel13, RP Singh1, J Huerta-Espino2, J Crossa1, J Yuen3, and A Djurle3
1International Maize and Wheat Improvement Center (CIMMYT), Mexico. 2Campo Experimental Valle de México-INIFAP, Chapingo, México. 3Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden
Sybil.Herrera{a}evp.slu.se
Leaf rust caused by Puccinia triticina is an important disease of wheat. Slow rusting is a kind of resistance that retards the rust progress despite a compatible (susceptible) reaction and is considered to be durable. Such resistance in bread wheat (Triticum aestivum) is often based on a few genes that have small to intermediate but additive effects. However, little is known about slow rusting in durum wheat (T. turgidum). Components of slow rusting, viz. latent period (LP), receptivity (R) and uredinium size (US), were determined for seven slow rusting and two susceptible durum wheats in three greenhouse experiments and for relative area under disease progress curve (RAUDPC) and final disease rating (FDR) in three field environments at two locations in Mexico. Disease epidemic was created by artificial inoculations with BBG/BN race, which recently caused major durum wheat yield losses in North-western Mexico. The RAUDPC and FDR of the slow rusting genotypes ranging between 14-48% and 22-59%, respectively, were significantly lower than those of the susceptible checks. The ranking of genotypes across environments was also consistent. Latent period and receptivity showed significant experiment x´genotype interaction. However, uredinium size was consistent across greenhouse experiments. The two susceptible checks had significantly shorter latent period (7.95-8.00 days) and larger uredinium size (17.3-23.8 ´10-2 mm2) compared to the slow rusting genotypes (8.48-10.31days, 8.1-14.8 ´10-2 mm2). The correlations between US vs. AUDPC and US vs. FDR were high in each environment and varied between 0.86 and 0.88. The correlations of LP with AUDPC and FDR were negative and varied between -0.67 and -0.78 and -0.60 and -0.80, respectively depending on the environment. Correlations of the receptivity with AUDPC and FDR were non-significant. These slow rusting durum wheats can be utilized in developing high yielding cultivars with durable resistance to leaf rust.
1.33 Race-specific aspects of QTLs for partial resistance to barley leaf rust
Thierry C. Marcel and Rients Niks
Wageningen University, The Netherlands
thierry.marcel{a}wur.nl
Partial resistance is governed by polygenes, is prehaustorial and is not based on hypersensitivity. Partial resistance was considered race-non-specific and more durable, fitting the concept of ‘horizontal’ resistance. However, detailed observations of the partial resistance to leaf rust (Puccinia hordei) in barley (Hordeum vulgare) revealed small cultivar x isolate interactions explained by assuming a minor-gene-for-minor-gene interaction model, similar to the so-called ‘vertical’ resistance system. Six quantitative trait loci (QTLs) have been mapped on a population of 103 recombinant inbred lines (RILs) obtained from the cross between the susceptible parent L94 and the partially resistant parent Vada. The most consistent QTLs, Rphq-2, Rphq-3 and Rphq-4, have been incorporated into L94 background to obtain near isogenic lines (NILs). Each NIL was tested with a set of 21 P. hordei isolates from which a final set of three isolates was selected. Those three isolates were used to map QTLs on seedlings of the RILs’ population and to evaluate the effect of each NIL-QTL on adult plants under agricultural conditions. Rphq-2 was effective in seedlings but not in adult plants to all the isolates tested, confirming previous results indicating that Rphq-2 is plant stage dependent, while Rphq-3 was effective in seedlings and in adult plants to the three isolates. However, Rphq-4 was effective in seedlings and in adult plants to two isolates but ineffective in both development stages to the third one, demonstrating a clear race-specific effect. Those results confirm the minor-gene-for-minor-gene model suggesting specific interactions between QTLs for partial resistance and P. hordei isolates.
1.34 Identification of partial resistance to powdery mildew in spring wheat from CIMMYT
Morten Lillemo1, Maarten van Ginkel1, He Zhong-hu2 and Chen Xinmin3
1CIMMYT, México. 2CIMMYT, c/o Chinese Academy of Agricultural Sciences, Beijing, China. 3Institute of Crop Breeding and Cultivation, Chinese Academy of Agricultural Sciences, Beijing, China
m.lillemo{a}cgiar.org
Powdery mildew is an important disease on wheat that can cause severe damage in temperate and maritime climates. It has previously not been given much attention in the wheat breeding program at CIMMYT since natural epidemics are not present in Mexico. As a result, most of the wheat germplasm from the breeding program in Mexico is highly susceptible, although powdery mildew is important in many areas where CIMMYT-based wheat varieties are grown, in particular China and the Southern Cone of Latin America. As an alternative to the commonly used race-specific resistance, partial resistance offers a much more sustainable way to prevent losses from biotrophic fungi like powdery mildew. This is especially true for resource-poor farmers in developing countries, who do not use fungicides and cannot easily get access to new varieties when resistances break down. Partial or horizontal resistance is characterized by the absence of immunity or hypersensitive response, and is generally governed by several additive race-non-specific genes that work on different epidemiological components to retard disease development.
Work started at CIMMYT in late 2001 to identify genotypes with partial resistance to powdery mildew, based on international data from 64 locations where nurseries from CIMMYT had recently been planted and data on powdery mildew was reported. Care was taken to select lines that did not show immunity or high severity at any location, and a subset of 50 high-yielding lines were selected for further testing in several global ‘hot spot’ locations for powdery mildew. A seedling test has also been carried out to detect the presence of possible race-specific genes that might hamper the breeding efforts for partial resistance. Lines like for example MILAN, SAAR, DULUS, FILIN and FILIN/MILAN have confirmed a reasonable level of partial resistance to powdery mildew, and now form the basis for breeding of resistance to this disease at CIMMYT. Genetic studies are under way to learn more about the genetic control of partial resistance in these lines, and a shuttle breeding program has been initiated between Mexico and China to improve the powdery mildew resistance in CIMMYT germplasm.
1.35 Genetic analysis of yellow rust resistance in the UK
Clare M Lewis and Lesley A Boyd
John Innes Centre, Norwich,UK
clare.lewis{a}bbsrc.ac.uk
Owing to the ability of the yellow rust pathogen (Puccinia striiformis) to mutate to virulence and overcome race-specific resistance genes in wheat cultivars there has been increased interest in recent years in the exploitation of partial, adult plant resistance (APR). APR is often considered to be durable, where durability is defined as the situation where a cultivar has been grown for many years over considerable acreage and maintained adequate resistance (Johnson, 1992). The cultivar Carstens V has been attributed as the source of some of the genes that confer durable yellow rust APR in modern cultivars. Of the resistance genes Carstens V carries (Chen & Line, 1993; Calonnec et al., 2002) two have been assigned a chromosomal location. A dominant, specific resistance gene YrCv (Stubbs, 1985) was first located on chromosome 2A (Afshari, 2000) and subsequently, a seedling, race-specific resistance gene Yr32 on 2AL (Eriksen et al., 2004). It is believed that Yr32 and YrCV may be the same gene, but this has yet to be confirmed.
The wheat cultivar Claire is very resistant to Puccinia striiformis and is believed to have Carstens V in its pedigree (P. Fenwick, personal communication). Claire is being studied to determine the genetics of its yellow rust resistance and trace this within its pedigree (Carstens V, Carstens VIII, Caribo and Buster). Claire has been postulated to carry the race-specific seedling resistance genes Yr2 and Yr3. Its adult plant resistance is first observed at growth stage 22 and has been tentatively attributed to QTLs located on chromosomes 2AS, 2DS, 3DL, 4AS and 5DL in a F2 population. These QTLs are currently being precisely mapped and will be confirmed in the F3. Buster, a distant sib of Claire has been postulated to have a non-race specific seedling resistance. The cultivars within Claire’s pedigree were postulated to carry the race-specific resistances YrCv (Carstens V), Yr3 (Carstens VIII) and Yr2 (Caribo). Preliminary segregation analysis of the yellow rust APR of Carstens V (x Lemhi/Kharchia local), Carstens VIII (x Kharchia local) and Caribo (x Kharchia local) in F2 populations suggests that at least one major gene confers resistance in each cross.
Afshari F, 2000. Studies on rust resistance in wheat with particular emphasis on stripe rust. Sydney, Australia: University of Sydney, PhD thesis.
Calonnec A, Johnson R, and de Vallavieille-Pope C, 2002. Genetic analyses of resistance of the wheat differential cultivars Carstens V and Spaldings Prolific to two races of Puccinia striiformis. Plant Pathology 51, 777-786
Chen XM and Line RF, 1993. Inheritance of stripe rust (yellow rust) resistance in the wheat cultivar Carstens V. Euphytica 71, 107-113.
Eriksen L, Afshari F, Christiansen MJ, McIntosh RA, Jahoor A, Wellings CR, 2004. Yr32 for resistance to stripe (yellow) rust present in the wheat cultivar Carstens V. Theoretical and Applied Genetics 108, 567-575.
Johnson R, 1992. Past, present and future opportunities in breeding for resistance, with examples from wheat. Euphytica 63, 3-22.
Stubbs RW, 1985. Stripe Rust. In: Roelfs AP and Bushnell WR eds.The Cereal Rusts Volume II. Diseases, Distribution, Epidemiology and Control. San Diego, US: Academic Press Inc, 61-101.
1.36 Identification of phenotypic and molecular markers associated with the slow rusting resistance gene Lr46
Garry Rosewarne, RP Singh, M William and J Huerta-Espino
CIMMYT Int., Mexico
g.rosewarne{a}cgiar.org
Rust diseases cause an important constraint in wheat production worldwide. To genetically manage these diseases, race specific resistance genes have often been used. However, as such genes are often rapidly overcome by the pathogen, recent focus has been on incorporating multiple slow rusting (non-race specific) resistance genes. These types of genes are generally additive in effect, with between 3-5 being required in a single genotype to confer adequate protection. However, the incorporation of multiple genes can be problematic as minor variations due to the addition of an extra gene can be difficult to detect in the field. Closely linked markers to slow rusting genes would enhance the ability of breeders to develop cultivars that have durable resistance and also enhance genetic diversity for such genes in wheat germplasm. Molecular markers for the slow rusting resistance gene complex Lr46/Yr29 were described by William et al. (2002) and we present further work on the identification of 3 more molecular markers that are associated with this gene. The new AFLP markers were identified on a population developed between a cross of Avocet S (susceptible) and Attila (multiple additive resistance genes). Through field analysis of this population, we were able to determine the level of reduction of leaf area infection contributed by each of the markers. Furthermore, the presence of Lr46/Yr29 is highly correlated with the phenotypic marker of leaf-tip necrosis (LTN). LTN is also associated with Lr34/Yr28 and is thought to be caused through pleiotropism or a closely linked gene. It is intriguing that 2 different non-race specific genes both show this effect. We have also used single chromosome recombinant inbred populations containing Lr46/Yr29 from 2 different sources to draw an accurate linkage map of the markers surrounding this gene complex. These markers should help with the development of positive PCR markers for use by breeders and in the cloning of Lr46 and Yr29.
William M, Singh RP, Huerta-Espino J, Ortiz Islas S and Hoisington D, 2002. Molecular marker mapping of leaf rust resistance gene Lr46 and its association with stripe rust resistance gene Yr29 in wheat. Genetics and Resistance, 93: 153-159.
1.37 Discovery of single nucleotide polymorphisms linked to the crown rust resistance gene Pc68 in the cultivated oat
Gang Chen, James Chong, Mark Gray, Suvira Prashar, and J. Douglas Procunier
Cereal Research Centre, Agriculture & Agri-Food Canada, Winnipeg, Canada
jchong{a}agr.gc.ca
Crown rust (caused by Puccinia coronata f. sp. avenae) is an economically important disease of oat worldwide. One strategy to attain more durable control of the disease is through resistance gene pyramiding. Various DNA markers linked to crown rust resistance genes, Pc38, Pc39, Pc48, Pc68, Pc71, Pc91, Pc92, and Pc94 have been developed during the past decade (reviewed in Wight et al., 2004), but only very few of these have been integrated into oat breeding programs to any major extent. This is because the application of molecular markers in breeding programs requires rapid, inexpensive, and highly automated methods. Single nucleotide polymorphisms (SNPs) are the most basic forms of DNA variations in both plant and animal genomes, and are found in both the coding and non-coding regions of the genes. They have rapidly gained popularity as markers of choice because of their high frequency in the genome and because they are amenable to automated, high-throughput analysis (Bhattramakki and Rafalski, 2001). The objectives of this study were to describe a procedure for SNP discovery in oat and to identify SNP markers linked to crown rust resistance. Ten existing restricted-fragment length polymorphism (RFLP) markers (Chen, 2000) were used as sources of sequence information for SNP discovery, including one (cdo309) located to be within 10 cM to a cluster of linked stem rust and crown rust resistance genes by comparative mapping (O’Donoughue et al., 1996). The crown rust resistance gene Pc68 was one of the genes in the cluster. Comparative sequence alignment of RFLP-derived polymerase chain reaction products revealed 12 putative SNPs among genotypes with and without Pc68. Seven were validated by the single base extension (SBE) assay (Bhattramakki and Rafalski, 2001), including the two derived from cdo309. Results showed that the cdo309 marker was first converted to a sequence-tagged site (STS), and then to two SNP markers. Only the STS and two SNP markers derived from RFLP marker cdo309 were shown to be linked to Pc68 (4.2 cM - 6.7 cM) in two segregating populations. Plants homozygous for Pc68 were readily identifiable from heterozygous and susceptible plants with these markers. This feature is useful in resistance breeding as heterozygous plants can be eliminated in the early generations. Large numbers of RFLP markers have been used to map the oat genomes and identify regions associated with disease resistance, quality, and agronomic traits. Our results demonstrated that we could exploit this existing mapping information and convert these marker loci to functional SNPs. The SBE method used to detect SNPs in this study is user-friendly, non-gel based, and cost-effective. To our knowledge, this is the first formal report of functional SNPs in oat and of applying them to conventional genetic studies.
Bhattramakki D, Rafalski A, 2001. Discovery and application of single nucleotide polymorphism markers in plants. In: Henry, RJ, ed. Plant Genotyping: the DNA Fingerprinting of Plants. Online Bookshop (http://www.cabi-publishing.org/Bookshop): CABI Publishing, 179-192.
Chen G, 2000. Identification of QTLs for horizontal resistance to crown rust in oat. Hangzhou, China: Zhejiang University, PhD thesis.
O’Donoughue LS, Chong J, Wight CP, Fedak G, Molnar SJ, 1996. Localization of stem rust resistance genes and associated molecular markers in cultivated oat. Phytopathology 86, 719-27.
Wight CP, O’Donoughue LS, Chong J, Tinker NA, Molnar SJ, 2004. Discovery, localization, and sequence characterization of molecular markers for the crown rust resistance genes Pc38, Pc39, and Pc48 in cultivated oat (Avena sativa L.). Molecular Breeding, in press.
1.38 Fine mapping of a QTL for leaf rust resistance in barley by haplotype analysis
Xiaoquan Qi1, Saleha Bakht1, Rients E Niks2, Pim Lindhout2 and Annie Osbourn1
1 Sainsbury Laboratory, John Innes Centre, Norwich, UK. 2Laboratory of Plant Breeding, Wageningen University, The Netherlands
xiaoquan.qi{a}sainsbury-laboratory.ac.uk
A set of 5 AFLP markers, previously mapped on the distal region of the long arm of barley chromosome 2, was chosen to analyse 57 barley cultivars. The 5 AFLP markers cover ca. 6 cM with ca. 1 cM between markers, providing an ideal marker density for a test of linkage disequilibrium (LD). Data analysis indicated that LD is preserved within ca. 2 cM in this region. Two ancestral haplotypes were identified. Recombination rates are 3 to 4 times higher in this subpopulation (57 cultivars) than in 103 recombinant inbred lines derived from the cross “L94” x “Vada”. This finding in barley is consistent with results from Arabidopsis thaliana in which LD decays within 1 cM, or 250 kb (Nordborg et al 2002). The slower decay of LD in barley and Arabidopsis thaliana compared with humans is largely due to a higher degree of inbreeding. Interestingly, a major-effect QTL for resistance to barley leaf rust that was previously mapped in this region (Qi et al, 1998) was also confirmed by LD analysis using the disease data that were collected in 1980 (Parleviet et al). Because of the higher recombination frequency in the 57 barley lines than the 103 RILs, it was possible to fine map the QTL to an AFLP marker within ca. 0.2 cM.
1.39 The development and application of near isogenic lines for the wheat stripe (yellow) rust pathosystem
CR Wellings1, RP Singh2, RA McIntosh1, ZA Pretorius3
1The University of Sydney, Plant Breeding Institute, Camden, Australia. 2CIMMYT, El Batan, Mexico. 3University of Orange Free State, Bloemfontein, South Africa.
colinw{a}camden.usyd.edu.au
The development of near isogenic lines (NILs) for host and pathogen studies in wheat stripe rust (Puccinia striiformis tritici, Pst) has been attempted in several laboratories. A NIL set developed at PBI Rust Laboratory has been based on Avocet S, a selection of the spring wheat cultivar Avocet that lacks the seedling resistance YrA. This recurrent parent combines moderate vernalisation response, daylength insensitivity, and resistance to leaf rust (Lr10,13) and stem rust (Sr5,26). This genotype has shown wide adaptability across international spring wheat locations, and has shown susceptible responses to regional populations of Pst. Resistance genes were backcrossed six times with selection based on seedling tests and adult field nurseries. Genes currently completed include Yr1, Yr5, Yr6, Yr7, Yr8, Yr9, Yr10, Yr15, Yr17, Yr18, Yr24, Yr26, Yr32. Where possible, the target gene was confirmed in the final selections using pathotypes with contrasting avirulence/virulence characteristics.
The application of the Avocet NIL set encompasses several possibilities:
1. Field based nurseries for assessment of pathogenic diversity in Pst. The requirement for specialist greenhouse facilities and experienced staff has limited the capacity to monitor Pst in many regions of the world. Avocet NILs grown in short rows or small plots has served to provide useful data on prevailing Pst populations. Sets were widely distributed, and data gathered from 1999 to 2001 provided evidence for regional differences in pathogenic variability. For example virulence for Yr1 was common in the eastern regions of Central Asia, China and certain locations in India. Virulence for Yr9 was more widespread in diverse locations including the Middle East (Iraq, Turkey, Syria), China, Africa (Uganda), Australasia (New Zealand) and South and Central America (Chile, Ecuador, Mexico).
2. Revised nomenclature for pathogenic diversity in Pst. Sets of differential testers, comprising various cultivars with nominated resistance genes, have been employed by regional rust laboratories. Difficulties associated with current differentials include: (i) complications associated with interpreting pathogenicity in the presence of multiple resistance genes, many of which remain undescribed; (ii) over reliance on seedling based greenhouse tests; (iii) the lack of routine data for assessing pathogenicity associated with adult plant resistances; (iv) the lack of international agreement for common differential sets. The Avocet NIL set offers an opportunity to revise pathotype nomenclature that reflects pathogenicity assessments for seedling and adult plant resistances using methods including greenhouse tests and field nurseries. Further modification of this set, especially the development of certain gene combinations, will be necessary to detect unique pathotypes among potentially mixed infections.
3. Reference stocks for gene designation. To date, symbols have been allocated to 33 genes for resistance to Pst. As this number increases, there will be a greater need to compare putative new genes with those previously described. The Avocet NIL set will provide a repository of single gene stocks to serve as a reference source for genetic studies that will include tests of allelism, linkage analyses and development of closely linked molecular markers.
1.40 Wheat Stripe Rust Control in China
A M Wan
Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China
anmin.wan{a}163.com
Wheat is the second main stable food crop in China. However, wheat stripe (yellow) rust, caused by Puccinia striiformis Westend f.sp.tritici Eriks., is the most important disease on wheat. Measures for controlling this disease in China are 1) Application of resistance based on traditional and modern methods. Exploitation and transfer of effective genes into commercial wheat, diversification of resistance genes and types of genes. Yr1, Yr2, Yr3, Yr4, Yr9, YrA and YrSu are ineffective while Yr5, Yr10, Yr11, Yr12, Yr13, Yr14, Yr15, Yr16 and Yr18 still have high resistance; 2) Seed coating and foliar spraying with fungicides. Triadimefon is the most widely and extensively used fungicide to combat stripe rust since late 1970’s. Dinicolazole, Propiconazole and Tebuconazole were used with limited amount;
3) Race and resistance monitoring. Information of pathogen’s variability and variation of resistance provide basis for resistance breeding and deployment, and disease occurrence prediction; 4) Agricultural practices. Elimination of volunteer wheat seedlings in over-summering regions reduces acreages of ‘green bridges’ and consequently decrease inoculum; 5) Further understandings of ‘hotspot’ region in southeast of Gansu and northwest of Sichuan which appears to be the center of stripe rust diversity, origin of new races, inoculum base for other regions in China. Effective control of this disease in this region is the first and key step to achieve the fundamental control of stripe rust in China; 6) Use of new and high technology (such as information and biotechnology), co-operation among breeders, pathologists, extension personnel, administrators, engineers and etc.
1.41 Evolution of resistance to strobilurin fungicides in fungi pathogenic to cereals
RA Wyand1, HN Slatter1, U Gisi2, JKM Brown1
1 John Innes Centre, Norwich, UK. 2 Syngenta Crop Protection, Basel, Switzerland
rebecca.wyand{a}bbsrc.ac.uk
The strobilurins are an important class of agricultural fungicide. They act by inhibiting the mitochondrial respiration of the fungus by binding to the cytochrome bc1 enzyme complex at the Qo site (Qo Inhibitors, QoIs). Strobilurins were first introduced in 1996, being extensively used to control fungal diseases of cereals, as well as grapevines, turf-grass, potatoes, fruit and vegetables. However, isolates resistant to strobilurins were discovered after only a short time period in field populations of a range of important fungal pathogens, including powdery mildew of wheat (Blumeria graminis f.sp. tritici, (Bgt), 1998) and barley (B. graminis f.sp. hordei, (Bgh), 1999).
In most fungal pathogens resistance is conferred by a single point mutation in the mitochondrial cytochrome b gene, which results in the amino acid substitution of glycine with alanine at position 143 (G143A) of the protein. The G143A substitution is thought not to affect the activity of the enzyme in Bgt (Fisher et al, 2004), suggesting that resistant isolates may not suffer a significant fitness penalty. Indeed, early investigations found no cost of resistance to strobilurins in Bgt (Heaney et al, 2000; Chin et al, 2001). However, only low numbers of single pustule isolates were used in these studies.
We investigated the affect of temperature on the cost of resistance to strobilurin fungicides in the asexual stage of the fungi Bgt and Mycosphaerella graminicola (the causal agent of Septoria tritici leaf blotch of wheat). We used a large segregating population of Bgt, derived from a cross between the strobilurin resistant parental isolate Fel09 and the sensitive parent JIW11. A mixed population of QoIR and QoIS progeny was cycled for one generation on untreated leaves, at different temperatures (20ºC, 15ºC, 10ºC and 5ºC), and the percentage of QoIR isolates in the mixture at generation 0 and 1 was calculated using a spore germination assay. A clear selection against QoIR isolates was observed at low temperature (e.g. at 5ºC, the selection coefficient s=0.30).
A cost of strobilurin resistance at low temperatures was also observed for M. graminicola. 39 single pycnidial isolates (21 QoIR and 18 QoIS) of M. graminicola, from four countries in north-west Europe, were separately inoculated on to detached leaves of two susceptible UK wheat varieties. The detached leaves were placed under the different temperature regimes (20ºC, 15ºC, 10ºC and 5ºC). The percentage leaf area with lesions bearing pycnidia was scored daily for 15 days, from the onset of pycnidial appearance, for each isolate at each temperature. Percentages were then used to calculate the Area Under the Disease Progress Curve (AUDPC). At 5ºC, QoIR isolates showed a 20% reduction in fitness (mean AUDPC) compared to QoIS isolates.
To our knowledge, this is the first time that a cost of resistance has been reported for more than one fungus to any fungicide. However, it does not necessarily explain the difference in the rate of evolution of QoI-resistance in the two fungi.
Chin KM, Chavaillaz D, Kaesbohrer M, Staub T, Felsenstein FG, 2001. Characterizing resistance risk of Erysiphe graminis f.sp. tritici to strobilurins. Crop Protection 20, 87-96
Fisher N, Brown AC, Sexton G, Cook A, Windass J, Meunier B, 2004. Modelling the Q(o) site of crop pathogens in Saccharomyces cerevisiae cytochrome b. European Journal of Biochemistry 271, 2264-2271.
Heaney SP, Hall AA, Davies SA, Olaya G, 2000. Resistance to fungicides in the QoI-STAR cross resistance group: current perspectives. BCPC conference: Pests and Diseases 2000, 755-762
1.42 Yield effects and control of powdery mildew in winter wheat in the presence of Septoria
Lise Nistrup Jørgensen, Hans Pinnschmidt
Danish Institute of Agricultural Sciences, Slagelse, Denmark
Lisen.jorgensen{a}agrsci.dk
Wheat powdery mildew (Blumeria graminis f.sp. tritici) has been a very common disease of winter wheat in Denmark for many years. The start of the epidemic is variable and depends on sowing time, cultivars, soil type and locality. Because of this variability, decision support systems like Crop Protection Online (CPO) still require input from individual fields in order to assess the risk and recommend the use of fungicides. In susceptible varieties control is recommended, for example, if 10-25% of the plants are infected with mildew at GS 30-31. However, experience from fungicide trials has shown that yield responses and margin over fungicide cost from control of mildew is often relatively low, indicating that mildew is considerably less yield reducing compared with, for example, septoria (Septoria tritici). A dataset of approximately 100 winter wheat trials from 1985-2003, carried out using mildew and septoria susceptible cultivars, has been analysed for responses to specific mildew fungicides as well as broad-spectrum fungicides applied twice in the season (GS 30-31 & 45-55). The aim was to analyse the factors that play a major role in yield responses when applying mildew specific fungicides.
Severity levels of powdery mildew were considerably lower than severity levels of Septoria diseases. Both diseases often occurred in combination and with high variation in their severity levels. Yield increases resulting from applying mildew specific fungicides ranged from -0.35 to 1.95 t/ha with an average of 0.57 t/ha. They were thus lower than yield increases due to applying broad-spectrum fungicides, that ranged from –0.04 to 3.39 t/ha with an average of 1.4 t/ha. In plots treated with mildew specific fungicides yield increases were primarily related to parameters of mildew severity, while in plots treated with broad-spectrum fungicides yield increases were primarily related to Septoria severity, but also to a significant extent to powdery mildew severity. The relationships between disease severity levels and yield increase were best described by monotonously increasing curves flattening with increasing disease severity levels. Regression and general linear modelling analyses confirmed that Septoria-induced yield losses were on average higher than mildew-induced yield losses. The higher yield gains in the broad-spectrum treated plots, as compared with plots treated specifically for mildew, were apparently due to the fact that the broad-spectrum fungicides gave superior control of Septoria and significantly reduced mildew as well. Mildew specific fungicides gave a better control of mildew than broad-spectrum fungicides, but had little effect on Septoria. A model containing fungicide treatment specific parameters for mildew and Septoria severity as covariates explained about 54% of the variation in yield increase of the plots treated with broad-spectrum fungicides, while explaining about 34% of the variation in yield increase of the plots treated with mildew-specific fungicides. All effects in the model were highly significant. The residuals of this model could be partly explained by significant effects of additional variables such as soil type and year. Our results show that multiple disease scenarios alter the economics of powdery mildew control and require adaptations of appropriate control tactics and strategies.
It is not possible from this dataset to separate yield responses from 1st and 2nd treatment, but other data has generally shown low yield responses from early fungicide treatments (T1), where mildew is often considered to be the major target. These results, as well as the data presented here, confirm that it is appropriate to recommend (as it is done in CPO) a broad-spectrum fungicide for mildew control after GS 32 rather than a mildew specific fungicide. A high level of Septoria is to be expected in most years and therefore this strategy ensures an overall acceptable yield gain. The results also indicate a large uncertainty regarding the use of the existing thresholds for control of mildew, as a poor correlation has been found between the level of attack (GS 30-31) and yield increases from using specific mildew products.
1.43 The effect of fungicides on the quality of malting barley
Keith Cottrell and S Rossall
University Nottingham
stxkdc{a}nottingham.ac.uk
Fungal diseases of barley can bring about a devastating loss in yield and diminish grain quality. Farmers have quality and quantity contracts to meet with maltsters, who in turn have quality contracts to comply with. Consequently, fungal diseases have the ability to damage contractual relationships between farmers and maltsters. Fungicides have the ability to limit such impact by preventing or reducing disease, thereby improving business stability and yield security.
The aim of the project is to investigate the effect modern azole and QoI fungicides have on the quality of malting barley. The first year field trial focused on the timing of fungicide application and its effects on grain and malt quality. A fungicide mixture of Acanto (Picoxystrobin), Sanction (Flusilazole) and Corbel (Fenpropimorph) was used at full field rates, with the aim to accentuate the effects of application timing. Barley cultivars susceptible to Rhynchosporium secalis and Powdery Mildew (Blumeria graminis) were grown and subjected to a factorial spraying regime of this mixture. The fungal pathogens to which cultivars are susceptible are different in their methods of causing disease; R. secalis is a necrotroph, which destroys photosynthetic area and B. graminis a biotroph, which transfers assimilates from the plant to the pathogen. Consequently, each may have different effects on barley quality. Plant growth analysis was conducted throughout development and post harvest grain analysis performed to determine any quality improvements. Micro-malting was performed on harvested grains and their potential value and viability to end users assessed.
In addition controlled environment work has investigated the effects of powdery mildew infection on barley hordein content. The ratio of hordeins has been shown to be related to malt quality, it is postulated that the biotroph will alter hordein ratios and therefore affect quality.
1.44 Foliar disease severity and stability of grain yield and quality of wheat cultivar mixtures in on-farm trials
Claude de Vallavieille-Pope1, Makram Belhaj Fraj1, Jean-Marc Meynard2, Hervé Monod3, and Bruno Mille1
1UMR INRA/INA-PG d’Epidémiologie Végétale et Ecologie des Populations, Thiverval–Grignon, France, 2UMR INRA/INA-PG d’Agronomie, Thiverval–Grignon, France, 3INRA, Unité de Biométrie et d’Intelligence Artificielle, Jouy-en-Josas cedex, France
pope{a}grignon.inra.fr
Mixtures of cultivars having different resistance genes can reduce the progression of foliar diseases (Finckh et al., 2000; Mundt, 2002). However, few studies have been conducted to assess cultivar mixture efficiency in agricultural conditions. A series of on-farm trials of four-cultivar mixtures of bread wheat and the corresponding pure stands, totalling 19 environments over 180 ha during 2 years, were grown under an integrated protection crop management system intended to reduce the use of both fungicides and N fertilizers by 30% compared to conventional wheat crops. Only one foliar fungicide spray was applied in most of the trials. The advantages of the mixtures over the means of the pure stands were: less foliar disease severity (mainly septoria blotch the dominant disease, and also brown rust and powdery mildew when present), greater grain yield (+ 0.32 t ha-1), and higher grain protein content (+ 0.54%). The performances of cultivar mixtures were close to that of the best cultivar grown in pure stand. The average baking score of the mixtures was as good as the mean of the pure stands. We can explain part of the advantage on grain quality by cultivar complementation for nitrogen use. The grain yield of the cultivar mixtures was more stable than that of the most productive cultivar, and was clearly more stable than all of its components grown in pure stands when the conditions were not optimal. Cultivars present in the harvest can be identified by microsatellite markers (Belhaj Fraj et al., 2003). This technique facilitates commercial controls and could be very useful for backing up legislation on the cultivation of cultivar mixtures.
1.45 How powdery mildew becomes virulent
Christopher J Ridout
John Innes Centre, Norwich, UK
christopher.ridout{a}bbsrc.ac.uk
Race-specific resistance genes in cereals are rapidly overcome by pathogens, limiting their value in breeding for disease resistance. In barley, race-specific Mla resistance proteins recognise avirulence (Avr) gene products from the powdery mildew fungus Blumeria graminis f. sp hordei. This event triggers hypersensitive cell death, preventing further growth of Bgh. The recent identification of a novel Avr gene family in Bgh will enable investigations into Avr function, recognition and evolution.
Two members of the Bgh Avr gene family (Avrk1 and Avra10) were isolated by map-based cloning, and their specific recognition in the barley cell demonstrated by a transient assay procedure. The gene family comprises over 30 homologues, their proliferation in the genome implying an essential role in the biotrophic lifestyle of Bgh. The proteins lack a signal peptide, but contain features that may enable them to cross the plant membrane. The Avr gene family was detected in other formae speciales of B. graminis infecting other grasses.
Bgh Avrs can be compared to bacterial effectors, which enter and manipulate host cells to enhance infection (van’t Slot & Knogge, 2002). In bacteria, pathogenicity results from the cumulative effect of several complemetary effectors, their individual contributions being hard to quantify (Dangl, 1994). If Bgh avirulence proteins are effectors that functionally complement each other, several can be lost by mutation without compromising the ability to infect. This would explain why virulence rapidly evolves without a fitness cost when individual avirulences are lost (Bronson, & Ellingboe 1986, Brown & Wolfe, 1990)
The isolation of Bgh Avr genes will enable investigations of how this novel class of protein promotes parasite success, and of how specific alleles trigger Mla-allele dependent resistance. Experiments to investigate the expression, delivery and localisation of Avr proteins are in progress. To establish whether the Avr proteins are also effectors, a search for pathogenicity targets has been initiated. The isolation of further homologues will enable studies on the evolution of the gene family within and between formae speciales. This novel gene family may reveal new targets for disease control, and possibly enable a more predictive approach to the selection of R genes in plant breeding.
Bronson CR, Ellingboe AH, 1986. The influence of 4 unnecessary genes for virulence on the fitness of Erysiphe graminis f. sp tritici. Phytopathology 76, 154-158.
Brown JKM, Wolfe MS, 1990. Structure and evolution of a population of Erysiphe graminis f. sp. hordei. Plant Pathology 39, 376-390.
Dangl JL, 1994. The enigmatic avirulence genes of phytopathogenic bacteria. Current Topics in Microbial Immunolology 192, 99-108.
van’t Slot KAE, Knogge W, 2002. A dual role for microbial pathogen-derived effector proteins in plant disease and resistance. Critical Reviews in Plant Science 21, 229-271.
1.46 Inheritance of avirulence in the wheat leaf rust fungus Puccinia triticina
Brent McCallum, Barbara Mulock, Daryl Somers and Guus Bakkeren
Cereal Research Centre, Agriculture and Agri-Food Canada, Winnipeg, Canada
bmccallum{a}agr.gc.ca
A series of crosses between different wheat leaf rust (Puccinia triticina) isolates was analyzed and stored in the late 1960's by researchers at the Agriculture and Agri-Food Canada Cereal Research Centre in Winnipeg. In 2000, we revived over 300 F1 and F2 progeny from four unique populations derived from the cross (race 9 (SBDG) x race 161 (FBDS)) and determined their virulence spectra on 28 near isogenetic wheat lines at the seedling stage. Segregation for virulence was observed on genes Lr1, Lr2a, Lr2b, Lr2c, Lr3, Lr3ka, Lr3bg, Lr11, Lr30, LrB, Lr14a, Lr14b, Lr18, and Lr28 in one or more of the populations. Avirulence to Lr1, Lr2a, Lr2b, Lr2c, Lr3, Lr3ka, Lr3bg, Lr11, Lr30, Lr14a, and Lr18 appears to be controlled by single dominant genes corresponding to each resistance host gene whereas avirulence to LrB, Lr14, and Lr28 appears to be controlled by single recessive genes. Segregation for avirulence to the adult plant resistance genes Lr22b and Lr12 was also observed when adult plants with these genes were inoculated. This series of crosses is currently the basis for a genomic study where we are generating molecular markers by amplified fragment length polymorphisms. A total of 128 primer combinations have generated 406 markers within the four F2 populations.
1.47 Generation of a wheat leaf rust, Puccinia triticina, EST database and microarray from stage-specific cDNA libraries
Guanggan Hu1, Rob Linning1, Andrena Kamp1, Chani Joseph1, Brent McCallum2, Travis Banks2, Sylvie Cloutier2, Yaron Butterfield3, Jerry Liu3, Robert Kirkpatrick3, Jeff Stott3, George Yang3, Duane Smailus3, Steven Jones3, Marco Marra3, Jacqueline Schein3, Jianming Pei4, Tim Westwood4 and Guus Bakkeren1
1Pacific Agri-Food Research Centre, Agriculture and Agri-Food Canada, Summerland, Canada
bakkereng{a}agr.gc.ca
We have constructed cDNA libraries from small amounts of mRNA representing several developmental stages of the wheat leaf rust fungus, P. triticina. These stages included mature urediniospores and urediniospores germinated on water or plant extract at 2 and 16 hrs. Compatible interactive stages on wheat encompassed appressorium (5 hrs post-inoculation) and haustorium formations (5 days pi, just before sporulation) as well as 24 hrs pi on an incompatible host. The transcripts were subjected to various treatments such as full-length cDNA production, subtractive and normalizing hybridizations, and size selection methods combined with PCR amplification or traditional cloning using adapters. Covering the first 4 stages, we sequenced over 14,500 clones from 13 cDNA libraries from either the 5’- or 3’-end, and 7727 clones from the incompatible stage. ‘Interactive libraries’ were subtracted in silico for wheat sequences using public cereal EST and genomic sequences, and for WLR sequences using public fungal genomic and EST sequences. A second approach exploited differences in codon usage. A non-redundant set of 4,278 ESTs putative fungal ESTs representing 2 Mbp of the transcriptome, was assembled. Eighteen percent of these ESTs exhibited homology via BLAST (e>10 -5) to known genes in public databases while an additional 25% match other ESTs or genome sequences of unknown function. Of the ESTs with similarity matches, 27% fit most closely sequences of fungal origin (63% of which from basidiomycetes). Information on gene categories will be presented. An extension of the set with 25,000 ESTs covering other stages (including on the alternate host) is in progress.
A microarray harboring approximately 4,800 sequences was constructed from cDNA inserts produced by PCR from standard forward and reverse primers. The analysis of global gene expression patterns during switches in life cycle stages is in progress. This research has been funded by the AAFC Crop Genomics Initiative
1.48 Isolation of genes expressed during compatible interactions between leaf rust (Puccinia triticina) and wheat using cDNA-AFLP and haustorial isolation
Lin Zhang1, Helen Meakin and Matt Dickinson
School of Biosciences, University of Nottingham, Loughborough UK
matthew.dickinson{a}nottingham.ac.uk
Puccinia triticina is an excellent model system for isolating genes expressed during compatible interactions with plants because of the synchronous infection that occurs when urediospores are inoculated onto a susceptible host. The fungus goes through a number of developmental stages to form intercellular hyphae and haustoria within the plant. We have developed and utilised two techniques to isolate fungal and plant genes expressed during this process. Using cDNA-AFLP, we have isolated fragments of wheat and rust genes that are expressed at specific defined time-points during the infection process. A number of these sequences have been used as probes in northern hybridisations and in real-time PCR to confirm their expression patterns, and have also been characterised by PCR analysis and Southern hybridisations to determine which are of fungal or wheat origin. A full-length cDNA library has been constructed from day 5 and 7 post-inoculation pooled cDNAs, and this library has been screened to isolate full-length cDNAs of selected fungal and wheat sequences. In the second approach, we have purified haustoria from infected plants, and used cDNA from these to isolate additional haustorial-specific sequences from the library. The results from the sequence analysis of these clones, which has revealed significant similarities amongst the fungal genes to a chitinase, an arabitinol dehydrogenase and a metallothionein, and in wheat to a katanin and a cell enlargement protein, will be discussed. Approximately 50% of the sequences obtained had no significant homology to sequences in databases.
1.49 Virulence specificities of Puccinia triticina (leaf rust) from durum wheat from world-wide collections
Maria E Ordonez1, James A Kolmer1,2, James V Groth1
1University of Minnesota, St. Paul, USA. 2USDA-ARS, Cereal Disease Laboratory, St. Paul, USA
jkolmer{a}umn.edu
Wheat leaf rust caused by Puccinia triticina is an important disease of wheat world-wide. The leaf rust resistance in tetraploid (AABB) durum wheat (Triticum turgidum) has generally been durable compared to the short-lived resistance of hexaploid (AABBDD) bread wheat cultivars. In recent years, durum leaf rust has become more prevalent in California, Mexico, France, and Spain. Collections of P. triticina from durum wheat from seven countries were analyzed for virulence specificities on 36 isogenic lines of Thatcher wheat that differ for single leaf rust resistance genes, and on 24 cultivars of durum wheat. Eight isolates from Argentina, 20 from France, 31 from Mexico, 11 from Spain and six from California had high infection types on all durum cultivars tested and on the Thatcher isolines with leaf rust resistance genes Lr10, Lr14b, Lr20, Lr23, Lr33, Lr41 and Lr44. These genes were probably derived from the AABB wheats, except for Lr41. Three isolates from Chile and four from the northern USA had virulence specificities similar to leaf rust races collected from bread wheats, and had lower infection types on the durum cultivars. Eight isolates from Ethiopia had low infection types on the susceptible cultivar Thatcher while two had high infection types on Thatcher. All isolates from Ethiopia had high infection types on the durum cultivars. There are at least two groups of P. triticina adapted to durum wheats that are distinct for virulences compared to the isolates that are found on hexaploid bread wheat. Molecular variation within and between the different collections from durum wheat will be assessed using AFLP variation, and will also be compared with isolates from bread wheat for molecular variation.
1.50 The biology of Puccinia striiformis on Hordeum spp. in Australia: the case for a new forma specialis
CR Wellings1, JJ Burdon2 and FJ Keiper3
1The University of Sydney, Plant Breeding Institute, Camden, Australia. 2CSIRO Division of Plant Industry, Canberra ACT, Australia. 3SARDI, Waite Campus, Adelaide SA, Australia.
colinw{a}camden.usyd.edu.au
The wheat stripe (yellow) rust pathogen Puccinia striiformis tritici (Pst) was first detected in Australia in 1979 and has continued a process of adaptive evolution in association with cultivated wheats that has resulted in a proliferation of pathotypes. The host range of Pst is predominantly wheat, although a range of genera in the Poaceae (Bromus, Phalaris and Hordeum) and occasional genotypes of barley (eg the Chinese cv. Fong Tien), rye and triticale are also considered of some importance in pathogen survival between and within seasons.
Although the predominant host of Pst is wheat, a noticeable increase in frequency of isolates recovered from wild Hordeum spp. stimulated an investigation of the evolutionary development of Pst on this host. Observations indicated that isolates of standard wheat pathotypes showed further differential variation on clones of Hordeum glaucum and Hordeum leporinum. However, it was concluded that pathotype evolution on the weedy Hordeum spp. was independent from, and therefore likely to have little impact on, that occurring on wheat.
An unusual variant of P. striiformis was first detected in Australia in 1998 (Wellings et al., 2000). The initial isolates were obtained from weedy Hordeum spp and were expected to yield various pathotypes of Pst. However, disease responses on the standard wheat differential genotypes indicated that these isolates were avirulent on many genotypes, including cvs. Morocco, Lemhi and Michigan Amber that are internationally regarded as standard susceptible genotypes for Pst. The only wheat that showed a moderately susceptible infection type in seedling tests was Chinese 166, which is used to assay for the response of resistance gene Yr1, and has been reported to be partially susceptible to isolates of P. striiformis hordei (Psh, barley stripe rust pathogen). The distinctive nature of these isolates led to the conclusion that they were distinct from Pst and Psh (currently not recorded in Australia) and are currently referred to as barley grass stripe rust (BGYR).
Greenhouse studies indicate that cultivars associated with pedigrees based on Skiff are vulnerable to BGYR. Recent evidence suggests that further pathotype evolution is occurring in BGYR populations, and hence it is likely that pathotypes will become progressively more adapted on commercial barley cultivars. Although BGYR has caused some concern in breeders selection nurseries, crop losses have not been recorded. Several molecular marker systems, including AFLP, SAM and S-SAP, and an isozyme marker revealed differentiation between Pst and BGYR isolates (Keiper et al., 2003). Pathogenic and molecular features strongly suggest that BGYR represents an undescribed forma specialis of P. striiformis.
Keiper FJ, Hayden MJ, Park RF and Wellings CR, 2003. Molecular genetic variability of Australian isolates of five cereal rust pathogens. Mycological Research 107, 545-556.
Wellings CR, Burdon JJ, McIntosh RA, Wallwork H, Raman H and Murray GM, 2000. A new variant in Puccinia striiformis causing stripe rust on barley and wild Hordeum in Australian Plant Pathology 49, 803.
1.51 In search of the correct name for leaf rust of cultivated wheat
Les J Szabo1, Jaroslava Marková2, Tamar Eilam3, Jacob Manisterski3, Pnina Ben Yehuda3
1USDA ARS Cereal Disease Laboratory, University of Minnesota, St. Paul, MN, USA. 2Charles University, Prague, Czech Republic. 3Institute for Cereal Crop Improvement, Tel Aviv University, Ramat Aviv, Israel.
lszabo{a}umn.edu
The taxonomic classification of leaf rust of cultivated wheat has been in flux for more than a century. In recent years the fungus has been referred to as Puccinia recondita f.sp. tritici, P. persistens f.sp. tritici and P. triticina. In 1857 Roberge described leaf rust on rye as P. recondita, which was later suggested by Cummins and Caldwell (1956) to be the valid name for leaf rusts of grasses including wheat. Plowright, in 1889, described leaf rust on Elytrigia (Agropyron) repens as P. persistens and demonstrated that the aecial host was Thalictrum flavum. In 1899, Eriksson described leaf rust on wheat as P. triticina and the aecial host was later shown to be Thalictrum species. From comparisons of host specificity, teliospore dimensions, amount of nuclear DNA and intercrossing ability, Anikster et al. (1997) concluded that leaf rust of wheat is a different species than leaf rusts of rye or several wild wheats. Consequently, “P. recondita” was deemed inappropriate for leaf rust of cultivated wheat. We have extended this study by comparing leaf rusts collected from El. repens, El. intermedia and Th. minus (P. persistens) to leaf rust collected from cultivated wheat. Inoculation studies indicated that leaf rust collected from wheat (‘wheat-type’) and El. intermedia (‘Elytrigia-type’) have distinctly different host ranges. The ‘wheat-type’ leaf rust readily infected cultivated wheat (Triticum aestivum and Tr. durum) as well as Tr. boeoticum, Tr. urartu and Tr. dicoccoides. The ‘Elytrigia-type’ leaf rust did not infect Tr. aestivum, Tr. durum or Tr. dicoccoides, but gave infection type 3-CL on Tr. boeoticum and 1,1+2CL on T. urartu. Conversely, ‘Elytrigia-type’ leaf rust readily infected El. intermedia while the ‘wheat-type’ did not. Of eight species of Aegilops tested, three (Ae. longissima, Ae. ovata and Ae. sharonensis) were susceptible to both leaf rust types, three (Ae. bicornis, Ae. speltoides, and Ae. squarrosa) were susceptible to only the ‘wheat-type’, and the remaining two (Ae. cylindrica and Ae. variabilis) were not susceptible to either type. Inoculations with germinating telia of ‘wheat-type’ and ‘Elytrigia-type’ each infected Th. minus and Th. speciosissimum. Intercrossing between these leaf rust types resulted in the formation of aecia, however the aeciospores did not infect either of the original telial hosts (wheat or El. intermedia). Teliospore measurements were similar between the two types. Morphology of substomatal vesicles of the two types are distinctly different. Those of the ‘wheat-type’ are single-celled oval shaped, while those of the ‘Elytrigia-type’ are elongate often with a septum dividing the substomatal vesicle into two cells. DNA sequence analysis of the internal transcribed spacer region of the nuclear ribosomal RNA repeat indicated that the ‘wheat-type’ and ‘Elytrigia-type’ are distinct, but closely related. The ‘wheat-type’ sequences formed a single cluster (bootstrap value of 72%). The majority of the ‘Elytrigia-type’ sequences formed two, well supported clusters (bootstrap values >93%) divided primarily along host lines (El. intermedia and El. repens). Our results indicate that the leaf rusts of wheat and Elytrigia are separated by telial hosts, differ in some morphological traits and apparently have been genetically isolated in nature. It is likely that these two leaf rusts began to diverge with the domestication of wheat, approximately 10,000 years ago, and the ‘wheat-type’ has become adapted to an agricultural environment. For wheat leaf rust, the choice between P. triticina and P. persistens ssp. tritici appears to be a matter of judgment about how complete the process of speciation has been, as well as a practical consideration for plant pathologists dealing with the biology and control of leaf rust of wheat.
Part 2 :
Posters and workshop presentations
2.1 Isolation of RGAs and disease related gene fragments from wheat stripe rust resistant differential lines
Mahinur S Akkaya, Xianming Chen, Osman Bozkurt, Figen Yildirim, Turgay Unver, Mehmet Somel
Middle East Technical University, Ankara, Turkey
akkayams{a}metu.edu.tr
Stripe rust, caused by biotrophic Puccinia striiformis f. sp. tritici, has been the most devastating wheat disease for the last 20 years in Turkey and the region. So far, only the sequence of Yr10 has been released as wheat stripe rust resistance gene (AF149114).
Several approaches were pursued for the identification of genes involved in the resistance against stripe rust. Firstly, RGA fragments amplified from 17 differential Avocet-Yr lines (ICARDA and C. Wellings) were investigated. Radioactively labeled PCR products, amplified using various RGA primers, corresponding to the p-loop, kinase and LRR domains, were separated on denaturing gels. Those fragments having unique profiles - present only in one differential line and absent in all the others including Avocet-S - were isolated, cloned and sequenced. One of the clones showed homology to rice Rim2 transcript, which accumulates in response to infection with Magnoporthe grisea. Also, the predicted protein sequence of another clone showed homology to the BIS1 protein of H. vulgare, which is up-regulated at rust infection sites.
Secondly, in order to identify those genes that are induced upon infection with Pst17 and Pst45 strains, presenting compatible interactions with Avocet-Yr10 and Avocet-Yr1 lines, respectively, differential display (DD) analysis was performed. Differentially expressed clones were isolated showing a high degree of homology to receptor like kinases and PR proteins.
Thirdly, the stripe rust differential lines used to isolate Yr10 gene homologs were investigated using primers to amplify the first 1500 bps of Yr10. PCR products of the expected size were sequenced from Avocet-Yr6 and Avocet-Yr11 lines. In addition, a longer PCR product was obtained from both lines, showing homologies to the 3’ end of the barley Rpg1 gene. Yr10 homologs from other Avocet differential lines will be sequenced.
Our findings from these three approaches will be presented.
2.2 Use of the Affymetrix microarrays to investigate the interactions between barley and powdery mildew
Andy Bailey, Ellen Colebrook, Lucy Weinert, Jane Coghill and Keith Edwards
School of Biological Sciences, University of Bristol, UK
andy.bailey{a}bris.ac.uk
Powdery mildew is an obligate biotroph, requiring living host cells to be able to grow and proliferate. We have performed a pilot study to investigate the transcriptional changes in barley leaves upon infection. This has made use of the recently released Affymetrix Barley1 22K GeneChip microarray. These arrays contain oligonucleotides representing 21,439 different genes, with 11 oligonucleotides probe pairs for each gene, allowing the transcription of all the target genes to be assessed simultaneously. In this study we investigated a compatible interaction, where infections had been able to establish for a period of 8 days. The arrays were hybridized to labeled cDNA, levels of transcript determined and compared to healthy control tissues. This showed the transcript levels of 9.5% of genes to be more than 7-fold altered upon infection, demonstrating the extensive reprogramming of host metabolism upon infection In any infection there are a proportion of host cells that resist infection, some cells that succumb to infection and some that the pathogen doesn’t attempt to infect. The results will be discussed in light of this complex situation. It was especially interesting to note that within particular gene families, especially within the phenylpropanoid/isoflavanoid pathway, some members were activated whilst others were repressed, showing that even when there may be no net change in transcripts for a particular class of enzyme, there may have been a significant alteration in the pattern of expression amongst family members.
This study highlights the utility of such microarrays for dissecting the molecular events during both successful and unsuccessful infections.
2.3 Search for durable resistance to wheat leaf rust in Brazil
Amarilis Barcellos, Camila Turra
OR Melhoramento de Sementes Ltda., Passo Fundo, RS, Brazil
amarilis{a}orsementes.com.br
Wheat breeding for resistance, the preferred approach to controlling leaf rust in Brazil, has been difficult because of the variability of the fungus. Resistance based on Lr genes effective throughout the plant cycle is not durable. The challenge to control the disease caused by Puccinia triticina is related to the high inoculum pressure, ‘green bridge’, early onset of epidemics and local wheat over- summering. The same races exist in all southern region of South America. In Brazil, high yield losses are suffered every year in susceptible cultivars grown without chemical control. After 1993, ten combinations of two Lr genes lost effectiveness for resistance, only four, Lr9, Lr16+24, and Lr19, remaining effective in combination. Individually, Lr24 and Lr26 have not been effective since 1980 and 1982, respectively, and Lr9 and Lr16 since 1986. Recent epidemics were probably caused by virulence on Lr26, 23+24 and Lr24+26 (Barcellos & Chaves, 2003). The seedling resistance genes most frequently identified in Brazilian genotypes were Lr26, 23, 10 and 24, in 1996-97 (Zoldan & Barcellos, 2002). An alternative is breeding for durable resistance, and our leaf rust program focuses on the resistance conditioned by genes more effectively expressed at the adult-plant stage, reducing the selection pressure for virulence. Most of the wheat cultivars, which probably originated in southern South America, with stable resistance to leaf rust worldwide have been those with adult-plant resistance. The Brazilian cv. Frontana (Lr13+34,+) is an international source for durable resistance. Toropi (Trp-1, Trp-2), another old Brazilian cultivar, expresses resistance essentially of the adult-plant type. Nowadays, the Brazilian wheat area is cultivated with susceptible genotypes, requiring fungicide, and resistant and slow-rusting cultivars. BR 23 (Lr13+34,+), a slow-rusting cultivar, has not been damaged significantly by leaf rust since 1987, despite occupying a large crop area, while, in the same region, several cultivars resistant throughout the plant cycle became highly susceptible. Lr34 seems to have contributed to the long-term cultivation of some Brazilian cultivars, such as BH 1146, IAC 5, Frontana, and BR 23 (Sousa & Barcellos, 2001). The presence of Lr13 in Brazilian cultivars is not rare, contributing in gene combinations to durable resistance (Sousa & Barcellos, 2000).
To search for more stable resistance among adapted wheat genotypes, the reaction of the first and flag leaves to 8 races of leaf rust was evaluated, under partially controlled conditions in the greenhouse. Spores in mineral oil were sprayed on the leaves and the plants incubated for 20-24 hours in a dark chamber at approximately 100% relative humidity and 16ºC. Lines expressing a susceptible reaction or X (susceptible and resistant on same leaf) at the first leaf stage were subsequently infected on the flag leaves. About 20 days later, the rust reaction of these Brazilian cultivars and lines, with genes for adult-plant resistance, was evaluated. Rust severity (1 to 100 percent of leaf area infected) and response (resistant to susceptible - R MR MS S) were estimated and transformed to CI, the coefficient of infection (severity x response; R = 0.2, MR = 0.4, MS = 0.8, S = 1). The average CI on flag leaves ranged from 0.2 to 40.6 (susceptible check). With respect to the mean reaction to all races evaluated, BR 23, Thatcher Lr12, Toropi , Frontana , Thatcher Lr34 and Manitou Lr13 showed an intermediate rust level on adult plants (CI 6.3 to 15.7). Large CI differences to races were expressed by Lr12, 13 and Lr22b.
Barcellos A, Chaves M, 2003. Epidemias de ferrugem da folha em cultivares brasileiras de trigo – Alterações na população do patógeno de 1993 a 2002. Seminario Internacional: Resistencia a royas en trigo. Resumenes. INIA La Estanzuela, Uruguay, 13.
Sousa CNA, Barcellos AL, 2000. Evaluation of leaf tip necrosis in Brazilian wheat genotypes. Annual Wheat Newsletter 46, 31.
Sousa CNA de, Barcellos AL, 2001. Resultados de testes para necrose híbrida (Lr13) em populações
F1 de cruzamentos de trigo na Embrapa Trigo. Embrapa Trigo – Comunicado Online, 61 [http:
www.cnpt.embrapa.br/biblio].
Zoldan SM, Barcellos AL, 2002. Postulation of genes (Lr) for resistance to leaf rust in Brazilian wheat cultivars. Fitopatologia brasileira 27(5), 508-516.
2.4 Map based cloning of the Run1 powdery mildew resistance gene from grapevine
Claire L. Barker1, Tamzin Donald1, 2, Anne-Françoise Adam-Blondon3, 4, Jerome Pauquet3, Alain Bouquet3, Mark Thomas1, Ian Dry1
1 CSIRO Plant Industry, Glen Osmond, Australia. 2 Current address: University of Western Australia, Nedlands, Australia. 3 UR-GAP Viticulture, ENSAM-INRA, Montpellier, France. 4 Current address: INRA-UGRV, Evry Cedex, France
claire.barker{a}csiro.au
Grapevine powdery mildew is caused by the biotrophic fungal pathogen, Uncinula necator and is the most economically important fungal disease of grapevines worldwide. The disease is of particular significance as all cultivated grapevines are of the species Vitis vinifera, which lacks natural resistance to the pathogen. In contrast, the wild North American grapevine, Muscadinia rotundifolia, is resistant to powdery mildew due to the action of a single, dominant gene termed Resistance to Uncinula necator 1 (Run1). Phenotypically, Run1 mediated resistance to Uncinla necator resembles that conferred by the barley powdery mildew resistance gene, Mla12, as in both incompatible plant-pathogen interactions a hypersensitive response is initiated in infected epidermal cells after formation of the first fungal haustorium.
The Run1 locus has been introgressed into Vitis vinifera using a pseudo-backcross strategy to generate segregating populations that can be used in mapping studies, with the aim of identifying the Run1 gene by map based cloning. To this end, eleven genetic markers have been identified that are tightly linked to the Run1 locus (Pauquet et al., 2001; Donald et al., 2002). These markers have been used to construct a physical map spanning the Run1 locus by isolation of Bacterial Artificial Chromosome (BAC) clones from a BAC library generated using the genomic DNA of a resistant individual from the fifth back-cross generation. The expansion of BAC contigs initiated from each of the genetic markers most closely linked to Run1 has provided valuable sequence information enabling many new markers to be designed in the vicinity of the resistance locus.
Interestingly, two of the genetic markers linked to the Run1 locus correspond to clusters of resistance gene analogues (RGAs) that encode proteins of the nucleotide binding site-leucine rich repeat class (Donald et al., 2002). One cluster of RGAs contains at least six members and encodes proteins with an N-terminal domain homologous to the Drosophila Toll and human Interleukin-1 receptors, whereas the other cluster contains numerous RGAs that encode proteins with an N-terminal coiled-coil domain. Analysis of recombinant plants is currently underway to determine whether one or both of these RGA clusters is a candidate for containing the Run1 gene.
Donald TM, Pellerone F, Adam-Blondon A-F, Bouquet A, Thomas MR, Dry IB, 2002. Identification of resistance gene analogues linked to a powdery mildew resistance locus in grapevine. Theoretical Applied Genetics 104, 610-618.
Pauquet J, Bouquet A, This P, Adam-Blondon A-F, 2001. Establishment of a local map of AFLP markers around the powdery mildew resistance gene Run1 in grapevine and assessment of their usefulness for marker assisted selection. Theoretical Applied Genetics 103, 1201-1210.
2.5 A glimpse into the metabolism of Blumeria graminis during development as inferred from transcript profiles
M Both, PD Spanu
Imperial College London, UK
m.both{a}imperial.ac.uk
cDNA microarrays of Blumeria graminis transcript profiles during development (asexual) cycle reveal the dynamics of gene expression as the fungus germinates, penetrates and feeds on it host and produces masses of conidia for dispersal. The analysis of the profiles of genes encoding enzymes involved in primary metabolism shows that there is a striking degree of coordinated regulation of some of the genes in similar pathways. In one example, genes encoding glycolytic enzymes are significantly up regulated as mature appressoria form and then again in haustoria. In another example, RNAs for lipid degrading enzymes are expressed at high levels in the conidia and the early germination stages and decrease significantly later, conversely RNAs for lipid biosynthetic are low at the early stages and increase markedly in the later stages of infection (nutrient assimilation and sporulation). We will discuss these results and draw inferences on the metabolic status of this obligate biotrophic fungus as it infects its host and completes its life cycle.
2.6 Identification of non-host resistance genes in wheat to barley yellow rust
Paula C. Rodrigues1,2, Jacqueline M. Garrood1, Qian-han Shen1,3, Phil H. Smith1 and Lesley A. Boyd 1
1John Innes Centre, Norwich, UK.2Departamento de Biologia, Escola Superior Agrária de Bragança, Bragança, Portugal. 3 Max-Planck Institut fur Zuchtungsforschung, Koln, Germany
lesley.boyd{a}bbsrc.ac.uk
Yellow rust, caused by Puccinia striiformis West., is an important foliar disease of wheat and barley throughout the world, and the development of resistant cultivars is the most economical and environmentally friendly method of control. Breeding for resistance to yellow rust has, for decades, been based on the use of race-specific resistance genes, which have proved to be short-lived. Non-host resistance has been studied as a possible source of durable resistance.
Two major genes, as well as an undetermined number of minor genes, for non-host resistance to the barley attacking form of yellow rust, P. striiformis f. sp. hordei, have been previously detected in the wheat cultivar ‘Lemhi’ (Johnson and Lovell, 1994). The present study aimed at quantifying and mapping those genes using QTL (quantitative trait loci) mapping procedures. For that purpose, an F2 population of 114 individuals resulting from the cross of resistant ‘Lemhi’ with ‘Chinese 166’, a wheat cultivar susceptible to barley yellow rust, was used as the mapping population. QTL effects and significance were estimated by means of interval mapping and MQM mapping procedures (Rodrigues et al., 2004).
A map for the F2 population was constructed which included 116 DNA markers (14 SSRs and 102 AFLPs). Two major QTLs have been mapped to chromosome arms 1DS (Psh1) and 2BL (Psh2), with significant LOD values. These two QTLs account for 76.7% of the phenotypic variance for resistance to barley yellow rust. Two other QTLs, with a minor effect, were mapped to chromosome arms 5AL (Psh3) and 6AL (Psh4), explaining 5.1% and 10.9% of the phenotypic variation, respectively. The QTL on 5A was derived from the susceptible variety, ‘Chinese 166’. In all cases the resistance towards P. striiformis f.sp. hordei was associated with a visual chlorosis/necrosis response typical of race-specific, host resistance.
Johnson R, Lovell NK, 1994. Genetics of resistance of wheat to barley-attacking races of Puccinia striiformis. Cereal rusts and Powdery Mildew Bulletin. 22, 32-40.
Rodrigues PC, Garrood JM, Shen Q-H, Smith PH, Boyd LA, 2004. The genetics of non-host disease resistance in wheat to barley yellow rust. TAG (in press).
2.7 The genome and chromosomes of Blumeria graminis
James KM Brown1, Elaine M Smith1, Qijun Xiang2, Christopher J Ridout1 and Xiayu Duan2
1 Department of Disease and Stress Biology, John Innes Centre, Norwich, UK
2 Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China
james.brown{a}bbsrc.ac.uk
In principle, the number of linkage groups in a comprehensive genetic map is an estimate of the number of chromosomes in an organism’s nucleus. In practice, however, it is rarely reliable to base an estimate of chromosome number on a linkage map alone. A genetic map is simply a statistical model fitted to mapping data. The estimate of chromosome number may therefore be too high, if the statistical parameters used to construct the map permit too few linkage groups to be joined together, or too low, if apparent linkages between markers which are in fact on different chromosomes are accepted in error. Most high-quality maps are in fact based on good knowledge of the organism’s cytology. In Blumeria graminis, however, as in other ascomycetes, the chromosomes are tiny, near the limit of what can be observed by light microscopy, so it is difficult both to obtain good preparations of condensed chromosomes and to estimate the chromosome number of the fungus from cytological data alone.
We have combined genetic linkage data and cytological photomicrographs to estimate the chromosome number of B. graminis. A genetic map was constructed of a cross of the B. graminis f.sp. hordei isolates DH14 and CC52 including eight avirulence genes, one gene for resistance to the fungicide ethirmol, a PCR marker linked to the mating type locus and over 300 AFLP markers. Examination of the strongest linkages between AFLP markers indicates that the mostly likely number of linkage groups is 11, although a slightly lower or higher number is also possible. Photographs of five nuclei of B. graminis f.sp. tritici in mitotic metaphase were obtained. These images can be interpreted as showing 11 chromosomes. There is no clear indication of a larger number of linkage groups, while lower estimates of the chromosome number would lead to inconsistencies in the interpretation of the cytological evidence. The larger chromosomes appear to be metacentric.
We conclude that the mostly likely chromosome number of B. graminis is 11. Although the mapping data were obtained from f.sp. hordei and the cytological data from f.sp. tritici, the two special forms diverged recently, probably only a few thousand years ago (Wyand & Brown 2003), so it is reasonable to assume that they probably have the same chromosome number.
Wyand RA, Brown JKM, 2003. Genetic and forma specialis diversity in Blumeria graminis of cereals and its implications for host-pathogen coevolution. Molecular Plant Pathology 4, 187-198.
2.8 Epidemiology of barley stripe rust and races of Puccinia striiformis f. sp. hordei: the first decade in the United States
Xianming Chen
USDA-ARS, Washington State University, USA
xianming{a}mail.wsu.edu
In the United States, barley stripe rust, caused by Puccinia striiformis f. sp. hordei, has spread in the south central and western United States and has caused localized damages in California and the Pacific Northwest since it was first reported in southern Texas in 1991. Barley stripe rust has been monitored using trap nurseries and field survey. Sixty-nine races of P. striiformis f. sp. hordei have been identified using a set of 12 barley genotypes. Races that were detected in California and/or Texas were also detected in the Pacific Northwest, indicating the spread of the races among the different regions and lack of selection from cultivars grown in various regions. Races with a relatively narrow virulence spectrum tended to become predominant. Major barley cultivars with non-race specific high-temperature, adult-plant resistance and the barley cropping system have played significant roles in reduction of damage by barley stripe rust.
2.9 Genetics and molecular mapping of resistance genes in wheat and barley against inappropriate formae speciales of Puccinia striiformis
Xianming Chen, Vihanga Pahalawatta
USDA-ARS and Department of Plant Pathology, Washington State University, Pullman, USA
xianming{a}mail.wsu.edu
Most wheat varieties are resistant to barley stripe rust caused by Puccinia striiformis f. sp. hordei (PSH) and most barley varieties are resistant to wheat stripe rust caused by P. striiformis f. sp. tritici (PST) (Chen et al., 1995b). The wheat variety Lemhi, which is susceptible to all PST (previously called CDL for Cereal Disease Laboratory) races except for PST-21, is resistant to all tested PSH races. Similarly, the barley variety Steptoe, which is susceptible to all PSH races, is resistant to all tested PST races. To determine genetics of the Lemhi resistance to PSH and the Steptoe resistance to PST, crosses were made between Lemhi and PI 478214, a wheat genotype originally from Ethiopia and susceptible to all tested PST and PSH races, and between Steptoe and Russell, a barley variety susceptible to some PST races and all tested PSH races. Seedlings of parents and F1, BC1, F2, and F3 progeny from the wheat cross were tested with races PSH-14, PSH-48, and PST-21, and those from the barley cross were tested with races PST-41 and PST-45 under controlled greenhouse conditions. Genetic analyses of infection type data showed that Lemhi had a dominant gene (provisionally designated as RpsLem) for resistance to the PSH races and the gene was closely linked to Yr21, a previously reported gene for resistance to PST-21 (Chen et al. 1995a); and that Steptoe had a dominant gene and a recessive gene (provisionally designated as YrStep1 and yrStep2, respectively) for resistance to races PST-41 and PST-45. For each of the crosses, genomic DNA was extracted from the parents and 150 F2 plants that were tested for rust reaction and which produced the F3 progeny that were tested with the races to confirm the phenotypes and determine the homozygosity/heterozygosity of the F2 plants. The phenotypic data and polymorphic markers identified using the resistance gene analog polymorphism (RGAP) technique (Chen et al. 1998a; 1998b) were analyzed with the Mapmaker computer program (Lander et al., 1987) to map the resistance genes. A linkage group for the genes in Lemhi was constructed with 11 RGAP markers and a linkage group for the dominant gene in Steptoe for resistance to PST was constructed with 12 RGAP markers. Using an RGAP marker that was linked to the resistant allele in repulsion and the set of nulli-tetrasomic Chinese Spring lines, the linkage group for RpsLem and Yr21 was mapped on wheat chromosome 1B, which confirmed the chromosomal location of Yr21 (Chen et al. 1995a). The results show that resistance in wheat and barley to inappropriate stripe rust pathogens is qualitatively inherited. These genes may provide effective resistance against appropriate pathogens when introgressed into appropriate hosts from inappropriate hosts.
Chen XM, Line RF, Jones SS, 1995a. Chromosomal location of genes for stripe rust in spring wheat cultivars Compair, Fielder, Lee, and Lemhi and interactions of aneuploid wheats with races of Puccinia striiformis. Phytopathology 85, 375-381.
Chen XM, Line RF, Leung H, 1995b. Virulence and polymorphic DNA relationships of Puccinia striiformis f. sp. hordei to other rusts. Phytopathology 85, 1335-1342.
Chen XM, Hayes PM, Toojinda T, Vivar H, Kudrna D, Kleinholfs A, Leung H, Line RF, 1998a.Genetic mapping of genes for stripe rust resistance in barley using resistance gene analog polymorphism and AFLP markers. Phytopathology 88, S16.
Chen XM, Line RF, Leung H, 1998b. Genome scanning for resistance-gene analogs in rice, barley, and wheat by high-resolution electrophoresis. Theoretical and Applied Genetics 97, 345-355.
Lander ES, Green P, Abrahamson J, Barlow A, Daly JM, Lincoln SE, Newberg L, 1987. Mapmaker: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics 1, 174-181.
2.10 Towards cloning wheat genes for resistance to stripe rust and functional genomics of Puccinia striiformis f. sp. tritici
Xianming Chen and Paul Ling
US Department of Agriculture, Washington State University, Pullman, USA
xianming{a}mail.wsu.edu
Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici, is one of the most important diseases of wheat in the United States. Use of genetic resistance is the most preferred approach to control the disease. In order to understand the molecular basis of the host-pathogen interactions, we are conducting research towards cloning and characterizing genes for resistance to stripe rust and understanding functional genomics of the pathogen. The dominant gene Yr5 confers resistance to all races of P. striiformis f. sp. tritici identified so far in the United States. Resistance gene analog polymorphism (RGAP) markers that are co-segregating with the Yr5 locus and have high homology with plant resistance genes have been previously identified (Yan et al., 2003). Based on a pair of the co-dominant and co-segregating RGAP markers, sequence tagged site (STS) and cleaved amplified polymorphic sequence (CAPS) markers also were developed, which could enable the incorporation of Yr5 into a wide range of wheat varieties (Chen et al., 2003). To clone Yr5, a genomic Hind III BAC library of hexaploid wheat (2n = 6x) was constructed using the wheat Yr5 near-isogenic line (NIL) (AVS/6*Yr5) developed in the Plant Breeding Institute, the University of Sydney, Australia. The BAC library consists of 410,000 clones with an average insert size of 130 kb, and covers approximately 3.3x wheat genome equivalents. Colony pools and high-density filters of the BAC library were made for identifying resistance clones. The STS markers were used to screen the multi-dimensional BAC clone pools. Twelve positive clones were identified using the Yr5 STS markers. Sub-clone libraries for each of the 12 positive clones were constructed. The average insert-size of sub-clones ranges from 4 kb to 12 kb. Each of the sub-clone libraries covers at least 10X of the original BAC insert. To isolate the expressed sequences from the Yr5 locus, full-length cDNA libraries were constructed from both the pathogen-challenged and non-challenged Yr5 NIL. Each of the libraries consists of about 35,000 clones. The Yr5 STS markers are used to screen the sub-clone and cDNA libraries. To understand the host-pathogen interactions, genomic and functional genomic studies of the wheat stripe rust pathogen were initiated. For physical mapping and gene isolation, a BAC library of P. striiformis f. sp. tritici was constructed with Hind III digested genomic DNA fragments cloned into the pIndigo BAC-5 cloning vector (Epicentre, USA). The BAC library that consists of 43,000 clones with an average insert size of 50 kb covers about 20x of the P. striiformis genome equivalent. To study functional genomics, especially genes involved in pathogenicity and genes of biologically importance, a full-length cDNA library of P. striiformis f. sp. tritici was constructed. This library consists of 17,280 clones representing general mRNA transcripts. An initial characterization and gene analysis were conducted by sequencing about 200 randomly selected clones. The results of the complete cDNA sequencing show approximately 98% of the cDNA clones in this library are of full length. These sequences are used in the BLAST search to characterize the genes.
Chen XM, Soria MA, Yan GP, Sun J, Dubcovsky J, 2003. Development of sequence tagged site and cleaved amplified polymorphic sequence markers for wheat stripe rust resistance gene Yr5. Crop Science 43, 2058-2064.
Yan GP, Chen XM, Line RF, Wellings CR, 2003. Resistance gene analog polymorphism markers co-segregating with the Yr5 gene for resistance to wheat stripe rust. Theoretical and Applied Genetics 106, 636-643.
2.11 Impact of wheat stripe rust and races of Puccinia striiformis f. sp. tritici in the United States
Xianming Chen1, Eugene A. Milus2, David L Long3, Lee F. Jackson4
1USDA-ARS, Washington State University, Pullman. 2University of Arkansas, Fayetteville, 3USDA-ARS, University of Minnesota, St Paul. 4University of California, Davis, USA
xianming{a}mail.wsu.edu
Stripe (yellow) rust, caused by Puccinia striiformis f. sp. tritici, is one of the most important diseases of wheat in the US. Historically, it was most destructive in the western US (Washington, Oregon, Idaho, California) and sometimes caused damage in the south-central states (Texas, Louisiana, Arkansas). Since 2000, it has become increasingly important in the Great Plains (Oklahoma, Kansas, Colorado, Nebraska) and the south-central states. In 2000, the disease occurred in more than 20 states and estimated yield losses were 250,000 t ($27,000,000). In 2001, stripe rust was more widespread and caused severe damage in the Great Plains, and estimated losses were 1,100,000 t ($119,000,000). In 2002, stripe rust caused considerable damage in the western and south-central states, and yield losses were estimated at 220,000 t ($24,000,000). The national yield loss in 2003 was estimated at 2,420,000 t ($267,000,000), the highest in the last decade. In addition to the above losses, millions of dollars were spent on fungicide applications each year. The recent epidemics were attributed to the rust-favorable weather conditions and new races that overcame resistance in wheat cultivars. Among the 109 races identified from the early 1960’s to 2003, 40 new races were identified in the last four years. Since first detected in 2000, a group of races that have common virulences on Lemhi, Lee, Fielder, Express, Yr8, Yr9, Clement, and Compair has become predominant. Races identified before 2001 were previously published (Chen et al., 2002; Line, 2002), and races identified since 2001 are presented in Table 1. High-temperature, adult-plant resistance, which is in major wheat cultivars in the Pacific Northwest (Washington, Oregon, and Idaho) and some cultivars in the Great Plains has prevented even greater losses.
Table 1. New races of Puccinia striiformis f. sp. tritici identified in the United States in 2001-03.
|
PST |
Virulence |
|
PST |
Virulence |
|
race |
formulasa |
|
race |
formulasa |
|
81 |
1,14 |
|
96 |
1,4,6,8,10,12,14 |
|
82 |
1,11,17 |
|
97 |
1,3,10,11,12,16,17,18,19,20 |
|
83 |
1,3,6,11 |
|
98 |
1,3,8,10,11,12,16,17,18,19,20 |
|
84 |
1,8,10,12,18 |
|
99 |
1,3,9,10,11,12,16,17,18,19,20 |
|
85 |
1,8,10,12,17,18 |
|
100 |
1,3,8,9,10,11,12,16,17,18,19,20 |
|
86 |
1,8,10,12,16,18,19 |
|
101 |
1,2,3,8,9,10,11,12,16,17,18,19,20 |
|
87 |
1,2,11,12,16,17 |
|
102 |
1,3,8,9,10,11,12,14,16,17,18,19,20 |
|
88 |
1,11,12,16,17,20 |
|
103 |
1,9,10,11,12,16,17,18,19,20 |
|
89 |
1,12,16,17,18,19,20 |
|
104 |
1,2,3,9,10,11,12,16,17 |
|
90 |
1,3,11,12,14,16,17,18,19,20 |
|
105 |
1,8,10,11,12,16,17,20 |
|
91 |
1,9,10 |
|
106 |
1,8,10,11,12,16,17 |
|
92 |
1,10,12 |
|
107 |
1,3,4,5,9,10,14 |
|
93 |
1,6,10,12 |
|
108 |
1,3,4,6,9,10,12 |
|
94 |
1,9,10,12 |
|
109 |
1,4,8,10,12 |
|
95 |
1,4,8,10,12,14 |
|
|
|
a1 = Lemhi, 2 = Chinese 166, 3 = Heines VII, 4 = Moro, 5 = Paha, 6 = Druchamp, 7 = Ribesel 47/51*, 8 = Produra, 9 = Yamhill, 10 = Stephens, 11 = Lee, 12 = Fielder, 13 = Tyee, 14 = Tres, 15 = Hyak, 16 = Express, 17 = Yr8 (Avocet S/6*Yr8), 18 = Yr9 (Avocet S/6*Yr9), 19 = Clement, and 20 = Compair
Chen XM, Moore M, Milus EA, Long DL, Line RF, Marshal D, Jackson L, 2002. Wheat stripe rust
epidemics and races of Puccinia striiformis f. sp. tritici in the United States in 2000. Plant Disease
86, 39-46.
Line, RF, 2002. Stripe rust of wheat and barley in North America: a retrospective historical review.
Annual Review of Phytopathology 40, 75-118.
2.12 Resistance to European isolates of Blumeria graminis f. sp. hordei in
selections from barley landraces collected in Pakistan
Jerzy H. Czembor ,Henryk J. Czembor , Loek J. M. van Soest
IHAR Radzikow, Poland
j.h.czembor{a}ihar.edu.pl
Seed samples of 133 barley landraces were used for screening for resistance to powdery mildew. These landraces were collected in Pakistan and originated from the Centre for Genetic Resources (CGN), Wageningen, the Netherlands. The infection types were scored according to a 0 - 4 scale and the cultivar Manchuria (CI 2330) was used as a susceptible control. In a preliminary study, about 30 plants per landrace were evaluated in the greenhouse with isolate 33. Isolate 33 represented the most avirulent isolate available, allowing the expression of the maximum number of resistance genes. 16 of the landraces tested were resistant to isolate 33. 20 single plant lines of each of the 16 landraces were selected and tested at the seedling stage with 19 differential isolates of powdery mildew. These isolates were chosen according to their virulence spectra on the Pallas isolines differential set and on 8 additional differential cultivars, and had virulences corresponding to all major resistance genes used in Europe. Only 1 line was resistant to all isolates used. Most of the lines tested were resistant to most of the isolates used. These results show that barley landraces collected from Pakistan represent a valuable source of resistance to powdery mildew. Resistant lines identified in this study should be used in barley breeding programmes.
2.13 Sources of powdery mildew resistance in barley landraces from Nepal
Jerzy H. Czembor, Henryk J. Czembor, Louis Jestin
IHAR Radzikow, Poland
j.h.czembor{a}ihar.edu.pl
In barley many race-specific resistance genes to powdery mildew are available. However, in the last twenty years several sources of powdery mildew resistance have been overcome by virulence in the pathogen. Barley breeders are constantly looking for gene pools from which new genes can be introduced into existing cultivars in order to improve resistance to powdery mildew. There are numerous reports on variation in barley landraces for reaction to powdery mildew, and many genes for resistance have been described. Based on these reports it may be assumed that barley landraces from Nepal may possess mildew resistance genes different from those which already have been introduced into modern barley cultivars.
In this study seed samples of 49 barley landraces were used for screening for resistance to powdery mildew. These landraces were collected in Nepal and originated from Gene Bank at INRA- Clermont-Ferrand, France. The infection types were scored according to a 0 - 4 scale. The cultivar Manchuria (CI 2330) was used as a susceptible control. In a preliminary study, about 30 plants per landrace were evaluated in the greenhouse for resistance to powdery mildew using isolate 33. This isolate was used because it represented the most avirulent isolate available, allowing the expression of the maximum number of resistance genes. 16 of the landraces tested were resistant to isolate 33. 18 single plant lines for each of the 16 landraces were selected, and tested at the seedling stage with 20 differential isolates of powdery mildew. These isolates were chosen according to their virulence spectra on the Pallas isolines differential set and on 8 additional differential cultivars. Seven lines were resistant to all isolates, and could be used in barley breeding programmes for resistance to powdery mildew.
This study showed that barley landraces collected from Pakistan possess effective genes for powdery mildew resistance.
2.14 Search for resistance to leaf rust in durum wheat germplasm
Ana I Del Olmo1, JC Sillero1, D Rubiales2
1CIFA, Dpto. de Mejora y Agronomía, Córdoba, Spain. 2CSIC, Instituto de Agricultura Sostenible, Córdoba, Spain
Ana_olmo{a}yahoo.com
A collection of 1100 accessions of durum wheat was screened for resistance to leaf rust (Puccinia triticina) under field conditions at 2 locations in Southern Spain (Córdoba and Conil) in the 2002-2003 season. Relative disease severity (the most susceptible accession being scored as 100%) was lower than 20% in about 13% of the accessions in Córdoba and in about 6% in Conil. Resistance of the 162 most resistant accessions was confirmed in a second season (2003-2004) at 2 locations (Córdoba and Jerez) and under controlled conditions in seedling plants with 2 isolates: DBBPPP (virulent on Lr2b, 2c, 10, 14a, 14b, 18, 20, 23 and B(I)) and DGHPPT (virulent on Lr2b, 2c, 10, 11, 12, 14a, 14b, 16, 18, 20, 23, 28, 30 and B(I)), collected at Conil and Córdoba, respectively, the previous year.
Most of the selected accessions with low AUDPC (about 43%) displayed a high infection type (IT) in seedlings to both isolates. No apparent signs of hypersensitivity had been observed in 35 of these accessions in the field at the two locations, which supports the view that they possess a significant level of partial resistance. In contrast, a MR type of reaction had been recorded in the field for 39 accessions that displayed high IT in seedlings, suggesting the presence of adult plant resistance. Some other accessions displayed low IT to one of the isolates in the seedling stage, suggesting the presence of genes for hypersensitive resistance, that could be Lr11, 16, 28 and 30 in 17 of the accessions. Resistance of another 3 accessions could not be explained by the presence of any of the 24 Lr genes studied.
2.15 Physiologic specialization of Puccinia triticina in Andalusia (Spain) in 2003
Ana I Del Olmo and D Rubiales
CIFA Córdoba, Spain
ana_olmo{a}yahoo.com
Leaf rust, caused by Puccinia triticina is the most widespread and regularly occurring rust on wheat. It is particularly severe on durum wheat, with little resistance available in commercial cultivars. In the present work we intend to determine the virulence of Andalusian (Southern Spain) isolates in 2003. Single uredinial isolates (23 in total) were derived from wheat leaf rust collections and tested for virulence phenotype on lines of Thatcher wheat that are near-isogenetic for leaf rust resistance genes Lr1, Lr2a, Lr2b, Lr2c, Lr3, Lr3bg, Lr3ka, Lr9, Lr10, Lr11, Lr12, Lr14a, Lr14b, Lr15, Lr16, Lr17, Lr18, Lr20, Lr23, Lr24, Lr26, Lr28, Lr30 and LrB(I). 8 virulence phenotypes were found. Virulence frequencies were high to Lr2c, 10, 12, 14a, 14b, 18, 20, 23 and B(I) and low to Lr2a, 26, and 3ka. All analysed isolates were avirulent on Lr9 and Lr24. The most common virulence phenotype was DBGPPP (virulent on Lr2b, 2c, 10, 11, 12, 14a, 14b, 18, 20, 23 and B(I)) which was found especially, in the East of Jaén province. Virulence phenotype DGHPPT (virulent on Lr2b, 2c, 10, 11, 12, 14a, 14b, 16, 18, 20, 23, 28, 30 and B(I)) was the second most common phenotype, which was spread all over the surveyed area.
An increase of virulence on Lr2b, 2c, 20, 28, 30 and B(I) and a decrease of virulence on Lr3, 3bg and 17 were observed in 2003 when compared to 1998-2000 (Martínez F, 2002. Mecanismos de resistencia a roya de la hoja en trigo y cebada: University of Córdoba, PhD thesis).
2.80 Pathogenicity target gene is the Achilles’ heel in host-pathogen interaction
Wubei Dong
Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany
wubei.dong{a}ipk-gatersleben.de
Host-pathogen interactions, especially those involving biotrophic parasites, are complex. Three major models related to this system have been published. They are the gene-for-gene (GFG) hypothesis, matching-allele (MA) model and guard hypothesis. In the GFG and MA models, the host-pathogen interaction is between pathogen genes and host resistance (R) genes. In the guard hypothesis, the host-pathogen interaction includes not only pathogen genes and host R genes but also pathogenicity target genes in the host. The pathogenicity target genes in the host are very important components of the interaction. To a pathogen, whether a plant is a host or a non-host is determined by the present or absent of pathogenicity target genes. Only when a pathogenicity target gene is present and an efficient R gene is not, or when the R gene has been overcome by the pathogen in the course of plant-pathogen coevolution, is the interaction of the plant and the pathogen compatible. Pathogenicity target genes in host plants are constitutively expressed, independently of all induced responses. By silencing or modifying the pathogenicity target gene, the host will be converted into a non-host, which may possibly generate durable resistance. This represents a potential new direction for crop breeding programmes.
2.16 Rust resistance genes in winter wheat cultivars registered in the Czech Republic
Veronika Dumalasová, Alena Hanzalová, Pavel Bartoš
Research Institute of Crop Production, Praha-Ruzyně, Czech Republic
blazkova{a}vurv.cz
Genes for resistance to rusts in winter wheat cultivars registered in the former Czechoslovakia and in the Czech Republic have been studied by testing with a set of races enabling postulation of resistance genes, by both standard genetic analysis and molecular methods. For the identification of Lr10, the molecular marker described by Schachermayr et al. (1997) was used and that of Robert et al. (1999) for Lr37. The most common gene for leaf rust resistance in cultivars registered in the years 1966-2003 was Lr3 (42% of cultivars), followed by Lr26 (28%), Lr13 (13%), Lr37 (9%), Lr10 (4%), Lr1 (1%), Lr14a (1%) and Lr17b (1%). Genes Lr13, Lr14a and Lr17b were postulated by R.Park (personal communication). The most effective leaf rust resistance gene is Lr37 carried by cultivars Apache, Bill, Clarus, Clever, Corsaire and the Czech cultivar Rheia. Genes Lr1, Lr10 and Lr13 are effective in combinations with other Lr genes, e.g. cv. Vlada (Lr1, Lr3, Lr13) and cvs. Siria and Alka (Moldau) carrying Lr10 and Lr13. According to Official Yield Trials (Přehledy odrůd 2003, Obilniny /Survey of cultivars 2003, Cereals, ÚKZÚZ) the best leaf rust rating was for cultivars Clever, Batis, Corsaire and Semper (8), Drifter, Complet, Apache, Bill (7), and Alka, Rheia, Contra (6) using the scale 1 = susceptible, 9 = resistant. Stem rust resistance of the Czech registered cultivars is based on genes Sr5, Sr11, Sr29, Sr31 and Sr38. Of the stem rust resistance genes, Sr31 has remained effective since the sixties when the first cultivar with a 1B/1R translocation was tested in Official Yield Trials. Breeding for yellow rust resistance has been directed towards field resistance and specific genes are no longer identified in registered cultivars. Leaf rust occurs every year and its economical importance is increasing, whereas the last epidemic of stem rust was in the year 1972. Yellow rust occurred in the years 1998-2001.
Robert O, Abelard C, Dedryver F, 1999. Identification of molecular markers for the detection of the yellow rust resistance gene Yr17 in wheat. Molecular Breeding 5, 167-175.
Schachermayr G, Feuillet C, Keller B, 1997. Molecular markers for the detection of the wheat leaf rust resistance gene Lr10 in diverse genetic backgrounds. Molecular Breeding 3, 65-74.
2.17 Development of resistance gene analog markers linked to the stripe rust resistance gene YrH52 derived from wild emmer wheat, Triticum dicoccoides
Tzion Fahima, Jianping Cheng, Zujun Yang, Jun Yan, Marion Röder, Eviatar Nevo
University of Haifa, Israel
fahima{a}research.haifa.ac.il
The wheat stripe rust resistance gene YrH52 derived from wild emmer wheat, Triticum dicoccoides, was previously identified and mapped on the short arm of chromosome 1B using microsatellite and AFLP markers. In this study, we have used disease resistance gene analog (RGA) markers to tag YrH52 and identify closely linked markers. Forty-three degenerate oligonucleotide primers, with a total of 402 primer combinations, were used to detect RGA markers flanking the YrH52 gene region. An improved technique for exploring RGAs was employed, based on the use of an automated laser fluorescence (ALF) sequencer. Using bulked segregant analysis to screen DNA pools of resistant vs. susceptible progenies, we were able to identify 17 RGA markers linked to YrH52. In addition, three new microsatellite markers were also mapped in this region. Of the 17 RGA markers, seven were clustered together and mapped as tightly linked to YrH52. These results demonstrate the usefulness of RGA sequences, when used in combination with bulked segregant analysis, to rapidly generate markers tightly linked to resistance loci in crop species. The molecular markers obtained in this study should be useful for marker-assisted selection and pyramiding of YrH52 with other stripe rust resistance genes and as start points for positional cloning of YrH52.
2.18 Novel stem rust resistance in barley lines with introgressions of Hordeum bulbosum chromatin
Thomas Fetch Jr1, Richard Pickering2, and Paul Johnston2
1Cereal Research Centre, Winnipeg, Canada. 2Crop and Food Research, Christchurch, NZ
tfetch{a}agr.gc.ca
Stem rust, caused by Puccinia graminis f. sp. tritici, is one of the most devastating diseases of cereals worldwide. In North America, this disease is usually controlled by host resistance. In cultivated barley (Hordeum vulgare L.), the resistance gene Rpg1 has provided durable resistance to most races of stem rust in North America (Steffenson 1992). However, since 1988, the barley crop in the Upper Midwest region of North America has been threatened by attack of race QCCJ, which is the most prevalent race found on cultivated barley during annual surveys (Fetch 2003). Of the four described barley stem rust resistance genes, only rpg4 is known to confer resistance to race QCCJ. However, this gene is temperature sensitive, thus new sources of resistance to QCCJ would be desirable. Pickering et al. (2000) have previously developed a number of barley lines with introgressions of Hordeum bulbosum chromatin into particular barley chromosomes. This study tested many of these lines to races MCCF and QCCJ at two temperatures (20 and 28C). Of 43 lines tested, two (119Y4 and 212Y1) conditioned resistance to race QCCJ, while all other lines were susceptible. Allelism tests between the rpg4 source Q21861 and 212Y1 segregated for stem rust reaction to race QCCJ, thus it appears that line 212Y1 may be a novel source of stem rust resistance. Allelism tests between Rpg1 and 212Y1 are in progress. The introgressions in the line 212Y1 are on chromosomes 6HS and 7HS, but further testing is needed to identify the location of the chromatin conferring the stem rust resistance. A population from the cross 119Y4/Emir was used to characterize the stem rust resistance found in barley line 119Y4. F2 and F3 data from 98 families indicate that resistance is likely conditioned by a single recessive gene. Further testing with additional families is underway to confirm our preliminary results. These two barley lines with introgressions of Hordeum bulbosum resistance may be useful in breeding efforts requiring new sources of stem rust resistance.
Fetch TG Jr, 2003. Physiologic specialization of Puccinia graminis on wheat, barley, and oat in Canada in 2000. Canadian Journal of Plant Pathology 25,174-181.
Pickering R, Johnston PA, Timmerman-Vaughan GM, Cromey MG, Forbes EM, Steffenson BJ, Fetch TG Jr, Effertz R, Zhang L, Murray BG, Proeseler G, Habekuß A, Kopahnke D and Schubert I, 2000. Hordeum bulbosum - A new source of disease and pest resistance genes for use in barley breeding systems. Barley Genetic Newsletter, 30, 6-9.
Steffenson Brian J, 1992. Analysis of durable resistance to stem rust in barley. Euphytica 63,153-167.
2.19 Evaluation of wild oat germplasm for resistance to oat stem rust race NA67
Thomas Fetch, Jr., Julie Gold Steinberg, and Jennifer Mitchell Fetch
Cereal Research Centre, Winnipeg, MB, Canada
tfetch{a}agr.gc.ca
Stem rust, caused by Puccinia graminis f. sp. avenae, is one of the most devastating diseases of oat (Avena sativa L.) worldwide. In North America, this disease is usually controlled by host resistance. Oat stem rust has caused significant yield losses in the eastern prairie region of western Canada. Currently, the predominant oat stem rust race in this region is NA67. Few genes confer resistance to NA67, and none are present in any oat cultivars registered for production in Canada. We evaluated 9,979 accessions from 22 Avena species in field nurseries from 2001 to 2003 to detect lines exhibiting resistance to race NA67. Thirty-six accessions were highly resistant and 12 were moderately resistant, comprising mostly the species A. strigosa. Seventy-one accessions had an intermediate response, comprising mostly A. abyssinica, A. barbata, A. sterilis, and A. vaviloviana. All other accessions were susceptible to race NA67. Some accessions are incorrectly classified, as some highly resistant lines identified as hexaploid species A. sativa or A. sterilis were confirmed to be diploid or tetraploid by chromosome counts. The most promising source of novel stem rust resistance is from the diploid species A. strigosa. Transfer of resistance from diploid and tetraploid species to A. sativa is extremely difficult. However, through the use of embryo rescue techniques and colchicine treatment, successful generation of F1 seed has been accomplished. These hybrids are currently being assessed for rust reaction and cytogenetic analysis. The diploid and tetraploid lines identified in this study should prove useful as new sources of resistance to oat stem rust.
Fetch TG Jr., 2003. Physiologic specialization of Puccinia graminis on wheat, barley, and oat in Canada in 2000. Canadian Journal of Plant Pathology 25,174-181.
Fetch TG. Jr., Gold J and Nevo E, 2002. Evaluation of wild oat germplasm for stem rust resistance. In: Swain D, Zydenbos S, eds. Proceedings of the Eighth International Congress Plant Pathology, 2003, Christchurch, NZ. Lincoln University Press, Canterbury, NZ, p195.
Zegeye T, Fetch T and Lamari L, 2004. Inheritance of stem rust resistance in four accessions of Avena strigosa Schreb. Canadian Journal of Plant Pathology 26 (In press, abstract).
2.20 Virulence and distribution of pathotypes in two biotrophic host-pathogen systems with high (wheat/yellow rust) and low (rye/leaf rust) selection pressure by the host
Kerstin Flath1, Bettina Klocke2, Thomas Miedaner3, Katinka Wilde3
1Federal Biological Research Centre for Agriculture and Forestry, Institute for Plant Protection in Field Crops and Grassland, Kleinmachnow, Germany. 2Martin-Luther-University Halle, Institute of Plant Breeding and Plant Protection, Halle-Wittenberg, Germany.3University of Hohenheim, State Plant Breeding Institute, Stuttgart, Germany
k.flath{a}bba.de
Race-specific resistances are known to be potent selection forces for biotrophic rust populations. In Germany, yellow or stripe rust (Yr, Puccinia striiformis) is confronted with a high selection pressure because 40% of the registered German wheat varieties carry effective seedling and/or adult-plant resistance. In contrast, host selection by cross-pollinating rye to leaf rust (Lr, Puccinia recondita) is low caused by a high host diversity and a low level of resistance in commercial varieties. Adaptability of rust populations to host resistance is affected by their virulence frequency, complexity, and diversity. To compare the population structure of both rusts, 226 yellow and 827 leaf rust isolates were analysed by two differential sets, each containing 17 genotypes in 2000-2002.
Eight of the Yr and seven of the Lr differentials showed effective resistances as shown by virulence frequencies ranging from 0 to 38%. All Yr isolates were avirulent for Yr5, Yr10 and Sp. Virulent isolates were found for all Lr resistances although they have never been used commercially. The percentage of complex isolates (9-14 virulences) was slightly higher for Lr (62%) than for Yr (57%). Both rust populations were highly diverse with Simpson indices of 0.95 and 0.98 for Yr and Lr, respectively. No dominant pathotype occurred in the Lr population, the Yr population was dominated by two pathotypes (32% of isolates).
Due to high diversity and complexity of the Lr population and the presence of corresponding virulences for all differentials, new effective race-specific resistances should be selected and combined with non-specific quantitative resistances. In Yr, the combination of effective race-specific resistance genes suffice for the control of the pathogen at present.
2.21 Breakdown of resistance of wheat cultivars and estimated losses caused by recent changes in the leaf rust population in South America
Silvia Germán1, Mohan Kohli2, Marcia Chaves3, Amarilis Barcellos4, Jorge Nisi5, Juan Annone6, Ricardo Madariaga7, Lidia de Viedma8
1INIA La Estanzuela, Colonia, Uruguay. 2CIMMYT, Consultant. 3EMBRAPA Trigo, Passo Fundo, Brazil. 4OR Melhoramento de Sementes Ltda., Passo Fundo, Brazil. 5INTA Marcos Juarez, Argentina. 6INTA Pergamino, Argentina. 7 INIA Quilamapu, Chillan, Chile. 8CRIA, Encarnación, Paraguay
sgerman{a}inia.org.uy
Approximately 9 million ha of wheat are sown in the Southern Cone region of South America (Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay) annually (FAO, 2004). Wheat leaf rust (Puccinia triticina) is one of the most important diseases in the region, causing yield losses of over 50% in severe epidemics, and is the primary reason for cultivar replacement. As the disease is present in most parts of the region, the utilization of susceptible or moderately susceptible cultivars (36 and 24% of the wheat area, respectively), allows the pathogen to over-summer in large areas, resulting in early onset of the epidemics. Severe leaf rust epidemics cause heavy economic losses if chemical control is not used. The high dynamism of races in the pathogen population results in short lived resistance in commercial cultivars. The economic impact of the loss of resistance of some cultivars since 1996 is analysed in the present study. Losses caused by leaf rust were estimated in terms of the cost of chemical control (average US$ 20/ha in Argentina, Chile, Paraguay and Uruguay, and US$25/ha in Brazil). In Argentina, where the use of chemical control is not widespread, losses in production were also considered. Approximately 40% of the total area under wheat in Argentina is affected by severe leaf rust epidemics. Out of the area seeded to susceptible cultivars in this country, only about 35% is chemically treated. The cultivar Klein Pegaso covered 20% of the Argentinean wheat area in 1999, when it became susceptible to leaf rust due to an increase in frequency of previously detected races LBP and LCT, or new races CHT, MHT and MCT (Antonelli, 2003). Leaf rust epidemics on that cultivar during 1999-2003 resulted in an estimated cost of US$ 20.3 million in chemical control and US$ 11.7 million in production losses. Another important cultivar ProINTA Puntal became susceptible in 2001. Similarly a severe epidemic on Klein Don Enrique, planted on approximately 25% of the wheat area in 2002, represented a US$ 15.9 millions increase in the cost of production due to fungicide applications and an additional loss of US$ 8.1 millions in production. Given that the figures of seeded area under different varieties are based on the proportion of commercialized seed, some of these losses may be overestimated. However, using the available data, the changes in the leaf rust races affecting these three cultivars resulted in an estimated loss US$ 73.9 millions to Argentine agriculture. Highly susceptible cultivars require at least two fungicide applications to control leaf rust in Brazil. Cultivar Embrapa 16 was seeded on 40% of the wheat area when it became susceptible to leaf rust in 1996 (races LFG and LFJ). Cultivar OR1 was released commercially and extensively used despite its high susceptibility to leaf rust (races MFK, MFR and MFT) and reached 21.5% of the wheat area in 1999. Cultivar BRS 49, susceptible to race MCJ, covered 12.6% of the area in 2001, the first year requiring chemical control. The cost of fungicide applications to control leaf rust in Embrapa 16, OR1 and BRS 49 has been estimated at US$ 33.0, 47.6 and 10.7 millions, respectively (total of US$ 90.3 millions). In Paraguay, US$ 3.2 millions were spent to control leaf rust on Itapúa 35, from 1996 to 1998. In Uruguay, race MCD, first detected in 1999, was virulent on previously resistant cultivars Estanzuela Pelón 90 and INIA Mirlo which represented 20 and 28% of the wheat area in that year. The cost of fungicide applications to control leaf rust on these cultivars was estimated at US$ 1.3 and 1.8 millions, respectively (total of US$ 3.1 millions). The importance of leaf rust is increasing in Chile, especially in late planted spring wheats. Since 1997 the control of leaf rust on cultivar Nobo INIA had a cost of US$ 0.6 millions. In summary, changes in the leaf rust population over the last years affecting 10 cultivars represented an estimated loss of US$ 172.2 millions to the wheat farmers in the Southern Cone region or South America.
2.22 Germin-like protein genes in the defence response of grapevine to powdery mildew
Dale Godfrey1, Amanda J Able2 and Ian B Dry1
1CSIRO Plant Industry Horticulture, Glen Osmond, Australia. 2University of Adelaide, Waite Campus, Glen Osmond, Australia
dale.mayer{a}csiro.au
We are interested in developing molecular strategies for the improved resistance of grapevine to the powdery mildew pathogen, Uncinula necator. Germin-like proteins (GLPs) have been implicated in the defence response of cereals against powdery mildew. Although many germin-like proteins (GLPs) have been identified in plants, the function of only a few have been investigated. Barley HvGLP4 is epidermal-specific and accumulation of transcript is significantly greater in cells containing effective papillae compared to infected cells. HvGLP4 exhibits superoxide dismutase activity, catalysing the conversion of superoxide to hydrogen peroxide. This protein is postulated to be involved in the production of cell wall appositions (papillae) that provide an effective physical barrier to penetration of epidermal cells by powdery mildew. We have cloned and characterised seven novel cDNA clones of the GLP gene family in grapevine. RT-PCR analysis revealed that the isolated GLP genes exhibit diverse patterns of expression in response to a variety of abiotic and biotic elicitors. Several grapevine GLPs were specifically induced by powdery mildew infection and appear to be localised to the epidermal tissue. The promoters that control pathogen-induced GLP gene expression have been isolated and the tissue-specific expression of grapevine GLPs in response to powdery mildew infection examined in the model species A. thaliana. Additionally, His-tagged pathogen-induced grapevine GLPs were introduced into A. thaliana to allow for biochemical analysis of protein function. The possible role that these grapevine GLP proteins may play in the defence response of grapevines against powdery mildew will be compared to the proposed models for barley HvGLP4.
2.23 Adaptation of Puccinia triticina populations to host cultivars for virulence and agressiveness
Henritte Goyeau1, Delphine Rimé1, Laurent Bouffier1,2, Claire Neema2, Christian Lannou1
1 INRA Pathologie Végétale 78850 Thiverval Grignon, France
goyeau{a}grignon.inra.fr
Adaptation of rust fungi populations to their wheat hosts cultivars has been widely documented concerning specific host-pathogen interactions. However it has already been suggested that rust populations are likely to be structured by selective forces other than selection for virulence to host specific resistance genes (Kolmer 1993, Martens 1985). Studying Puccinia triticina populations in France, we have looked in a first step to the part of diversity that could be explained by interactions between specific resistance genes and avirulence genes, and then started to investigate about diversity in agressiveness as a life trait possibly modeling population structure.
Distribution frequency of pathotypes, identified on a differential set with 18 lines, was established in 1999-2002 on the five most widely grown bread wheat cultivars (two susceptible and three resistant) in France. Sample size each year for each cultivar was between 18 and 63, collected from a national trial network on unsprayed plots. Population on the susceptible cultivars, Soissons (Lr14a) and Isengrain (Lr14a), was composed of one dominant pathotype (frequency 50 to 60%), several minor pathotypes (frequency less than 10%), and several rare pathotypes found only once. This structure was confirmed for cv. Soissons on commercial plots, located in three different areas (South, Paris and North areas), with samples of 60 to 200 isolates : virulence as well as AFLP data showed that the same structure was observed at the national scale.
On the cultivars Trémie (Lr10, Lr13 + Adult Plant Resistance), Apache (Lr13, Lr37) and Orvantis (Lr10, Lr13, Lr37), some slight sporulation could be observed in the field, and isolates thus collected belonged to 3 to 5 dominant pathotypes (10 to 25%), and to some minor or rare pathotypes. This structure was confirmed for cv. Trémie on commercial plots, for virulence and AFLP markers.
Dominant pathotypes were not the same between the three resistant cultivars, and dominant pathotypes of resistant cultivars were absent or rare on the susceptible cultivars. The population structure was thus strongly influenced by host. Most of the minor and rare pathotypes had the virulence genes required to overcome the host resistance, but still remained at a low frequency. This low frequency of minor and rare pathotypes could not be explained by specific interactions.
Our hypothesis was that differentiation in agressiveness between the different pathotypes might help to explain the observed structure. Agressiveness, measured as spore efficacy and weight of spores produced by unit of leaf area, is being evaluated for isolates collected from cv. Soissons, belonging to either dominant or rare pathotype. The first results on agressiveness measures will be presented. Consequences for management of resistance are potentially important, as diversity for agressiveness in populations could allow selection for increased levels of agressiveness, under the selection pressure possibly exerted by cultivars with partial resistance, thus leading to the erosion of this resistance (Lehman & Shaner 1997).
Kolmer JA, 1993. Selection in a heterogeneous population of Puccinia recondita f. sp. tritici. Phytopathology 83, 909-914.
Martens JW, 1985. Oat stem rust. In: Roelfs AP, Bushnell WR, eds. The cereal rusts. London, UK: Academic Press. II: 103-129.
Lehman JS, Shaner G, 1997. Selection of populations of Puccinia recondita f. sp. tritici for shortened latent period on a partially resistant wheat cultivar. Phytopathology 87, 170-176.
2.24 Antioxidants in Blumeria graminis and Magnaporthe grisea
Catherine Henderson, Ziguo Zhang and Sarah Gurr
University of Oxford, UK
catherine.henderson{a}plants.ox.ac.uk
Phytopathogenic fungi elicit a range of defence responses in their hosts, the oxidative burst being one of the most rapid. Reactive oxygen species (ROS), in particular H2O2, accumulate at sites of pathogen invasion and are thought to have a number of functions including being directly antimicrobial, signalling, and bringing about the cross-linking of cell wall proteins. I am interested in the pathogen’s perspective on this defensive action. Since it is faced with .O2- and H2O2 during invasion of host tissue, the evolution of reactive oxygen detoxifying systems may arm it with a major selective advantage. My work compares antioxidant strategies in both the genetically intractable obligate biotroph Blumeria graminis, and the more amenable Magnaporthe grisea. I will discuss use of a combination of immunolocalisation, real-time RT-PCR, mutant analysis and a novel H2O2 scavenging assay to investigate the role of a secreted catalase, CATB, and how I am now extending the work to encompass further putative antioxidant genes.
2.25 The modern epidemiological situation of cereal rusts in European Russia
Sergey S Sanin, Larisa N Nazarova, Tagir Z Ibragimov
ALL-Russia Scientific Research Institute of Phytopathology, Russia
itz{a}vniif.rosmail.com
The principal grain producers in Russia, where 75% of the grain-growing area is concentrated in 6 regions, are:
North Caucasus region. Brown rust (Puccinia triticina) spreads everywhere in this region. Its share of the population structure decreased from 47 to 65%. Epiphytotics have occurred in 2-3 years out of 10, crop losses 30-35%. In recent years a marked increase in yellow rust (Puccinia striiformis) development has been observed. Its share of the population structure has increased from 10% to 39%.
Central-Chernozem region:. Brown rust of wheat (Puccinia triticina) spreads over the entire region. Its share is 22% of the population structure. Epiphytotics have occured in 3-4 years out of 10, crop losses 15-20%. Dwarf rust (Puccinia hordei ) has been found in recent years.
Central region: Brown rust of wheat (Puccinia triticina) spreads absolutely everywhere. Its share in the population structure has decreased from 55% to 36%. In epiphytotics, which have occurred in 5-7 years out of 10, crop losses were 20-30%. Brown rust of rye (Puccinia dispersa) occurs annually. Its share of the population structure is 49%. Epiphytotics have occurred in 5-7 years out of 10, crop losses 20%. In some years stem rust (Puccinia graminis) has been observed in rye. Dwarf rust of barley (Puccinia hordei) amounts to 15% of the pathogen complex.
Povolzhski region: The area sown with winter wheat has increased, but spring wheat has decreased. Brown rust of wheat (Puccinia triticina) appears annually and virtually everywhere in the region. Its share of the population structure amounts to 55%. Epiphytotics have occurred in 6-7 years out of 10, crop losses 20-30%. Brown rust of rye (Puccinia dispersa) occurs annually all over the area. Its share of the population structure is 67%. Epiphytotics have occurred in 5-6 years out of 10, crop losses 20-25%. Stem rust (Puccinia graminis) has weak or moderate development.
Volgo-Vyatski and Uralski regions: Brown rust (Puccinia triticina) spreads on spring wheat every where. Brown rust (Puccinia dispersa) of rye also occurs annually in some degree, amounting to 49% of the population structure. Epiphytotics have occurred in 4-6 years out of 10, with crop losses of 15-20%. The share of stem rust of rye (Puccinia graminis) in the population structure reaches 22%. Epiphytotics have occurred in 4-5 years out of 10, crop losses – 30-50%.
2.26 Do HVNAC-like transcription factors regulate host defence against the powdery mildew (Blumeria graminis f.sp. hordei) in barley (Hordeum vulgare)?
Michael K Jensen1, Torben Gjetting2, Peter Hagedorn2, Michael Lyngkjaer2, Sisse Gjetting1, Jesper H Rung1 and David B Collinge1
1Royal Veterinary and Agricultural University (RVAU), Frederiksberg, Denmark. 2Risoe National Laboratory, Roskilde, Denmark
mkrj{a}kvl.dk
NAC proteins constitute a large family of plant-specific transcription factors. They are believed to be involved in the floral and lateral root development in A. thaliana (Aida et al, 1999; Xie et al, 2000). NAC transcripts have furthermore been shown to be upregulated by different abiotic and biotic stresses, including pathogen attacks and abscisic acid treatment (Hegedus et al, 2003, Greve et al, 2003). Furthermore yeast two-hybrid analyses have revealed the interaction between a viral capsid protein and an A. thaliana NAC member (Ren et al, 2000).
Colleagues at Section for Plant Pathology at RVAU have isolated a NAC transcript from barley by differential display of powdery mildew-infected barley epidermis, 72 hai, and obtained the full length cDNA by RACE PCR (Gregersen, P., pers. comm.). The clone was named HvNAC6 (H. vulgare) for its high sequence identity to the rice NAC protein OsNAC6 (O. sativa). HvNAC6 was used as bait to screen a cDNA library for other HvNACs, and three other full length clones were obtained, representing three other NAC subfamilies.
Our group is currently working on the biological and biochemical function of these transcription factors. The biological function of HvNAC6 is studied by the use of a transient expression assay in detached barley leaves. In this assay, RNA interference (RNAi) is used to determine the role of HvNAC6 upon powdery mildew inoculation. Cell specific transcript analysis have so far indicated an upregulation of HvNAC6 gene expression in barley epidermal cells containing haustoria from the compatible interaction between a near-isogenic Pallas line and the powdery mildew isolate A6. Northern blots have supported these indications. The biochemical function of HvNAC6 will be analysed by yeast two-hybrid analyses, gel overlay assays and immunoprecipitation. Hence, the aim of the current research is to delineate the signalling pathway involving this family of transcription factors and their function in the mediation of defence responses and/or suppression of the plants innate immunity toward pathogens.
Aida et al. 1999. Shoot apical meristem and cotyledon formation during Arabidopsis embryogenesis: interaction among the CUP-SHAPED COTYLEDON and SHOOT MERISTEMLESS genes. Development. Apr;126(8),1563-70.
Xie et al. 2000. Arabidopsis NAC1 transduces auxin signal downstream of TIR1 to promote lateral root development. Genes Development, Dec 1;14(23),3024-36.
Hegedus et al. 2003. Molecular characterization of Brassica napus NAC domain transcriptional activators induced in response to biotic and abiotic stress. Plant Molecular Biology, 53(3),383-97.
Greve et al. 2003. Interactions between plant RING-H2 and plant-specific NAC (NAM/ATAF1/2/CUC2) proteins: RING-H2 molecular specificity and cellular localization. Biochememical Journal, Apr 1;371(Pt 1), 97-108.
Ren et al. 2000. HRT gene function requires interaction between a NAC protein and viral capsid protein to confer resistance to turnip crinkle virus. Plant Cell, Oct;12(10), 1917-26.
2.27 Differences in the genetic basis of complete resistance to Puccinia triticina
Zoran Jerkovic, Marina Putnik-Delic
Institute of Field and Vegetable Crops, Sad, Serbia and Montenegro
Jerkovic{a}ifvcns.ns.ac.yu
Five new Novi Sad (NS) lines were crossed with each of six lines from the local gene bank, all eleven lines showing complete seedling resistance (only rare minute chlorotic flecks) to a Puccinia triticina population. Three different combinations of the resistance genes in the new lines were recognized from an analysis of F2 resistance data. The large differences in frequencies of resistant plants in the progenies of the crosses Purdue 3120 x NS 1565, ZGKT 238&82 x NS 1565, Szegedy 768 x NS 1551 and ZGKT 238 x NS 1551, showed that the resistance of these two new lines did not have the same basis as NS 1536, NS 1549 and NS 1572. The results indicated that Purdue 5392 and UPI 301did carry the same resistance genes while the ZGKT lines were only partially identical in this respect. According to statistically confirmed coincidences between the experimentally achieved and theoretically correspondent dihybrid and threehybrid segregation ratios, complete resistance was to gene amount and inter-allelic interactions related There was evidence for the effect of at least two complementary genes for resistance in new NS lines.
2.28 Evolution of virulence in Puccinia striiformis f.sp. tritici
Annemarie F Justesen and Mogens S Hovmøller
Danish Institute of Agricultural Sciences, Research Centre Flakkebjerg, Slagelse, Denmark.
AnnemarieFejer.Justesen{a}agrsci.dk
Puccinia striiformis f.sp. tritici, which causes yellow (stripe) rust of wheat, is not known to complete a sexual life cycle, at least in north-west Europe. However, new pathotypes causing previously resistant cultivars to become susceptible evolve rapidly, possibly owing to mutation from avirulence to virulence. In a previous study of 100 P. striiformis f.sp. tritici isolates comprising 11 pathotypes from NW-Europe, 21 PstI/MseI +2 primer combinations were used to study genetic diversity. These were selected as the most informative based on an initial screening by 240 primer combinations of four isolates of different Danish origin. The isolates were grouped in 20 AFLP phenotypes based on a total of 28 polymorphic bands. Three of the groups possessed more than a single pathotype, suggesting evolution of virulence within a group. To investigate a hypothesis of single mutations leading to gain or loss of specific virulence traits, an extended AFLP screening by 256 primer combinations, revealing approximately 18,000 fragments, was carried out. Pairs of isolates of similar pathotypes and identical AFLP phenotype, based on the 21 primer combinations selected initially, were investigated. A pair was sampled at the same location and time, and when possible within the same field trial. Once a new polymorphism was discovered between any pair of isolates, all isolates belonging to the same group was investigated by the corresponding primer combination. Whereas almost all isolates in one of the groups remained identical, the two other groups diversified to some extent. However, in all groups, pairs of isolates of identical AFLP phenotype and different at a single virulence locus were identified, e.g. pairs varying in virulence for Yr2, Yr6, Yr7 and Yr17, respectively. Gain or loss of virulence for Yr6 was observed in two relatively distant groups. The data are consistent with the hypothesis of pathotype evolution by mutation at individual virulence loci in different clonal lineages.
2.29 Identification of molecular markers for wheat stripe rust resistance genes using RAPD and RGAP
Mansureh Keshavarzi, Mohammad Tahir Hallajian
University Azad, Tehran, Iran
mansureh_1343{a}yahoo.com
The resistance gene analogue polymorphism (RGAP) and random amplified polymorphic DNA (RAPD) techniques were used to identify molecular markers associated with resistance genes to yellow rust (Puccinia striformiis f.sp. tritici) in wheat. Genomic DNA of the five most resistant and five most susceptible F3 families from a cross between cv. Flanders (resistant parent) and cv. Morocco (susceptible parent) was bulked and, together with genomic parental DNA, amplified with RGAP and universal primers. 18 RGAP primers were designed based on LRR, NBS and kinase domains of known resistance genes. The amplified RAPD and RGAP products were detected on agarose and PAGE gels, respectively. Eight RGAP and two RAPD markers were amplified in the resistant, but not susceptible, bulks. Of these, RGAP marker (P210) and RAPD marker (OPF3) showed polymorphism between the susceptible and resistant parents. The results suggest a linkage between P210 and yellow rust resistance gene(s) in Flanders.
2.30 Genetics of leaf rust resistance in the Americano wheat landraces from Uruguay
James A. Kolmer and Lisa M Oelke
USDA-ARS, St.Paul, Minnesota, USA
jkolmer{a}umn.edu
The wheat landraces, Americano 25e (Am 25e), Americano 26n (Am 26n), and Americano 44d (Am 44d) were selected in Uruguay prior to 1918, and were used as leaf rust resistance sources in the initial crosses for the development of modern wheat cultivars for Uruguay and Argentina. These early landraces may contain leaf rust resistance genes that have not been previously characterized. The three landraces were crossed and backcrossed with the leaf rust susceptible wheat Thatcher (Tc), and approximately 80 BCF2 families were obtained from each cross. The BCF2 families were tested for segregation of leaf rust resistance at the seedling and adult plant stages with different races of Puccinia triticina. BCF2 plants selected for rust infection type were progeny tested as BCF3 lines. The backcross families of Tc / Am25e segregated for two independent seedling resistance genes to race BBBD, one of which may be Lr3; the second may be a previously uncharacterized resistance gene; and segregated for a third previously uncharacterized gene to race SBDG. Adult plants from Tc / Am25e BCF2 families that were homozygous susceptible to BBBD, segregated for a single resistance gene to BBBD that had a distinct infection type compared to the currently known adult plant Lr genes. BCF2 families from Tc / Am26n segregated for a single gene to race BBBD, which may be Lr11, and for a single independent unknown gene to race SBDG. Tc / Am26n BCF2 families that were homozygous susceptible to BBBD as seedling plants, segregated for two genes to BBBD as adult plants, which were likely Lr13 and Lr34, based on infection types. BCF2 families from Tc / Am44d were homozygous susceptible as seedlings to BBBD, and segregated as adult plants for a single gene that had a unique infection type compared to known adult plant Lr genes.
2.31 Introduction, spread, and yield loss caused by new races of Puccinia triticina on wheat in the U.S.A.
James A Kolmer, David L Long
USDA-ARS Cereal Disease Laboratory, University of Minnesota, St. Paul, USA
jkolmer{a}umn.edu
In 1996, new races of Puccinia triticina were detected for the first time in the Great Plains of the U.S.A. These races, designated as MBDS and MCDS, were virulent to the leaf rust resistance gene Lr17, which is present in the hard red winter wheat Jagger, which has been widely grown in Texas, Oklahoma, Kansas, and Nebraska. Races MBDS and MCDS increased quickly in the southern Great Plains region and have been the predominant races in this region since the late 1990s. By 2003 these races had also spread to the northern spring wheat area of Minnesota and the Dakotas, the soft red winter wheat region of the eastern U.S, and California. Since 1997, yield losses due to leaf rust have been estimated up to 4.0% in the hard red winter wheat grown in Oklahoma, Kansas, Nebraska, and South Dakota. In the spring wheat region of Minnesota and the Dakotas, losses due to leaf rust have also been up to 4.0% since 1997. In addition to virulence to Lr17, races MBDS and MCDS differ from the other races in the Great Plains region by having virulence to genes LrB and Lr3bg, and avirulence to Lr28. Races MBDS and MCDS also had very different molecular phenotypes as determined with AFLP markers compared to the other races in the Great Plains. Races MBDS and MCDS were likely introduced to the southern Great Plains region in the mid 1990s. The origin of these races has not yet been determined.
2.32 Postulated resistance genes in cultivars and lines with alien genes to wheat leaf rust
TM Kolomiets1, ED Kovalenko1, AI Zhemchuzhina1, LF Pankratova1, IF Lapochkina2
1All Russian Research Institute of Phytopathology, Vyazemy, Russia. 2 Agricultural Research Institute of Non-Chernozem Zone, Moscow region, Russia
kolomiets{a}vniif.rosmail.com
Twelve leaf rust resistance genes, Lr1, Lr2a, Lr3, Lr10, Lr14b, Lr16, Lr19, Lr23, Lr25, Lr26 and Lr 27+31, were revealed in common spring wheat cultivars by means of phytopathological testing with 15 pathotypes of leaf rust. Gene Lr1 was found in cvs. Moskovskaya 39, Ivolga, Bezenchukskaya 616 and Lutescens 661, Lr2a in cv. Trizo and Lr3 in cvs. Mironovskaya 808 and Zarya. Lr10 was discovered in cvs. Smena, Noris, Vadimovka, Nasledniza, Kurskaya 2038, Malahit and Svetoch, Lr14b in cvs. Zhemchuzhina Zavolzhya, Tulaykovskaya belozernaya and Tulaykovskaya stepnaya, and Lr16 in cvs. Voronegskaya 14, Priokskaya and Krestiyanka. Lr19 was identified in the cvs. Ekada 6, Aestivum 276, Ulia, Volgouralskay and Samsar. Cvs. Batiko, Pyramida and Tulaikovskaya 1 carried Lr23, Amir, Milturum, Ester, MIS, Lada carried Lr25 and Alisa and Mironovskaya 61 Lr26. The combination of Lr14b with Lr10 was found in cv. Zhiguleovskaya and that of Lr 27+31 in cv. Enita.
Eight leaf rust resistance genes: Lr1, 10, 16, 27, 28, 31, 43 and 44, have been found in fixed soft spring wheat lines with alien genes.
Most cultivars and lines have from one to four extra unidentified leaf rust resistance genes.
2.33 IGS, SSR and SRAP analysis of Puccinia striiformis isolates
Hedvig Komjáti, Matias Pasquali, Amelia Hubbard, David Lee, Rosemary Bayles
Szent István University, Hungary
komjatih{a}fau.gau.hu
Puccinia striiformis f. sp. tritici is the causal agent of yellow rust on wheat (WYR). Large numbers of virulence phenotypes have been identified over the years in the United Kingdom. The traditional method of pathotype identification, based on infection of differential cultivars, is time and labour consuming. Molecular techniques offer the potential for a quick and reliable method for pathotype characterisation.
The aim of the work was to explore the level of molecular variability of P. striiformis using different techniques. Twelve isolates of P. striiformis f. sp. tritici belonging to different pathotypes, one isolate of P. striiformis f. sp. hordei (barley yellow rust - BYR) and one isolate of P. triticina (wheat brown rust - WBR) were included in the study. All isolates were obtained from the NIAB collection. DNA was prepared from single spore isolates using DNeasy Plant Mini Kit (QIAGEN GmbH, Hilden, Germany). The analyses of the intergenic spacer (IGS) sequences based on the paper Rose-Amsaleg et al. (2002) and simple sequence repeats (SSRs) applying the conditions described by Enjalbert et al. (2002) showed inter-specific variation with the WBR and BYR isolates: no variation was observed between the isolates of WYR. Using the technique of Li & Quiros (2001), sequence related amplified polymorphisms (SRAP), polymorphic bands were observed between WYR isolates paving the way for the identification of pathogenicity-related markers.
2.34 Analysis of the genetic relatedness of German leaf rust isolates (Puccinia hordei Otth) by AFLPs
Doris Kopahnke, Hans-Ulrich Leistner, Ilona Krämer, Edgar Schliephake and Frank Ordon
Federal Centre for Breeding Research on Cultivated Plants, Institute of Epidemiology and Resistance, Aschersleben, Germany
d.kopahnke{a}bafz.de
World-wide surveys show that cereal rust fungi populations are often highly polymorphic for virulence characteristics and first results obtained by RFLP and RAPD analyses provide hints that in contrast to the formerly assumed uniformity a large degree of diversity exists. In order to get detailed information on the genetic relatedness of Puccinia hordei, 24 races present in the collection of 35 isolates of the Institute of Epidemiology and Resistance at Aschersleben and exclusively collected in Central Germany since 1950 were analysed by AFLPs. All races are extensively characterised for their virulence on 18 differential lines. Yearly observations of the leaf rust population in Saxonia-Anhalt since 1974 revealed that since 1985 mainly two highly virulent races have been present in this region. Out of the 35 races 24 were chosen representing pathotypes with different virulence patterns.
For molecular analyses 50 mg of rust spores were mixed with an equal volume of sand and homogenised by mortaring in liquid nitrogen. DNA was extracted using the CTAB-method followed by an additional purification step. AFLP analysis was carried out using EcoR1 and Mse1 digestion followed by a two step pre-amplification (+0, +1) and a selective amplification by EcoR1+2/Mse1+2-primers. Out of 32 tested primers the ones giving clear cut banding patterns were selected for AFLP-analysis of 24 leaf rust races which are biologically defined by the presence of different virulence genes. As an outgroup one race of yellow rust was included.
Based on these data genetic similarity was estimated and UPGMA-cluster analysis revealed differentiation between the leaf rust races and the out-group and also distinct grouping of the leaf rust races. However, no clear correlation between the differentiation on the basis of virulence genes and the applied AFLP primer combinations has been found up to now. In order to get a more detailed view additional AFLP-primers will be tested in the future.
2.35 Molecular markers with rust and powdery mildew fungi: Calculating similarity of banding profiles
Evsey Kosman, KJ Leonard
Tel Aviv University, Israel
kosman{a}post.tau.ac.il
We show that there are no acceptable universal approaches for measuring dissimilarity between individuals with molecular markers. Different measures are applicable to dominant and co-dominant DNA markers depending on ploidy of organisms. We show that the Dice coefficient is the suitable measure for haploid powdery mildew fungi with co-dominant markers and it can be applied directly to {0,1}-vectors representing banding profiles of individuals. Neither the Dice, Jaccard nor simple mis-match coefficient is appropriate for dikaryotic rust fungi with co-dominant markers. By transforming multi-allelic banding patterns at each locus into the corresponding homozygous or heterozygous states, a new measure of dissimilarity within loci was developed and expanded to measuring dissimilarity between multi-locus states of two individuals by averaging across all co-dominant loci tested. There is no rigorous well-founded solution in the case of dominant markers. The simple mis-match coefficient is the most suitable measure of dissimilarity between banding patterns of closely related haploid forms. For distantly related haploid individuals, the Jaccard dissimilarity is recommended. In general, no suitable method for measuring genetic dissimilarity between diploids with dominant markers can be proposed, and rough estimates might be all that is possible. Banding patterns of diploids with dominant markers represent individuals’ phenotypes rather than genotypes.
2.36 A computational tool for the analysis of plant pathogen populations
A Dinoor, A Herrmann, E Kosman, GA Schachtel
Tel Aviv University, Israel
kosman{a}post.tau.ac.il
The analysis of plant pathogen populations is normally based on experimental data sets that are organized in large tables with two entries (differentials in columns, isolates in rows). Several computer programs for processing this kind of data were developed, e.g. VIRULA by Welz and Ellmer (1991); HaGiS by Herrmann et al. (1999); and KOIND by Kosman (2001), each focusing on only a few aspects of the data. Our collaborative project aims at supporting a more exhaustive, more effective, and more compatible evaluation and presentation of such data by developing a new unified package of tools that contains most functions of the mentioned programs but extends them with a number of additional features and some recently developed methods. Our software will include tools for the basic routine steps such as data entry, dichotomization, identification of phenotypes, and characterization of samples by graphical means and by indices. Inference-statistical procedures will provide estimates of various diversity indices and other parameters for sexually and for asexually reproducing populations. These estimates obtained by bootstrap methods will allow further statistical evaluations. Recommendation concerning minimal sample sizes for reliable estimations in specific experimental situations will be offered. To make results of different researchers more compatible a tool to convert pathotype names from one nomenclature to another will be installed (e.g. from binary/octal to binary/hexadecimal).
Herrmann A, Löwer CF, Schachtel GA, 1999. Plant Pathology 48, 154-158.
Kosman E, 2001. In: Sustainable Systems of Cereal Crop Protection against Fungal Diseases as the Way of Reduction of Toxin Occurrence in Food Webs. L. Tvarůžek, ed. Agricultural Research Institute Kromĕříž, Ltd, Kromĕříž, 195-197.
Welz G, Ellmer J, 1991. In: Jørgensen JH, ed. Integrated Control of Cereal Mildews: Virulence Patterns and Their Change. Roskilde, Denmark: Risoe National Laboratory, 123-33
2.37 The main parameters of durable resistance to leaf rust in wheat
ED Kovalenko1, MI Kiseleva1, DA Solomatin1, AI Zhemchuzhina1, IF Lapochkina 2
1 All Russian Research Institute of Phytopathology, Vyazemy, Russia. 2 Agricultural Research Institute of Non-Chernozem Zone, Nemchinovka, Russia
kovalenko{a}vniif.rosmail.com
For the creation of wheat cultivars with durable protection against leaf rust, genes for adult-plant resistance and partial resistance or “slow rusting” are widely used. We have used eight test-pathotypes of the fungus, with virulences to adult plants of a Thatcher near-isogenic series with the individual resistance genes Lr12, 13, 34, 37 and combinations, to identify the genotypic basis of varietal adult-plant resistance. Adult–plant resistance genes were found in 20 wheat cultivars and lines arising as a result of interspecific hybridization with Ae. speltoides. The resistance gene Lr13+ was found in winter wheat cv. Mironovskaya 29, Lr37+ in cv. Mirleben and Lr12 in cv. Rodina. Resistance genes Lr12, Lr13, Lr34 and Lr37 and combinations were present in some lines with alien genes. The latent period was determined for 13 wheat cultivars described as “slow rusting” under field conditions. Specific interactions between the resistance of cultivars and the aggressiveness of pathotypes of P. triticina were revealed.
2.38 Significance of wheat yellow rust (Yr) genes in Chile
R Madariaga1, M Mellado1 and V Becerra1
1National Institute of Agricultural Research (INIA),Quilamapu, Chillan, Chile
rmadaria{a}quilamapu.inia.cl
Stripe rust caused by Puccinia striiformis West is the principal biotic factor causing the withdrawal of wheat cultivars from commercial use in Chile. Until the advent of EBI fungicides, susceptible cultivars reaching 20 MS were discarded from cultivation. However, with the protection of EBI’s or EBI’s + strobilurin fungicides, the life expectancy of cultivars has been chemically enlarged. Wheat breeding for stripe rust resistance has been in use since 1960 in Chile, with ephemeral success. The improvement method used consisted of a blind approach of Mendelian crosses between local agronomically adapted genotypes and foreign sources of resistance such as Heines VII, Selkirk, Cappelle-Desprez or Clement. More recently we have started to study specific genes such as those included in the Avocet isogenic lines for stripe rust. Field experiments were planted using standardised protocols, 2 m x 2 rows, 180 kg seed/ha at locations between Santiago, latitude 34 38 57 S and Osorno 40 56 36 S . Greenhouse tests of the lines showed similar responses to disease reactions observed in field studies. Avocet S and Yr9 showed the highest susceptibility, while Avocet isogenic lines carrying Yr5, YrSk, Yr10, Yr15 and YrSp pass the 12 qqm/ha Avocet S yield by 43; 56; 57; 61 and 66 qqm/ha respectively showing low or absence of disease. In addition Jupateco R carrying Yr18 showed moderate susceptibility but Jupateco S was as highly susceptible as Avocet S. An important contribution to identifying single effective Yr genes to pathogen virulences in Chile has been obtained from three years studies on the Avocet isogenic lines received from the Project Sydney University – CIMMYT.
2.39 Does the mlo resistance gene increase the susceptibility of spring barley to spotting diseases?
Joanne C Makepeace1, James KM Brown1, Simon JP Oxley2, James I Burke3
1 John Innes Centre, Norwich, UK. 2 Scottish Agricultural College, Edinburgh, UK. 3 Teagasc, Oak Park Research Centre, Carlow, Ireland
joanne.makepeace{a}bbsrc.ac.uk
Mutant alleles of the Mlo gene have been widely used in breeding spring barley as they confer durable resistance to Blumeria graminis (powdery mildew). However, these alleles have undesirable pleiotropic effects, including spontaneous necrotic flecking of leaves which leads to reduced yield. Recently, it has also been shown that barley plants with the mlo5 allele are more susceptible than wild-type (Mlo) plants to two necrotrophic pathogens, Magnaporthe grisea (blight; Jarosch et al. 1999) and Cochliobolus sativus (spot blotch; Kumar et al. 2001).
We are investigating whether plants with mutant mlo alleles are more susceptible than Mlo wild-type plants to three faculatative pathogens which are important in barley producing areas of Europe, namely Rhynchosporium secalis (scald), Pyrenophora teres (net blotch) and Ramularia collo-cygni. We are comparing near-isogenic lines of cultivars Ingrid and Pallas which have wild-type or mutant alleles of the Mlo gene. In both growth room experiments and field trials, lines with mutant mlo alleles were in fact more resistant to R. secalis, showing reduced disease symptoms and reduced levels of penetration by the fungus through the host’s cuticle. The effect of increased resistance to R. secalis was consistent across different mlo mutant alleles in the Ingrid background. It was also consistent across the two cultivar backgrounds, Ingrid and Pallas, for the mlo5 allele.
We have carried out a series of field trials in which all three diseases have been studied. Analysis of the data is in progress and conclusions will be reported at the conference.
Jarosch B et al., 1999. The ambivalence of the barley Mlo locus: Mutations conferring resistance against powdery mildew (Blumeria graminis f. sp, hordei) enhance susceptibility to the rice blast fungus Magnaporthe grisea. Molecular Plant-Microbe Interactions 12, 508-514.
Kumar J et al., 2001. A compromised Mlo pathway affects the response of barley to the necrotrophic fungus Bipolaris sorokiniana (teleomorph Cochliobolus sativus) and its toxins. Phytopathology 91, 127-133.
2.40 Detection of race-nonspecific resistance against leaf rust of wheat at seedling stage
K Manninger, G Máté, G Kátay, D Magyar and B Barna
Plant Protection Institute, Hungary
sman{a}nki.hu
Race-specific resistance of wheat (Triticum aestivum) to leaf rust (caused by Puccinia triticina) is often short-lived. Race-nonspecific resistance to disease at adult stage has shown more durability in many widely grown cultivars. This resistance causes a reduction of epidemic development despite a susceptible infection type. This resistance can be characterised by the following components: latency period, infection frequency and spore production.
Incubation time (latency period) and infection frequency were examined in greenhouse on different stages of seven wheat genotypes (Mv Emma, Mv Mezõföld, Mv Mambo, Mv 12-02, Mv22-2001, Mv336-02, Mv C1535-02) with leaf rust. In wheat genotypes the increase in latency period was pronounced in Mv Mambo which genotype showed a higher level of race-nonspecific resistance in the field. Variation in infection frequency was high in wheat genotypes, but not significant. Mv Mambo showed reduction in infection frequency.
Infected leaf segments were stained and observed by UV microscope 5 days after infection. Among the genotypes Mv 336-02 expressed strong hypersensitive reaction to leaf rust, in Mv Mambo colony diameter was smaller than in other susceptible genotypes.
The incubation time (latency period) and staining method are useful tools in the detection of race-nonspecific resistance against leaf rust in wheat genotypes at seedling stage.
2.41 Virulence survey for wheat rusts in Hungary during 2000-2003
Klára Manninger, Plant Protection Institute, Hungary
sman{a}nki.hu
Wheat leaf rust (Puccinia triticina) occurred annually, but its severity was variable in Hungary during 2000-2003. The wheat leaf rust population was virulent on Lr1, Lr2a, Lr2b, Lr2c, Lr3, Lr3ka, Lr3bg, Lr10, Lr11, Lr14a, Lr14b, Lr15, Lr16, Lr26, Lr30, LrB at seedling and adult stage. No virulent isolates were found on the lines carrying the resistance genes Lr9, Lr19, Lr24, Lr29, Lr38, Lr44, LrW, which were effective against leaf rust in the field, too. Lr12, Lr13, Lr17, Lr18, Lr20, Lr21, Lr22, Lr34, Lr35, Lr37 resistance genes were effective or moderately effective at adult stage only. Varieties with Lr26 and Lr34 are widely grown in Hungary. Rust isolates collected in Hungary are virulent on Lr26 and Lr34 in recent years. During the past 25 years Lr26 gene has lost its effectiveness. Lr34 was effective up to 1999, but now provides little protection in Hungary.
Yellow rust (Puccinia striiformis) was widespread in Röjtökmuzsaj (West Hungary) in 2000. There was an epidemic explosion all around the country in 2001, and it occurred sporadically during 2002-2003. Two pathotypes were mostly responsible for the 2001 epidemic. They were virulent for Yr2, Yr3, Yr6, Yr7, Yr8, Yr9, Yr17, YrCV and YrSD, and avirulent for Yr1, Yr4, Yr10, Yr15, YrSU and YrSPA. Among 78 Hungarian varieties 29 were strongly infected by yellow rust in 2001. Nevertheless, results of seedling stage and adult stage tests showed that some unknown effective resistance genes protected some of the Hungarian varieties against yellow rust.
2.42 Effects of meteorological conditions on uredo- and teliospores of rusts
Klára Manninger and D Magyar
Plant Protection Institute, Budapest, Hungary
sman{a}nki.hu
Hirst-type air samplers were used to register the daily concentration of airborne spores. The samplers were located in a traditional vineyard in Umbria country of central Italy, in 1994, 1995 and 1996, between May and June. The spore traps were placed in an instrumented tower at height of 30 m above ground level. For microscopical work we used 4000X object lens of DIALUX 20 microscope. Two longitudinal transverses along the length of the silicone-coated slide were scanned to determine spore concentration of the air samples. Concentration was expressed in spore/m3. Spearman’s Correlation Analysis was applied to clarify the relations between changes of daily airborne fungal propagule concentration and meteorological factors.
Uredospore counts of Puccinia spp. correlated positively with high temperature, number of sunny hours, evaporation and solar radiation. High relative humidity, cloud cover, fog, number of rainy days, duration of precipitation, rain on the previous day, had negative effect on the spore concentration. Intensive rainstorms with strong winds removed the airspora more efficiently. The presence of airborne sand particles showed positive correlation with the uredospore counts. Puccinia teliospores were observed rarely in the monitored period. Sudden change of wind direction caused spore release. High air pressure decreased the number of spores. Phragmidium teliospores were sparsely detected. Uredospores of Cronartium melampsoridium spp. were frequent in air samples.
2.43 Genetic diversity of Puccinia striiformis f. sp. tritici in the United States
Samuel G Markell1, Eugene A Milus1 and Xianming Chen2
1University of Arkansas, Fayetteville. 2USDA-ARS, Washington State University, Pullman, USA.
smarkel{a}uark.edu
In North America, epidemics of stripe rust, caused by Puccinia striiformis f. sp. tritici, have historically occurred in several regions of Mexico, and in western United States (Washington, Oregon, Idaho, California). Since 2000, stripe rust has emerged as a severe disease in south-central states (Arkansas, Louisiana, Texas) and in the Great Plains (Oklahoma, Kansas, Nebraska, Colorado). Recent epidemics in the south-central states and the Great Plains have been attributed to widespread and consistent overwintering of the pathogen in south-central states, favorable weather, susceptible cultivars, and new races that overcome the resistance gene Yr9 that had been effective against all races until 2000. The pathogen has no known sexual stage or alternate host to aid survival between wheat crops, and environmental conditions in the south-central states and Great Plains are not favorable for survival of the uredial stage over summer. The source of inoculum that overwinters in south-central states and the mechanism for evolving new races of the pathogen are not determined. Until recently, virulence/avirulence on differential lines was the only type of marker available to track inoculum dispersal and to determine how new races evolve. Polymorphic AFLP (Justesen et al., 2002) and SSR (Enjalbert et al., 2002) markers for P. striiformis f. sp. tritici were identified recently and may be useful in epidemiology and genetic studies of the pathogen. The objective of this study was to determine if the AFLP and SSR markers were useful for differentiating US isolates and to identify additional polymorphic markers. For each isolate, each polymorphism was scored as present or absent, and the data were analyzed with the Numerical Taxonomy System-pc software. Both AFLP and SSR markers clearly distinguished pre-2000 isolates from isolates collected in 2000 and later. These molecular markers likely would be useful for understanding genetic relationships among isolates, migration patterns among regions that may serve as donors or recipients of inoculum in North America, and determining the mechanism for the evolution of new races.
Enjalbert J, Duan X, Giraud T, Vautrin D, de Vallavielle-Pope C, Solignac M, 2002. Isolation of twelve microsattelite loci, using an enrichment protocol, in the phytopathogenic fungus Puccinia striiformis f. sp. tritici. Molecular Ecology Notes 2, 563-565.
Justesen AF, Ridout CJ, Hovmøller MS, 2002. The recent history of Puccinia striiformis f.sp. tritici in Denmark as revealed by disease incidence and AFLP markers. Plant Pathology 51, 13-23.
2.44 Catalogue of rust fungi in the Czech and Slovak Republics to be published
Jaroslava Marková
Charles University, Prague, Czech Republic
markovaj{a}natur.cuni.cz
Recently a catalogue of all rust species collected or recorded in the territory of former Czechoslovakia is being prepared. It is based on the material of the late Professor Urban who had begun to collect data and prepare the list of rust species. The catalogue will consist of approximately 370 species. Each species will be presented under the correct name (including the infraspecific taxa), main synonyms and characterized by life cycle and the host species. Additional list of localities with the date of collection, the name of the collector and herbaria where the specimen is preserved will be arranged according to the geographical areas. The localities of the commonly distributed species will be summarized. Short remarks concerning the differences between the related rust species will be added. An elaboration of the check list will be based on the present knowledge of the rust taxonomy, nomenclature and chorology. A list of excerpts and an alphabetical index of the host plant species and rust species will be included as well. A comparison of the listed species with the original work of Bubák (1906) may lead to an evaluation of the changes in the occurrence of certain species of rusts. The publication of the catalogue of rust fungi in the Czech and Slovak Republics is scheduled for 2006.
2.45 Orientation of mycelium growth of Puccinia triticina in wheat seedling leaves
Fernando Martinez, D Rubiales and RE Niks
University of Wageningen, The Netherlands
fernan{a}us.es
The wheat leaf rust fungus tends to grow deep into the mesophyll of the host plant leaf. A wheat leaf rust isolate was inoculated on two wheat cultivars on the adaxial or on the abaxial side of the first seedling leaf. After incubation, some leaves were fixed adaxial side up and others abaxial side up. Latency period and infection frequency were measured on both sides of each leaf. Pustules tended to appear earlier (shorter latency period) and were more frequent (higher infection frequency) in the side of the leaf that faced down, which could be either the adaxial or the abaxial side. We conclude that P. triticina tends to grow downwards into the leaf (negative phototropism or positive geotropism) especially when the adaxial leaf side is facing down.
2.46 Changes in the wheat leaf proteome caused by compatible and incompatible interactions Puccinia triticina (wheat leaf rust)
Christof Rampitsch1, Natalia Bykova1, Brent McCallum1, Werner Ens2
1Cereal Research Centre, Agriculture and Agri-Food Canada, Winnipeg, Canada. 2 University of Manitoba, Winnipeg, Canada
bmccallum{a}agr.gc.ca
Protein phosphorylation is invisible to nucleic acid-based investigations, but plays a major role in regulating many biochemical processes, including plant resistance to pathogens. Yet phosphorylation is difficult to study because the main players are rare proteins with a low copy number. Changes in the wheat (Triticum aestivum) leaf proteome and phosphoproteome resulting from leaf rust (Puccinia triticina) challenge to susceptible and resistant cultivars >Thatcher= and >Thatcher Lr1= were investigated. Responding proteins were identified as follows: (1.) By two-dimensional electrophoresis (2DE) and MALDI-QqTOF MS/MS; (2.) By enrichment of phosphoproteins using Ga (III) or iron (III) IMAC in a gel free strategy combined with anion-exchange chromatography (off-line) prior to analysis by nanoflow LC-ESI QTOF MS/MS and off-line LC-MALDI MS/MS; and (3.) By direct labelling of kinase targets in vitro with [γ32P]ATP, subsequent separation by 2DE and identification by MALDI QqTOF MS/MS. Some proteins from 2D gels were also sequenced de novo by tandem MS. The putative identity of all responding proteins and the possible roles of some of these in cellular recognition and signal transduction during disease resistance are discussed. In cases where data from wheat is unavailable, data from tomato (Lycopersicon esculentum) is presented.
2.47 Microsatellite tagging of the leaf rust resistance gene Lr16 on wheat chromosome 2BS
Curt McCartney, Daryl Somers, Brent McCallum, Julian Thomas, Gavin Humphreys, Jim Menzies, and Doug Brown
Agriculture and Agri-Food Canada, Cereal Research Centre, Winnipeg, Canada
bmccallum{a}agr.gc.ca
Leaf rust, caused by Puccinia triticina, is one of the most damaging diseases of wheat on a worldwide basis. Lr16 is a widely deployed seedling leaf rust resistance gene. Lr16 provides a level of partial resistance in the field, although virulence to Lr16 exists in the P. triticina population. The primary objective of this study was to identify markers linked to Lr16 that are suitable for marker-assisted selection. Lr16 was tagged with microsatellite markers on the distal end of chromosome 2BS in three mapping populations. Seven microsatellite loci mapped within 10 cM of Lr16, with the map distances varying between populations. Xwmc764 was the closest microsatellite locus to Lr16. Xwmc764 mapped 1, 9, and 3 cM away in the RL4452 x AC Domain, BW278 x AC Foremost, and HY644 x McKenzie mapping populations, respectively. Lr16 was the terminal locus in all three populations. Xwmc764, Xgwm210, and Xwmc661 were the most suitable markers for selection of Lr16 because they had simple PCR profiles, numerous alleles, high polymorphism information content, and were tightly linked to Lr16. Twenty-eight spring wheat lines were evaluated for leaf rust reaction with the Puccinia triticina virulence phenotypes MBDS, MBRJ, and MGBJ, and analysed with five microsatellite markers tightly linked to Lr16. There was good agreement between leaf rust infection type data and the microsatellite allele data. Microsatellite markers were useful for postulating Lr16 in wheat lines with multiple leaf rust resistance genes.
2.48 Temporal gene expression of the wheat leaf rust pathosystem using cDNA microarray
Bourlaye Fofana1, Stephen Strelkov2, Travis Banks1, Brent McCallum1 and Sylvie Cloutier1
1Cereal Research Centre, Agriculture and Agri-Food Canada, Winnipeg, Canada.2 University of Alberta, Edmonton, Canada
bmccallum{a}agr.gc.ca
The wheat-leaf rust (Puccinia triticina) interaction is a genetically well defined system but much remains unknown regarding the molecular basis of the disease development. To investigate patterns of gene expression in wheat seedlings (Triticum aestivum L. cv. Thatcher Lr1) a microarray made of 7728 cDNAs representing a unigene set from four different cDNA libraries was constructed. A total of 54 hybridizations were performed using total RNA isolated from leaf tissues that were Mock inoculated or inoculated with P. triticina race BBB or TJB and collected at 3, 6, 12 hours, and 1, 4 and 9 days postinoculation, in three independent biological replications. The preliminary results from the incompatible reactions (BBB) at early stage of disease development (3, 12 and 24 HPI) indicated a network group of genes that are up regulated compared to compatible reactions (TJB). These are essentially signal recognition proteins, transduction protein kinases (CDPK, MAPK, Ser/Thr PK), calmodulin, transcription factors, PR proteins, defensin, secondary metabolism genes, stress response genes. While expression of some disease resistance genes (LRR, Leaf rust resistance genes, Ser/Thr PK diamine resistance gene) are up regulated expression of some others present in the microarray remained unchanged. Interestingly, expression of some of the HR genes (ascorbate peroxidases, metallothionein proteins, catalase) were shown to be up regulated, while others like gluthatione s- transferase and super oxide dismutase were down regulated. High expression levels of transcription factors, PR-proteins, chitinase, ß-glucanase were observed by 24 HPI and expression profiles using cluster analysis revealed a well coordinated expression of signal recognition and transduction proteins, HR and PR- proteins, and DR genes. Expression data from this study will be discussed in relation to leaf rust pathogenesis and its regulation.
2.49 Genetic and physiological analysis of mutations in wheat showing enhanced adult plant resistance to yellow rust
James PE Melichar¹, Simon Berry² and Lesley A Boyd¹
¹John Innes Centre, Norwich, UK. ²Advanta Seeds UK Ltd, Docking, Norfolk, UK
james.melichar{a}bbsrc.ac.uk
Puccinia striiformis f.sp. tritici is a wholly asexual, biotrophic pathogen. It is the causal agent of yellow rust of wheat, and a serious economic foliar disease of most temperate wheat growing areas of the world. It has a distinctive phenotype of intervenial-stripes of yellow pustules (uredia). Control in the UK is based on an integrated approach of resistance breeding with rotation of fungicides of differing modes of action.
Mutagenised populations of the wheat varieties Guardian and Hobbit ‘sib’ have been screened in field tests, and mutant lines selected that show enhanced yellow rust adult plant resistance. These include the Guardian-derived mutants M66 and M257 (Boyd and Minchin, 2001) and the Hobbit ‘sib’ mutant line I3-54 (Smith et al, MPMI in press). M66 and M257 also show broad-spectrum resistance to powdery mildew and brown rust (Boyd et al., 2002), while I3-54 is resistant to powdery mildew but not brown rust. The M66 mutant also shows evidence of spontaneous necrotic flecking analogous to the lesion mimics found in mlo barley lines.
Pathology studies have examined the resistance mechanisms in these mutants at three distinct growth stages: seedling (GS 12-13), tillering (GS 26-27) and heading (GS 49-51), in order to elucidate the timing and phenotype of the adult plant resistance development in these mutants.
Preliminary histological examination of pathogen arrest in M66 and M257 suggests that the enhanced resistance is not due to an early hydrogen peroxide mediated HR, with resistance operating at the point of sporulation. In contrast, pathogen arrest in I3-54 occurs in the stomatal cavity, but again is not associated with a hydrogen peroxide mediated defence reaction.
Boyd LA, Minchin PN, 2001. Wheat mutants showing altered adult plant disease resistance. Euphytica 122, 361-368.
Boyd LA, Smith PH, Wilson AH, Minchin PN, 2002. Mutations in wheat showing altered field resistance to yellow and brown rust. Genome 45, 1035-1040.
Smith PH, Howie JA, Worland AJ, Stratford R, Boyd LA, 2004. Mutations in wheat exhibiting growth stage specific resistance to biotrophic fungal pathogens. MPMI (in press).
2.50 New races of Puccinia striiformis f. sp. tritici more aggressive than older races at 18oC
Eugene A Milus and Esra Seyran
University of Arkansas, Fayetteville, USA
gmilus{a}uark.edu
Stripe rust (yellow rust) of wheat is caused by Puccinia striiformis Westend. f. sp. tritici Eriks. and Henn. and has been one of the most important diseases wherever wheat is grown in cool environments. In the United States in 2000, stripe rust occurred in more than 20 states and was unusually severe in Arkansas and surrounding states (Chen et al., 2002). These epidemics were attributed to favorable weather and the occurrence of new races. Sixteen new races with virulence on yellow rust resistance genes Yr8 and/or Yr9 were identified, and this was the first report of virulence on these genes in the United States (Chen et al., 2002). However, Yr8 is not known to be in any wheat cultivar, only a portion of the susceptible cultivars had Yr9, and the weather in 2000 was not dramatically different from previous years. Furthermore, the new races completely replaced the old races that were found before 2000 in states east of the Rocky Mountains, and stripe rust continued to develop long after temperatures were presumed to be too warm for disease development. These data and observations suggested that the new races may be more aggressive than old races. The objective of this study was to determine if increased aggressiveness may be a contributing factor in recent stripe rust epidemics. Six isolates collected before 2000 and 14 isolates collected since 2000 were considered representative of “old” and “new” races, respectively. Isolates were evaluated at 12°C and 18°C for two components of aggressiveness, latent period (time from inoculation to sporulation) and spore germination rate (area under the germination curve and percentage germinated at 12 hours). There were significant (P<0.05) temperature by isolate interactions for latent period and spore germination rate. All new isolates had significantly shorter latent periods at 18°C than at 12°C. Of the six old isolates, four had similar latent periods at both temperatures, one had a shorter latent period at 18°C, and one had a shorter latent period at 12°C. As measured by area under the germination curve, eight new isolates and one old isolate had significantly faster spore germination rates at 18°C than at 12°C, and two old isolates had significantly faster spore germination rates at 12°C than at 18°C. Results were similar for the percentage of spores germinated at 12 hours. The results of this study indicated that new races appeared to be more aggressive than old races at warmer temperatures and that this increased aggressiveness likely contributed to the expanded geographic range and increased severities of stripe rust that have been observed east of the Rocky Mountains since 2000.
Chen XM, Moore M, Milus EA, Long DL, Line RF, Marshall D and Jackson L, 2002. Wheat stripe rust epidemics and races of Puccinia striiformis f. sp. tritici in the United States in 2000. Plant Disease 86,39-46.
2.51 The virulence of leaf rust population and resistance of spring wheat varieties and breeding lines in Northern Kazakhstan and Siberia
A Morgounov, M Koishibayev, Yu Zelenskiy, V Zykin, M Berdagulov, Sh Rsaliev, J Kolmer
CIMMYT, Almaty, Kazakhstan
a.morgounov{a}cgiar.org
Spring wheat is grown in the territory of Northern Kazakhstan on an area of 10 million ha. The adjoining area of Russia grows an additional 10-15 million ha of spring wheat, making the region a relatively uniform environment of dryland grain production. Leaf rust along with Septoria spp. represents a major biotic constraint especially in moist years accounting for up to 20-30% of yield losses. The Kazakhstan-Siberia Network on Spring Wheat Improvement (KASIB) unites 14 research and breeding institutions in germplasm exchange and evaluation. It also monitors the leaf rust population through evaluation of the Thatcher Lr isogenic lines. The data collected in 2001-2004 demonstrated that the following Lr genes provide protection from the pathogen population in the region: Lr 9, 12, 13, 20, 22A, 23, 24, 25, 26, 28, 29, 34, 35, 36. The majority of the varieties presently cultivated are susceptible to leaf rust. The new spring wheat varieties and breeding lines resistant to disease across locations are: Kazakhstanskaya 15, 19, E 736, 756, 387 MC, Duet, Kvinta, Lutescens 148-97-16, Sonata, Udacha, Aria, Tertsia, Fora. The presence of the Lr genes in the resistant varieties is being studied. Regional cooperation and multilocational testing allowed identification of resistant germplasm with high grain yield.
2.52 High-yielding winter wheat varieties resistant to yellow and leaf rust in Central Asia
A Morgounov, M. Yessimbekova, Sh. Rsaliev, S. Baboev, H. Mumindjanov, M. Djunusova
CIMMYT, Almaty, Kazakhstan
a.morgounov{a}cgiar.org
Winter wheat occupies 4 million ha of irrigated and rainfed land in the countries of Central Asia (Kazakhstan, Kyrgyzstan, Tajikistan, Turkmenistan, Uzbekistan). Yellow rust was a dominant disease in the late 90s and early 2000s accounting for yield losses of 20-40% during the epidemic years (1999, 2000). In 2002-2004 leaf rust started to appear on wheat and some varieties resistant to yellow rust became susceptible to leaf rust. Regional cooperation on winter wheat variety exchange and evaluation through multilocational testing allowed identification of genotypes combining resistance to two rusts in adapted, high-yielding background. The origin of the selected resistant and high-yielding varieties is Central Asia, Russia, Turkey-CIMMYT-ICARDA breeding program, Eastern Europe, USA.
2.53 Genetic analyses of adult-plant resistance to wheat stripe rust, Puccinia striiformis f.sp. tritici, in seven Australian wheat cultivars
K Nazari, CR Wellings, HS Bariana
The University of Sydney, Plant Breeding Institute, Camden, Australia
kumarse{a}camden.usyd.edu.au
Stripe rust, caused by Puccinia striiformis f.sp. tritici (Pst), is one of the most important diseases of wheat in Australia. Genetic analyses of adult-plant resistance (APR) to Pst were conducted in seven Australian wheat cultivars. The test-cross approach was chosen in this study in order to identify the number of resistance gene(s) and to develop reference stocks for Yr-genes conditioning APR in these cultivars. A total of 281 testcross F2 (BC1F2) families were screened for disease response in field nurseries using Pst pathotype 110 E143A+ in 2002. Stripe rust resistance appeared to be controlled by 2 to 4 genes. F3 (BC1F3) progeny tests were conducted in 2003 on those F2 families which were previously selected for single gene segregation ratio (3R:1S). Although unexpected natural infection of pathotype 134 E16A+ in F3 families did not allow correlation with the 2002 F2 data, single gene families were identified with similar segregation pattern in both years. In some crosses entire families that were predicted to segregate for a single gene to Pst 110 E143A+ (e.g. cv. Goldmark) became susceptible when progeny tested against Pst 134 E16A+. This strongly suggests race-specificity for at least one adult-plant resistance gene. Future work is planned to confirm this hypothesis. Multipathotype tests at the seedling stage indicated that the cultivars Mawson and Batavia carry seedling resistance genes Yr9 and Yr33, respectively. Therefore deviation from single gene ratios in field tests among Mawson F3 families was possibly due to selection for Yr9 in F2 lines against pathotype 110 E143A+. However, Batavia populations selected for single APR gene failed to show evidence of the low seedling infection type reported for Yr33, even when tested at different temperatures. Other cultivars (Goldmark, Sunland, Snipe, Giles, and Whistler) were susceptible to both pathotypes in seedling tests. Single gene F3 families among each cross that were effective against both pathotypes were selected to isolate and further characterise candidate APR genes. For each single gene family, homozygous susceptible (HS) and homozygous resistant (HR) lines were harvested individually for Bulk Segregant Analysis using microsatellite molecular markers.
2.54 Interaction between bacterial inoculum from a previous crop with fungal disease development on a subsequent crop
Adrian C Newton, Lizbeth J Hyman and Ian K Toth
Scottish Crop Research Institute, Invergowrie, Dundee, UK
a.newton{a}scri.sari.ac.uk
Winter wheat and winter barley were grown on soil contaminated with different levels of a marked bacterial potato pathogen Erwinia carotovora subsp. atroseptica (Eca) from a previous blackleg-infected potato crop, to determine whether the presence of this pathogen could affect disease development on wheat and barley. Wheat diseases were greater on the areas with high levels of Eca contamination than on areas with low level contamination. Rhynchosporium on barley was not affected overall, although it decreased on the high level contamination areas early in the season. The Eca strain was detected on the upper foliage of both wheat and barley and both biotrophic and necrotrophic pathogens were affected. Increased S. tritici symptoms in the field may be due to ‘synergistic’ interactions between the Eca and the fungal mycelium as reported in previously published laboratory experiments. The initial infection by the fungus itself may not be very damaging but bacterial rot promoted by increased availability of nutrients may be more damaging and plant resistance could be compromised. Induced susceptibility is also possible, especially in the case of the biotrophic infections such as Blumeria graminis.
These data have implications for crop management in rotation systems, particularly if organic matter high in bacterial inoculum is incorporated. It may also affect the efficacy of crop protection measures, particularly the choice and application of fungicides. Furthermore, practically valuable active ingredients may be eliminated in early fungicide screens, and new resistance sources missed where only single host-pathogen combinations are tested. The importance of assessing disease complexes and the implications for disease management, including the roles of organisms not pathogenic on the crop being assessed, will be discussed.
2.55 Introgression of non-host resistance to Puccinia hordei from Hordeum bulbosum into cultivated barley
Rients E. Niks1 and Richard Pickering2
1Laboratory of Plant Breeding, Wageningen University, The Netherlands; 2New Zealand Institute for Crop and Food Research Limited, Christchurch, New Zealand
rients.niks{a}wur.nl
Hordeum bulbosum is not known to be a host to the barley leaf rust fungus Puccinia hordei. This wild barley species can be crossed with cultivated barley and introgression of chromosome fragments has been achieved. Exploitation of genes for non-host resistance of H. bulbosum may be an interesting approach to barley breeding. Such genes may confer a more durable type of resistance than the genes available in the cultivated host species.
Twelve introgression lines of barley, each containing a chromosome fragment of H. bulbosum, were evaluated in the seedling stage for resistance to barley leaf rust. The non-host resistance of H. bulbosum to barley leaf rust was complete, and mainly non-hypersensitive and prehaustorial. This resistance was also displayed by the interspecific hybrids of H. bulbosum with two susceptible barley cultivars.
The introgression lines varied from susceptible to completely resistant. In some lines, complete or incomplete resistances were based on a hypersensitive reaction. In other lines, however, the introgressed H. bulbosum fragment caused a non-hypersensitive partial resistance of a similar level to that in the partially resistant cultivar Vada. One line in particular, 182Q20, clearly surpassed the already high level of partial resistance in cv Vada. The fragment introgressed into this line was located distally on the long arm of chromosome 2H. This fragment is being backcrossed into experimental barley lines to determine the rust species specificity of this non-hypersensitive resistance.
2.56 An Arabidopsis Mlo knock-out mutant phenocopies the barley mlo broad-spectrum powdery mildew resistance phenotype
Chiara Consonni, H. Andreas Hartmann, Paul Schulze-Lefert, and Ralph Panstruga
Max-Planck-Institute for Plant Breeding Research, Köln, Germany
panstrug{a}mpiz-koeln.mpg.de
Recessively inherited loss-of-function alleles (mlo) of the barley Mlo gene confer resistance that is effective against all known isolates of the barley powdery mildew fungus, Blumeria graminis f. sp. hordei. In susceptible Mlo wild type plants, the fungus potentially manipulates the protein encoded by this gene for plant cell invasion. Until recently, it was unclear whether mlo resistance represents a species-specific phenomenon restricted to barley. Although Mlo homologs are found in all flowering plants examined to date it was uncertain whether powdery mildews target MLO proteins in other plant species for plant cell invasion. Our results demonstrate that mlo resistance can be induced in the model plant Arabidopsis by inactivation of a particular of the 15 Mlo homologs. The respective Arabidopsis insertion mutants were found to be highly resistant against the virulent powdery mildew fungus, Golovinomyces orontii, while infection phenotypes to the bacterial pathogen Pseudomonas syringae, and the oomycete Peronospora parasitica appeared unaltered. Similar to barley mlo resistant plants, the powdery mildew sporelings fail to switch from surface to invasive growth in the Arabidopsis mutants. No recognizable pleiotropic effects are detectable in the mutants. These data demonstrate that mlo resistance is effective in both major clades of flowering plants, suggesting that the role of MLO proteins for colonization by powdery mildews is ancient and evolutionarily conserved.
2.57 Pathogenicity of Blumeria graminis f.sp. hordei, the causal organism of barley powdery mildew, in Iran
Mehran Patpour
Seed and Plant Improvement Institute(SPII), Iran
patpour{a}hotmail.com
To determine the genetics of pathogenicity and annual changes of virulence factors in the population of Blumeria graminis f.sp. hordei, the causal organism of barley powdery mildew, 18 Near Isogenic Lines (NILs) containing different Ml genes were planted in 11 locations in Iran. Susceptible Cultivars, Afzal and Zarjo were planted among the lines and also around the nurseries. To study the response of NILs at the seedling stage, the method of Mains and Deitz (1930) was used. Infected barley samples were collected from different locations and transferred to Karaj. After isolation and multiplication of the spores, they were inoculated on NILs, separately in the greenhouse. Data on disease severity and response of NILs were taken according to Saari and Prescott (1975), modified by Eyal et al., (1987). In general, the results showed that, the virulence frequencies of Blumeria graminis f.sp. hordei varied in different locations and years. In all locations, virulence for Mlg, MlCP and Mla6 was not observed. One of the isolates was virulent on Mlp in the seedling stage. Virulences for Mla in Ahvaz, for Mla7, MlAb and Mla3 in Dezful, for Ml(La) in Ahvaz, Karaj and Deazful, for Mla9 in Karaj, Dezful, Gorgan, Mashhad, Ahvaz and Ardabil, for Mlk in Firoozkandeh, Mashhad, Karaj, Gorgan, Ahvaz and Dezful, for MlSpiti in Dezful and Ahvaz for Miln’s Golden Promise line in Mashhad, Gorgan, Dezful, Ahvaz, Karaj, Miandoab and Gharakhil, for Mla13 in Ahvaz and Dezful, for Mla16 in Gorgan, for Mla17 in Minoodasht and Ardabil, for Mla18 in Gharakhil, Gorgan and Minoodasht, for Mla19 in Karaj, Ahvaz and Dezful was observed.
Eyal Z, Scharen AL, Prescott JM and van Ginkel M, 1987. The septoria diseases of wheat: Concepts and methods of disease management. Mexico, D.P.: CIMMYT.
Mains EB and Dietz SM,1930. Physiological forms of barley mildew, Erysiphe graminis hordei Marchal. Phytopathology 20, 229-239.
Saari EE and Prescott JM, 1975. A scale for appraising the foliar intensity of wheat diseases. Plant Disease Reporter 59 (5), 377-380.
2.58 Studying the powdery mildew fungus at genome, transcriptome and proteome level
Carsten Pedersen, Ziguo Zhang, Gerhard Saalbach, Peter Hagedorn and Hans Thordal-Christensen
Risø National Laboratory, Denmark
carsten.pedersen{a}risoe.dk
Powdery mildew caused by Blumeria graminis is one of the most severe diseases in cereals, especially wheat and barley. The plant-pathogen interaction is controlled by specific resistance genes in the plant and matching fungal avirulence genes according to the gene-for-gene hypothesis. B. graminis is an obligate fungus that develops haustoria inside the epidermal cells and it is important for the fungus to keep the cells alive in order to obtain nutrients from the plant. We are studying the fungus and the interaction at the genome level, the transcriptome level and the proteome level.
A detailed study at the genome level has been carried out by sequencing and analysing 74 kb of genomic DNA. It showed a complex mixture of genes, various kinds of retrotransposable elements and other types of repetitive DNA elements. The study gives a glimpse of what to expect of a whole-genome sequencing project.
At the transcriptome level we are looking at expression profiles of a unigene-set of about 1520 genes from an EST-project using high-density arrays on filters. We have compared 5 isolates at three different stages and are now undertaking a more detailed study of expression during the infection process.
Our proteome analysis is focusing on the intimate interaction. We have developed a method to isolate haustoria from infected leaves and are now doing proteome analysis on this material aiming at identifying proteins involved in uptake and transport of nutrients as well as proteins involved in signalling and communication with the plant.
Results from our studies on the three levels will be presented.
2.59 Defence responses activated by Iodus 40®, Milsana®, salicylyl heptanoate and trehalose during the wheat/Blumeria graminis f.sp.tritici compatible interaction
Delphine Renard 1, Béatrice Randoux 1,2, Philippe Reignault 1, Jean Sanssené 2 and Roger Durand 1
1 Université du Littoral Côte d’Opale, Calais, France. 2 Institut Supérieur d’Agriculture de Beauvais, France
delphine.renard{a}univ-littoral.fr
The negative impact of the occurrence of compatible interactions between wheat and Blumeria graminis f.sp.tritici leads to the intensive use of chemical fungicides. Such a strategy results in damages to both the environment and public health and alternative means of disease control have to be considered. We studied the induction of plant resistance through the elicitation of defence responses on the interaction between wheat and Blumeria graminis f.sp. tritici by treatments with four natural products or eliciting molecules: Iodus 40®, Milsana®, salicylyl heptanoate (SH) and trehalose. Our work focused on the obtained protection level and, at the cellular level, on active oxygen species (AOS) accumulation, lipid peroxidation and phenolic compounds autofluorescence.
Plants treated once showed quantitative differences in protection level obtained with the different tested products or molecules. Since plant defences involve the accumulation of AOS, these early markers of defence elicitation have been histochemically analysed and we focused on the hydrogen peroxide (H2O2) accumulation near the sites of penetration by the fungal appressorial germinative tube (AGT). Treatments resulted in an increased frequency of high DAB staining intensity. Moreover, as lipid peroxidation is expected as a major consequence of this AOS accumulation, the rate of lipid peroxides was also measured. Finally, we assessed autofluorescent phenolic compounds accumulation in both the papillae and in the cell wall in response to the applied treatments.
The extent of defence responses activated by the four different elicitor products or molecules tested in this work will be discussed.
2.60 Increased gene expression in leaf epidermis of barley leaves following attack by barley powdery mildew
Jesper H Rung1, Micheal Krogh Jensen1, Sisse Gjetting1, Per L Gregersen2 and David B Collinge1
1Royal Veterinary and Agricultural University, Thorvaldsensvej, Frederiksberg, Copenhagen. 2Danish Institute of Agricultural Sciences, Flakkebjerg, Denmark.
jhr{a}kvl.dk
A number of physiological processes are activated in plants during attack by potential pathogens. The defence response of barley to the barley powdery mildew fungus Blumeria graminis f. sp. hordei has been studied intensively and several induced defence related gene transcripts have been identified (Collinge et al., 2002). Nevertheless, several transcripts still remain unidentified. Recently 27 transcripts were isolated by a differential display of epidermis mRNA from powdery mildew inoculated barley leaves. None of these represent transcripts identified by classical differential or subtractive hybridization (Gregersen et al., 2001). Four of the identified transcripts encode a S-adenosyl-L-methionine synthetase (SAM–synthetase), a putative Dioxygenase involved in the methionine salvage pathway (IDI1), a Receptor-like kinase (HvRLK1) containing a novel extra cellular domain, and a putative transcription factor (NAC domain protein). SAM is the precursor for the synthesis of secondary metabolites like ethylene and polyamines. We will investigate the role of SAM synthesis and recycling in the defence response. The enzymatic activity of IDI1 will be characterised and the biological function of IDI1 and SAM synthetase will be studied by a transient expression assay based on post-transcriptional gene silencing. The RLKs constitute a growing protein family and are thought to function as receptors that regulate downstream phosphorylation cascades (Tichtinsky et al., 2003). Our aim is to identify downstream interacting proteins by Yeast Two Hybrid screening and gel overlay assays to elucidate these signalling cascades.
Collinge DB, Gregersen PL, and Thordal-Christensen, H, (2002). The nature and role of defence response genes in cereals. In: RR Belanger and WR Bushnell (eds.), The Powdery Mildews: A Comprehensive Treatise. APS Press, St. Paul, Minnesota, USA.
Gregersen PL and DB Collinge, (2001). Penetration attempts by the Powdery Mildew fungus into Barley leaves are accompanied by increased gene transcript accumulation in the epidermal cell layer. Journal of Plant Pathology 83, 257-260.
Tichtinsky G, Vanoosthuyse V, Cock JM, Gaude T (2003), Making inroads into plant receptor kinase signalling pathways. Trends In Plant Science 8 (5), 231-237
2.61 The distribution of brown and yellow rusts of wheat on different scales is related to the monocyclic processes of infection and dispersal
Ivan Sache
INRA, Thiverval-Grignon, France
sache{a}grignon.inra.fr
In this presentation, I will compare the geographical distribution of yellow and brown rusts of wheat as well as the distribution of the two diseases in a wheat field. I will explain the striking differences in the distribution of the yellow and brown rust by a few contrasted monocylic processes, which were assessed both in growth chamber and in the field. The overall geographical distribution of the two rusts is related to the temperature and wetness requirements for the establishment of infection. The weather-dependent success of infection explains why yellow rust is considered as adapted to temperate, oceanic climates whereas brown rust is considered as adapted to warmer climates. The contrasted within-field patterns of the two rusts, focal for yellow rust and fairly homogeneous for brown rust, are related to the dispersal ability of the fungal spores. The aerodynamical features of the spores of the two rusts being similar, the difference in mean dispersal distance must be caused by the difference in the size of the dispersal unit. Several experiments done on both spore and disease dispersal consistently ranked the two rusts according to their relative dispersal ability, which is higher in brown rust than in yellow rust. Generalization of these findings must, however, be done with caution, since there is circumstantial evidence of epidemics which do not fit this framework. Consideration of the genetic and ecophysiological interaction between crop and pathogen populations is often required to get a better understanding of rust epidemics. Moreover, these results have an explanative value but are of limited help to predict where and when a rust epidemic will start.
2.62 Efficient low cost methods in epidemiology of airborne pathogens
Erik Schwarzbach
Miroslav, Czech Republic
eschwarzbach{a}iol.cz
Efficient low cost tools and methods for epidemiological studies of airborn pathogens, such as powdery mildew or leaf rust, are described. Young experimental plants can be grown infection free safely under cellophane bags and irrigated from the bottom by capillarity. An infection free workplace can be constructed by assembling cheap air filters used for truck engines and an air blower. Long-term storage of cultures can be done by maintaining inoculated intact seedlings in test tubes on agar at a temperature close to zero. Contamination of agar medium or substrate by bacteria or moulds can be largely prevented by colloid silver in extremely low concentration. A uniform distribution of spores even in a small settling tower is achieved, if the spores are first mixed in a small volume of air, which is then pushed into the main tower with still air, where spores settle by gravity. Representative sampling of airborn inoculum is possible by an expensive jet spore trap mounted on the roof of a car, but also by periodically exposing infection-free plants for a few days at a well chosen elevated strategic point, far from diseased crops. In this way the course of an epidemic throughout the whole growing season can be monitored, as is demonstrated in the example below.
2.63 Natural variation of non-host disease resistance in Arabidopsis against wheat leaf rust and powdery mildew
Reza Shafiei and Garry J Loake
Institute of Molecular Plant Science, University of Edinburgh, Edinburgh UK
s0234671{a}sms.ed.ac.uk
Developing durable disease resistance remains a major challenge in agriculture. In this context, we are exploring the molecular basis of non-host disease resistance (NHR), which is durable and broad-spectrum in nature. We have assessed the responses of 83 Arabidopsis ecotypes to Blumeria graminis f.sp. tritici (Bgt) and Puccinia graminis f.sp. tritici (Pgt). Significant differences in responses to these pathogens were identified among the Arabidopsis accessions. In response to Bgt, accessions were classified into two major classes based on their relative resistance. The frequency of fungal penetration, haustorium formation and subsequent hypersensitive cell death in the Wc-1 accession were significantly greater than in other accessions tested. In Wc-1, haustoria were bilateral, with several well developed projections, similar to those found in the host plant (wheat). NHR in the Arabidopsis mutant enhanced disease susceptibility (eds) 1, was significantly reduced against Bgt (Yun et al. 2003). Surprisingly, the level of NHR determined in Wc-1 was less than that expressed in eds1 plants. With respect to attempted Pgt infection, the Wa-1 accession exhibited the greatest frequency of penetration, hypersensitive cell death and formation of intercellular hyphae. The identification of this accession may help shed light on the genetic basis of the chemical and topological signals required to guide rust germlings towards their target cells.
Yun B-W, Atkinson HA, Gaborit C, Greenland A, Read N, Pallas JA and Loake GJ, (2003). Plant Journal 34, 768-777.
2.64 Threat to stable wheat production in Eastern Africa and Asia from a new race of Puccina graminis tritici
Ravi P Singh1, Julio Huerta-Espino2 and Miriam G Kinyua
1International Maize and Wheat Improvement Center (CIMMYT), Mexico. 2INIFAP-CEVAMEX, Chapingo, Mexico. 3Kenya Agricultural Research Institute, National Plant Breeding Research Centre, Njoro, Kenya.
R.Singh{a}cgiar.org
Stem rust, caused by Puccinia graminis, is known historically for causing severe losses to wheat (Triticum aestivum) 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 developing countries’ spring wheat area is currently sown to cultivars either derived directly from CIMMYT-germplasm or CIMMYT germplasm used as parent. For more than 30 years, a major proportion of the CIMMYT wheat germplasm has remained resistant to stem rust and this disease is often not considered important any more. In fact in many countries wheat breeding is currently done in the absence of stem rust. In 1998, high susceptibility of CIMMYT germplasm was noted in Uganda and the race was later identified to carry a combined virulence for several genes present in CIMMYT germplasm including two genes, Sr31 and Sr38, of alien origin. Gene Sr31, located in 1B.1R translocation, occurs at high frequency in CIMMYT’s spring wheat germplasm and is also common in several winter wheat cultivars grown in Asia, Europe and USA. High susceptibility of Kenyan wheat cultivars and CIMMYT wheat germplasm was observed at Njoro, Kenya in 2001 indicating that either the Sr31 virulent race from Uganda is now widespread in Eastern Africa or alternatively a new Sr31-virulent race has evolved. Recent evidence has indicated that an Yr9-virulent P. striiformis race first evolved in Eastern Africa and then migrated to South Asia through Middle East and West Asia in about 10 years and caused severe epidemics in its migration path. Several important cultivars grown in the Middle East and Asia are now susceptible to stem rust in Kenya and could suffer severe losses if the Sr31-virulent P. graminis race follows a migration pattern similar to that of P. striiformis. Germplasm, identified to be resistant to stem rust in Kenya during the last two years, can be valuable source to diversify resistance.
2.65 Partial virulence for mlo resistance to barley powdery mildew in UK in 2003
SE Slater, JDS Clarkson
NIAB, Cambridge, UK
Spring barley cultivars carrying the mlo gene for resistance to powdery mildew (Blumeria graminis f.sp. hordei) have been grown commercially in the UK since 1980. Unlike the majority of resistance genes used in commercial cultivars, mlo resistance has remained effective for more than 20 years. Occasionally isolates have given limited infection on Apex and Riviera, the mlo differential cultivars, in detached leaf tests but failed to produce infection in further tests, suggesting that mlo resistance is influenced by environmental factors. In 1998, however, a group of isolates continued to give low levels of infection on mlo-carrying cultivars in a series of tests. Further tests from 1999 onwards have detected additional isolates carrying partial virulence for mlo. However, although numbers of isolates carrying mlo partial virulence have risen since 1999, the infection level given by partially virulent isolates has not increased.
Isolates were again screened for partial virulence to mlo in 2003. Of the 413 isolates tested by the UK Cereal Pathogen Virulence Survey (UKCPVS), 43% and 27% respectively gave limited infection on Apex and Riviera. A selection of these isolates (Apex + isolates) was further tested on a range of mlo-carrying cultivars, together with isolates which had given no infection on Apex and Riviera in the original differential tests (Apex 0 isolates). Apex + and Apex 0 control isolates from previous years were also included. Two identical experiments were carried out in March and May 2004.
In the first test, the 2003 Apex + isolates failed to produce higher infection levels than the 2003 Apex 0 isolates, although both sets of isolates produced more infection on the mlo cultivars than the Apex 0 controls. The Apex + controls gave the highest levels of infection. However, in the second test, 2003 Apex + isolates gave levels of infection in excess of the levels produced by Apex 0 isolates. The highest levels of infection were again given by the Apex + controls, with the lowest infection levels given by the Apex 0 controls.
Although isolates with low levels of partial virulence for mlo continue to be found regularly in the powdery mildew population, the level of partial virulence in these isolates does not appear to have increased since 1998.
2.66 Comparison of virulence frequencies in populations of cereal powdery mildew from Cambridgeshire, Suffolk and Lancashire in 2003
J D S Clarkson and S E Slater
NIAB, Cambridge, UK
john.clarkson{a}niab.com
Virulence surveys of wheat and barley powdery mildew (Blumeria graminis f.sp tritici and B. graminis f.sp hordei) populations have routinely been carried out by the UK Cereal Pathogen Virulence Survey (UKCPVS) since 1967. In addition to collection of samples from UK trial sites and crops, in recent years random collection of airborne spore samples has been limited to one site in Cambridge. However, populations of powdery mildew in trial areas may not be representative of populations in general where large areas are sown with relatively few cultivars carrying resistance genes in common
To examine the differences in population structure, random samples of airborne spores were collected from five locations in 2003, three in Cambridgeshire (eastern England) and one each in Suffolk (eastern England, approximately 20 miles from Cambridge) and Lancashire (north west England, 200 miles from Cambridge). Seedlings of the susceptible cultivars Cerco (wheat) and Golden Promise (barley) were exposed for 7 day periods in June. Single colony isolations were made from the resulting infections and subsequently tested on a range of differential cultivars to determine virulence frequencies and pathotypes present in the populations.
Although small differences in virulence frequency of barley powdery mildew were detected between the samples from the five sites, overall the populations appeared similar. Va9 and Va3 were less frequently identified in samples from Cambridgeshire, while Vk1 occurred more frequently in the sample from Suffolk. The Lancashire population did not appear particularly different from those collected in eastern England. Over half the isolates of barley powdery mildew tested carried nine virulence factors. The Lancashire population appeared more complex, with the simplest pathotype carrying eight virulence factors, compared with six in the eastern populations.
Wheat powdery mildew virulences V2, V4b, V6, corresponding to the resistance genes in current popular cultivars, were detected in all isolates tested. VTo and VBr were present in 11% of the isolates from one Cambridgeshire population, but in over 20% in the remaining four populations, with the highest frequency (27%) in the Lancashire sample. Virulence for Mld and MlAx was lower in one of the Cambridgeshire samples. All the populations were dominated by a single common pathotype, V2,4b,5,6,8,Ta2, although this pathotype was less common in one Cambridgeshire sample. Pathotype V2,4b,5,6,8,Ta2,To,Br was more common in the Lancashire population.
However, as sample sizes were small, variation between populations may not be significant. In general it appears that the virulence structure of geographically separated populations of wheat and barley powdery mildew in the UK is similar.
2.67 Identification of molecular markers linked to the wheat leaf rust resistance gene Lr20
Melinda Tar, Laszlo Purnhauser, Maria Csosz, Akos Mesterhazy
Cereal Research Non-Profit Company, Szeged, Hungary
tarm{a}gk-szeged.hu
Wheat leaf rust caused by Puccinia recondita is one of the most important fungal diseases of wheat worldwide. Although fungal diseases can be controlled with fungicides however breeding for resistance is considered to be the most economical and environmentally appropriate strategy to reduce damages due to this disease. However, the traditional way of transferring one or more resistance genes to a single wheat cultivar is very laborious and time consuming process. In recent years, DNA-based markers (RAPD, AFLP, STS and SSR) have shown great promise in lessening the time and expense for pyramiding resistance genes. To use the advantages of marker assisted selection it is important to develop more molecular markers closely linked to the different leaf rust resistance genes. The aims of this work were to identify molecular markers linked to the Lr20 leaf rust resistance gene of wheat.
The near isogenic line (NIL) of Lr20 and its recurrent parent Thatcher, were used to identify AFLP and SSR markers linked to Lr20. An F2 population from a cross between the resistant NIL Lr20 and the susceptible cv ‘GK Delibab’ (Triticum aestivum) was used to map the linked markers. Segregation of Lr20 for resistance was evaluated using artificial infection. Out of 135 AFLP primer combinations tested, 32 showed polymorphism between the parental lines. However no linkage was found between of these bands and the resistant phenotype when tested in the F2 segregating population. In addition 15 microsatellite primers were tested also between the parental lines. Three were polymorphic but only one of them was linked to the Lr20.
Acknowledgements
The research was supported by the grants of the Hungarian Ministry of Education (grant No. OTKA TS 40887 and NKFP Wheat Consortium, project No. 4/038/13).
2.68 Stabilisation of polymorphism in gene-for-gene relationships through host-mediated interactions between parasites
Aurélien Tellier and James KM Brown
John Innes Centre, Norwich, UK
aurelien.tellier{a}bbsrc.ac.uk
The gene-for-gene relationship, which operates in many host plant–parasite interactions (1) describes the interaction of host plants and biotrophic parasites. Interactions are asymmetric i.e. the host’s defences are only qualitatively effective when it has a resistance gene that matches an avirulence gene in the parasite. A major challenge for theoretical biologists has been to account for the maintenance of polymorphism at resistance and avirulence loci in hosts and parasites, for which there is much evidence from field and molecular data. Constitutive costs of resistance and virulence are the most obvious explanations, but evidence for such costs is limited (2,3) and the known costs are generally not high enough to maintain polymorphism (4,5; c.f. 6).
We have taken a new theoretical approach to this problem, involving host-mediated interactions between parasites. The pathogen undergoes several generations per plant generation and resistance and virulence have realistically small costs. Each plant is infected by a succession of virulent and avirulent pathogens. We then introduce induced (or acquired) resistance, in which a resistant plant attacked by an avirulent pathogen not only expresses strong defence against the avirulent pathogen itself, but also weaker, systemic resistance against any further pathogen attack (virulent or avirulent). Activation of induced resistance involves a cost to the plant (7).
We reach three main conclusions from our model.
i) Stable polymorphism, i.e. long-term maintenance of resistant and susceptible plants as well as virulent and avirulent pathogens, is achieved with realistic values of the cost and effectiveness of induced resistance, and realistic values of constitutive costs of gene-for-gene resistance and virulence.
ii) The stability of the polymorphic state depends on the global cost of resistance (constitutive and induced defences) compared to the cost of disease.
iii) The relative importance of two different ways of maintaining polymorphism can be determined: constitutive gene-for-gene resistance, characterized by qualitative responses and tiny costs, and inducible defences with quantitative effectiveness and a higher cost of expression.
From these results we propose a new hypothesis for maintenance of polymorphism in gene-for-gene interactions. This may be achieved when there is frequent competition between virulent and avirulent pathogens on a host plant. Interactions between parasites, either within species (as investigated here) or across species, may be a major driving force for coevolution of plants and parasites. To test this hypothesis, the relative values of constitutive and inducible costs and the severity of damage caused by the pathogen should be determined experimentally in different pathosystems. In addition, further information is required about allele frequencies and super-infections occurrence in natural pathosystems.
1. Crute IR, Holub EB, Burdon JJ, 1997. The Gene-for-Gene Relationship in Plant-Parasite Interactions (CAB International).
2. Bergelson J, Purrington CB, 1996. American Naturalist 148, 536.
3. Brown JKM, 2002. Current Opinion in Plant Biology 5, 339-344
4. Leonard KJ, 1977. Annals of the New York Academy of Sciences 287, 207
5. Brown JKM, 2003. Trends in Genetics 19, 667
6. Tian D et al., 2003. Nature 423, 74–77
7. Baldwin IT, 1998. Proceedings of the National Academy of Sciences 95, 8113
2.69 Powdery mildew control in winter barley pure stands and cultivar mixtures using different timing and doses of fungicides
Anna Tratwal ¹, Jadwiga Nadziak ²
¹Plant Protection Institute, Poznań, Poland. ²Plant Breeding Smolice” Bąków Division, Poland.
Crop monoculture is successful in obtaining maximum yield in high-input agriculture under near-optimal environmental conditions. Monoculture of modern cereal crops is popular for technical and organizational reasons. However, a negative consequence of genetic uniformity is an increase in genetic vulnerability to diseases. Experimentally and practically it has been proven that cultivar and species mixtures can constitute an alternative to cultivar growing in pure stands. Cultivar mixtures can provide functional diversity that limits the expansion of pathogens and this makes use of knowledge about interactions between hosts and their pathogens to direct pathogen evolution. It has previously been shown that different epidemiological and ecological factors operate in mixtures, leading to considerable disease reduction, pest and weed control, and finally result in higher and more stable grain yields, than in varieties grown in pure stands.
The results of two years of field experiments, designed to evaluate the epidemiological and economical effects of winter barley cultivar mixtures, are presented in this paper. The impact of reduced dosages of fungicides on disease incidence and grain yield in the mixtures was also evaluated.
2.70 Analysis of mutual occurrence of two diseases: powdery mildew and leaf rust on winter wheat cultivars with susceptible, partially resistant and specific resistant reactions to powdery mildew
Lubomir Vechet
Research Institute of Crop Production, Drnovska, Prague-Ruzyne, Czech Republic
vechet{a}vurv.cz
In four years experiments the development and severity of powdery mildew (Blumeria graminis f.sp. tritici) and leaf rust (Puccinia triticina f.sp. tritici) was analysed on three groups of winter wheat cultivars: Kanzler (the control – susceptible standard to powdery mildew; Lr1; Lr3a) Mikon and Ramiro – partially resistant standard to powdery mildew and cultivars with specific genes of resistance Asta (Pm2, Pm6; Lr3) and Vlasta (Pm2 and Pm6 and probably minor genes of resistance to powdery mildew).
Development of powdery mildew was earlier whereas that of leaf rust was later. Disease severity of leaf rust was higher than powdery mildew. The leaves most affected by powdery mildew were the lower leaves whilst those most affected by leaf rust were the upper leaves. Higher severity of both diseases on a leaf did not reach fifty percent of the leaf surface. The cv. Vlasta was very resistant to powdery mildew but more susceptible to leaf rust. Cultivars with partial resistance and specific resistance had lower disease severity than the cultivar susceptible to powdery mildew. Differences in disease severity of leaf rust among three studied cultivars were not so unambiguous as in powdery mildew. The cv. Kanzler had higher disease severity in 2001 and 1999. Disease severity on the cv. Vlasta rapidly increased in the last period of evaluation at the end of June only. Very low leaf rust severity on the cv. Mikon was in 1999 while on the cvs. Kanzler and Asta was medium disease severity. The cv. Mikon had lower disease severity than the others cultivars.
The highest severity of powdery mildew was on the lower leaves mostly on the fourth or third leaves from the top whereas severity of leaf rust was the highest on upper leaves in most cases on the second or the first leaf. On the most affected leaf proportion of both diseases was different in all cultivars but leaf rust predominated in opposite to powdery mildew.
2.71 Efficient substances of biological and synthetic texture induced resistance to powdery mildew (Blumeria graminis f.sp. tritici) on winter wheat
Jana Martinkova, Lubomir Vechet, Marie Sindelarova, Lenka Burketova
Research Institute of Crop Production, Drnovska, Prague, Czech Republic
vechet{a}vurv.cz
The effect of several compounds of natural origin: curcuma (Curcuma longa L.), ginger (Zingiber officinale Roscoe), Reynoutria sacchaliensis L., preparation from oak bark (Quercus robur L.) and synthetic compounds: benzothiadiazol – BTH and glycine betaine were examined for their ability to reduce disease severity of Blumeria graminis f.sp. tritici on leaf segments of the susceptible cv. Kanzler. Leaf segments (length 3 cm) were prepared from the first leaves of young plants seven days after treatment by inductors and inoculated by mixture of powdery mildew (virulent to genes of resistance Pm2, 6; Pm4a, Pm4b) in a growth chamber. 12 days after inoculation disease severity of the pathogen was evaluated in four repetitions. Five experiments in four replications were done and disease severity was assessed on a 9 point scale. All used inductors reduced severity of powdery mildew. Polyacrylamide gel electrophoresis (PAGE) was used for separation of extracellular proteins. Gels were silver stained. The most pronounced effect on disease development was found in BTH (14% of the control) and curcuma (25% of the control) treated plants. The least effective was salicylic acid (only 78% of the control). Simultaneously, the role of the inducers in leaf extracellular protein synthesis was analyzed. Increased production of extracellular proteins was found after application of all inducers but mainly in plants treated with BTH and ginger extract.
2.72 Virulence of Puccinia triticina population in the North-Caucasian Region, Russia
Galina V Volkova
All-Russian Research Institute of Biological Plant Protection, Russia
volkova1{a}mail.kubtelecom.ru
The population of Puccinia triticina in the North-Caucasian region of Russia is characterized by its high heterogeneity. The identified pathotypes differ in their qualitative and quantitative composition of virulence alleles. The pathotypes with 23-28 virulence alleles are the most dangerous ones for the host plant. These make up 12.6% of the fungus population. An analysis of the qualitative composition showed high virulence frequencies (40-90%) to the genes Lr1, Lr3a, Lr10, Lr11, Lr12, Lr13, Lr14a, Lr14b, Lr16, Lr17, Lr22a, Lr22b, Lr30, Lr34, Lr35, Lr37, Lr40 at the germination stage. Low virulence frequencies (up to 11%) were identified to the genes Lr21, Lr24, Lr32, Lr39, LrB, LrW. The genes Lr9, Lr19, LrTr demonstrated high performance against brown rust at the first ontogeny stage. At the adult plant stage the varieties and lines Lr9, Lr19, Lr25, Lr29, LrTr had high performance in controlling the North-Caucasian Puccinia triticina population. The genes which were effective during the whole growing season of wheat can be recommended for practical breeding in the North-Caucasian Region in Russia.
2.73 Studies of interaction between pathogens
Jeanette H. Vollmer, Hanne Østergård
Risø National Laboratory, Roskilde, Denmark
jeanette.vollmer{a}risoe.dk
A fundamental ecological observation is that activity of any organism affects the environment in which it lives. This has consequences not only for that organism or members of that species, but for other species inhabiting the same environment. It means that the dynamics of a population of a species living in a given environment cannot be evaluated without considering other species in the same environment and the interactions with and between these. Two species may experience several potential interaction types, e.g. competition (A and B have a negative effect on each other), mutualism (A and B have a positive effect on each other) or exploitation (A has positive effect on B, while B has negative effect on A), which may be either direct or indirect.
Pathogen interactions and their effects on disease levels are often ignored in plant pathology, where pathogens are most often studied in isolation. Here we will discuss how to infer interaction from data. We will present a list of studies that consider two diseases simultaneously and discuss the use of different approaches of analysis to demonstrate interaction. Among these approaches is the use of the Lotka-Volterra model for competition between two species, the implications of which will be considered. We will discuss the difference between interaction and density dependence.
2.74 Pathogen dynamics associated with historic stripe (yellow) rust epidemics in Australia in 2002 and 2003
CR Wellings and KR Kandel
The University of Sydney, Plant Breeding Institute, Camden, Australia.
colinw{a}camden.usyd.edu.au
Following the first occurrence of wheat stripe rust (caused by Puccinia striiformis tritici = Pst) in eastern Australia in 1979, severe epidemics were experienced in 1983 and 1984. Epidemics continued to be occasional problems in certain seasons and regions, but these were essentially localised events. Wheat growing regions in Western Australia (WA) remained free of stripe rust due to the physical separation afforded by the Nullabor Desert, and the predominant west to east movement of weather patterns. The first record of wheat stripe rust in WA occurred on 20 August 2002. The disease spread rapidly under conducive conditions of temperature, moisture and widespread cultivation of susceptible cultivars. During this period, eastern Australia experienced a severe drought, although stripe rust was common in cultivars vulnerable to certain pathotypes. The 2003 season was almost a complete contrast: stripe rust incidence in WA was very low, whereas a return to better seasonal conditions in the east, combined with the first detection of the new WA Pst pathotype resulted in an epidemic reminiscent of those of the mid eighties. Industry sources estimate fungicide control costs in eastern Australia in 2003 was in excess of $A40 million.
Rust epidemics are an interplay of several factors which, when combined, will determine the extent of crop losses. These factors in 2002 and 2003 included:
1. Early epidemic onset. The most severe epidemics in the 1980’s began in mid winter, allowing sufficient inoculum load to fuel widespread infection in spring. The 2002 epidemic in WA began in mid August (late winter), and the 2003 epidemic in eastern Australia began in late July (mid winter).
2. Epidemic development. Where sufficient inoculum and susceptible cultivars are present, seasonal conditions in spring will further determine epidemic development. The severe regional epidemics in 2002 and 2003 were characterised by extended cool temperatures with adequate rainfall throughout spring.
3. Pathogen change. The predominant pathotypes of Pst during this period included:
· 110 E143 A+, first detected in 1986, and periodically recovered over the years. This pathotype became increasingly important in eastern Australia when cultivar H45 (carrying Yr7) became widely adopted in recent years.
Virulence/avirulence formula: Yr2, Yr3, Yr4, Yr6, Yr7, YrA / Yr1, Yr5, Yr8, Yr9, Yr10, Yr15, Yr27.
· 104 E137 A-, Yr17+ was first detected in 1999 and has remained at low levels in the pathogen population. It is relatively avirulent, but has a particular advantage on cultivars such as Trident, Camm, QAL2000 which are principally protected by Yr17. Virulence/avirulence formula: Yr2, Yr3, Yr4, Yr17 / Yr1, Yr5, Yr6, Yr7, Yr8, Yr9, Yr10, Yr15, Yr27, YrA.
· 134 E16 A+ was first detected in WA in 2002 and spread to the east in 2003. This pathotype represents a foreign incursion to Australia since it has pathogenicity features that contrast widely to prevailing pathotypes in the east. It remains the sole pathotype of Pst in WA, and rapidly became the dominant pathotype in the east during its first year in 2003.
Virulence/avirulence formula: Yr6, Yr7, Yr8, Yr9, YrA / Yr1, Yr2, Yr3, Yr4, Yr5, Yr10, Yr15, Yr27.
2.75 Resistance of Ethiopian barley landrace lines to Puccinia hordei under field conditions
Getaneh Woldeab1, Chemeda Fininsa2, and Jonathan Yuen3
1Ethiopian Agricultural Research Organization, Plant Protection Research Center, Addis Ababa, Ethiopia. 2Alemaya University, Dire Dawa, Ethiopia. 3Swedish University of Agricultural Sciences, Uppsala, Sweden
getaneh.woldeab{a}yahoo.com
In the 2003/04 cropping season, 481 single plant derived lines of 12 Ethiopian barley (Hordeum vulgare L.) landraces as well as local, susceptible and resistant checks were evaluated at Adet (Gojam zone) in the north, Ambo (Shewa zone) in the central and Sinana (Bale zone) in the southern parts of the country, for partial resistance to barley leaf rust (Puccinia hordei). The experiments were laid out in triple lattice design in a plot size of 0.4m2 with two rows per entry. Two rows of leaf rust susceptible cultivar L94 were planted between and around the replications to initiate infection. Incidence of the disease reached 100%. Epidemics of Leaf rust were slight (area under disease progress curve (AUDPC) for the susceptible check, L94 = 164.1) at Ambo, moderate (AUDPC = 460.6) at Adet and severe (AUDPC = 587.2) at Sinana. In all the test sites, no effective major resistance genes to barley leaf rust could be identified. Conversely, the variation between and within the landraces in mean area under the curve was large. Many landrace lines possess lower mean AUDPC than the susceptible check. Moreover, landrace lines 1637-09, 1686-56,1686-79, 3255-35, -50, -65, 3262 -49, -64, -69, -70 and -76 originating from Shewa, Arsi and Bale zone at altitude of about 2400m. have significantly lower mean AUDPC than the other lines at each and across the test sites. These and many other Ethiopian barley landrace lines tested seem to carry genes for partial resistance to barley leaf rust.
2.76 Linkage analysis of powdery mildew resistance gene Pm16 in common wheat (Triticum aestivum L.) using molecular markers
Xinmin Chen, Zhonghu He, Xianchun Xia
Institute of Crop Breeding and Cultivation/National Wheat Improvement Center, Chinese Academy of Agricultural Sciences,Beijing, China
xiaxianchun{a}caas.net.cn
Application of resistant varieties is a most economical way to control powdery mildew (Blumeria graminis f. sp. tritici) in wheat (Triticum aestivum L.). Identification of molecular markers closely linked to resistance genes can largely increase the efficiency for pyramiding resistance genes in wheat varieties. The objective of our study was to identify molecular markers closely linked to powdery mildew resistance gene Pm16 that is resistant to almost all isolates of B. graminis tritici in China at present. A F2 population with 156 progenies produced from the cross ‘Chancellor (susceptible) × 70281 (resistant)’ was used for mapping the powdery mildew resistance gene. The resistant line 70281 in the cross was derived from Erlikang that possessed the resistance gene Pm16. A total of 45 SSR markers on Chromosomes 4A and 5B of wheat and 15 SSRs on chromosome 3 of rice were employed to test the parents as well as the resistant and susceptible bulks, and 7 resulted polymorphic markers on Chromosomes 4A and 5B of wheat were employed to genotype the F2 population. The results indicated that the SSR marker Xgwm159, located on the short arm of chromosome 5B, is closely linked to the resistance gene Pm16 with a genetic distance of 5.3 cM. In combination with the analysis of Chinese Spring nulli-tetrasomic lines N4AT4D-N4AT4B and N5BT5D, it is confirmed that powdery mildew resistance gene Pm16 is located on the short arm of chromosome 5B in wheat instead of chromosome 4A as reported previously by Reader and Miller.
2.77 Seedling and adult-plant resistance to powdery mildew in Chinese bread wheat cultivars and introductions
ZL Wang1, LH Li1, ZH He1, XY Duan2, YL Zhou2, XM Chen1, M Lillemo3, RP Singh3, XC Xia1
1Institute of Crop Breeding and Cultivation/National Wheat Improvement Center, Chinese Academy of Agricultural Sciences, Beijing, China. 2Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China. 3International Maize and Wheat Improvement Center (CIMMYT), Mexico
xiaxianchun{a}caas.net.cn
Powdery mildew caused by Blumeria graminis f. sp. tritici is a widespread wheat disease in China. Identification of race-specific genes and adult-plant resistance (APR) is of major importance in wheat breeding for an efficient genetic control strategy. The objectives of this study were to identify genes that confer seedling resistance to powdery mildew in Chinese bread wheat cultivars and introductions used by breeding programs in China, and evaluate their APR in the field. Twenty isolates of B. graminis tritici with different virulence patterns collected from various regions of China were used for the identification of resistance genes at seedling stage in 192 Chinese wheat cultivars and introductions from CIMMYT, US and European countries. Twenty differential lines with known Pm genes were included in each test and the low and high infection types displayed by them with the B. graminis tritici isolates were used for postulation of resistance genes in the wheat cultivars tested. Isolate E20 with a broad spectrum of virulence was used for evaluating APR to powdery mildew in field trials. The results indicated that (i) 98 out of 192 tested wheat cultivars and lines carried one or more resistance genes to powdery mildew, (ii) Pm8 and Pm4b are the most common resistance genes in the Chinese wheat cultivars, while Pm8 and Pm3d are present most frequently in the wheat cultivars introduced from CIMMYT, US and European countries, (iii) Genotypes with Pm1, Pm3e, Pm5 and Pm7 were susceptible, whereas, genotypes with Pm12, Pm16 and Pm20 were highly resistant to almost all isolates of B. graminis tritici tested, and (iv) 21 genotypes expressed APR at adult stage. Our data showed that area under the disease progress curve (AUDPC), maximal disease severity (MDS) on penultimate leaves and the index of disease are good parameters for the description of APR in the field.
2.78 Resistance gene analog markers co-segregating with powdery mildew resistance gene Pm31, derived from wild emmer wheat
Weilong Xie1, Bin Zeng1, Ori Orion1, Amos Dinoor2, Qixin Sun3, Marion S. Röder4, Eviatar Nevo1, Tzion Fahima1
1Institute of Evolution, University of Haifa, Mount Carmel, Israel. 2Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot, Israel. 3China Agricultural University, Beijing, China. 4Institute for Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany
fahima{a}research.haifa.ac.il
Wild emmer wheat, Triticum dicoccoides, the tetraploid progenitor of cultivated wheat, is a valuable source for resistance to powdery mildew. Pm31 is a novel gene for powdery mildew resistance transferred from Israeli wild emmer accession ‘G-305-M’ into hexaploid wheat by crossing with breeding line 781, then backcrossing into Chinese elite wheat cultivar ‘Jing 411’ (G-305-M/781//Jing 411*3). This gene was previously located on chromosome 6AL using microsatellite markers. In order to identify closely linked markers for marker-assisted selection and map-based cloning, the resistance gene analogs (RGAs) approach was used. A large scale screening of degenerate or specific oligonucleotide primers, based on the conserved domains of cloned plant disease resistance genes, was conducted to test for polymorphism between DNA bulks of homozygous resistant and homozygous susceptible plants. Association of polymorphic bands with Pm31 was determined using a segregating mapping population containing 162 individuals. Twelve RGA markers were identified to be linked to Pm31 within 1.6 cM. Five RGA markers co-segregated with the gene and seven RGA markers clustered together at a distance of 1.6 cM to the gene. The clustering of RGA markers may indicate the existence of either a cluster of resistance genes or suppressed recombination around the Pm31 region. Three co-segregating RGA markers were cloned and sequenced. Marker Xuhw202 showed significant homology in translated amino-acid sequence with many cloned plant disease resistance genes. Five STS (sequence-tagged site) markers from three RGA markers were developed, and could be detected on agarose gel. These direct or closely linked RGA markers and the STS markers should be useful in marker-assisted selection to tag the Pm31 gene and in cloning of the gene for wheat improvement.
2.79 Syntaxin required for penetration resistance
Jin-Long Qiu, Ziguo Zhang, Helge Tippmann, Karen L Olesen, Hans Thordal-Christensen
Risoe, Denmark
ziguo.zhang{a}risoe.dk
The molecular mechanism of penetration resistance (PER) remains one of the major unknown in the study of plant-pathogen interactions. We used a genetic approach to study PER in Arabidopsis. Mutants, easily penetrated by the non-host barley powdery mildew fungus (Blumeria graminis f.sp. hordei, Bgh), have been identified. Four recessive mutations occur in the PEN1 gene. This gene was isolated by map-based cloning and found to encode a syntaxin (AtSYP121) (Collins et al., 2003). Syntaxins belong to the eukaryotic t-SNAREs that are part of protein complexes driving vesicle and target membranes to merge during vesicle trafficking. The fusion protein GFP-PEN1 complements the mutations, and reveals that PEN1 is located at the plasma membrane. Furthermore, the fusion protein accumulates around papillae, which are formed at the sites of penetration attempts. A mutation in the PEN1 gene also results in increased penetration by the host powdery mildew fungus (Erysiphe cichoracearum), suggesting the existing of a basic resistance mechanism common to hosts and non-hosts. Knock-out of PEN1 cause a delay in papilla formation, suggesting that the vesicle trafficking, involved in building up this cell wall structure, is hampered. More evidence for the importance of vesicle trafficking in PER, come from tests with Brefeldin A (a vesicle trafficking inhibitor) and cytochalasin E (an actin polymerization inhibitor). Both can phenocopy the pen1 mutations in wild-type plants. Phenotypes related to SAR and ABA signalling are uncovered in pen1 mutants. We speculate what additional function these may suggest for SYP121 in plants.
Abstract 2.80 follows 2.15
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