Genome-wide association study identifies novel loci and candidate genes for rust resistance in wheat (Triticum aestivum L.).

BACKGROUND: Wheat rusts are important biotic stresses, development of rust resistant cultivars through molecular approaches is both economical and sustainable. Extensive phenotyping of large mapping populations under diverse production conditions and high-density genotyping would be the ideal strategy to identify major genomic regions for rust resistance in wheat. The genome-wide association study (GWAS) population of 280 genotypes was genotyped using a 35 K Axiom single nucleotide polymorphism (SNP) array and phenotyped at eight, 10, and, 10 environments, respectively for stem/black rust (SR), stripe/yellow rust (YR), and leaf/brown rust (LR). RESULTS: Forty-one Bonferroni corrected marker-trait associations (MTAs) were identified, including 17 for SR and 24 for YR. Ten stable MTAs and their best combinations were also identified. For YR, AX-94990952 on 1A + AX-95203560 on 4A + AX-94723806 on 3D + AX-95172478 on 1A showed the best combination with an average co-efficient of infection (ACI) score of 1.36. Similarly, for SR, AX-94883961 on 7B + AX-94843704 on 1B and AX-94883961 on 7B + AX-94580041 on 3D + AX-94843704 on 1B showed the best combination with an ACI score of around 9.0. The genotype PBW827 have the best MTA combinations for both YR and SR resistance. In silico study identifies key prospective candidate genes that are located within MTA regions. Further, the expression analysis revealed that 18 transcripts were upregulated to the tune of more than 1.5 folds including 19.36 folds (TraesCS3D02G519600) and 7.23 folds (TraesCS2D02G038900) under stress conditions compared to the control conditions. Furthermore, highly expressed genes in silico under stress conditions were analyzed to find out the potential links to the rust phenotype, and all four genes were found to be associated with the rust phenotype. CONCLUSION: The identified novel MTAs, particularly stable and highly expressed MTAs are valuable for further validation and subsequent application in wheat rust resistance breeding. The genotypes with favorable MTA combinations can be used as prospective donors to develop elite cultivars with YR and SR resistance.

Dissecting the causal polymorphism of the Lr67res multipathogen resistance gene.

Partial resistance to multiple biotrophic fungal pathogens in wheat (Triticum aestivum L.) is conferred by a variant of the Lr67 gene, which encodes a hexose-proton symporter. Two mutations (G144R and V387L) differentiate the resistant and susceptible protein variants (Lr67res and Lr67sus). Lr67res lacks sugar transport capability and was associated with anion transporter-like properties when expressed in Xenopus laevis oocytes. Here, we extended this functional characterization to include yeast and in planta studies. The Lr67res allele, but not Lr67sus, induced sensitivity to ions in yeast (including NaCl, LiCl, and KI), which is consistent with our previous observations that Lr67res expression in oocytes induces novel ion fluxes. We demonstrate that another naturally occurring single amino acid variant in wheat, containing only the Lr67G144R mutation, confers rust resistance. Transgenic barley plants expressing the orthologous HvSTP13 gene carrying the G144R and V387L mutations were also more resistant to Puccinia hordei infection. NaCl treatment of pot-grown adult wheat plants with the Lr67res allele induced leaf tip necrosis and partial leaf rust resistance. An Lr67res-like function can be introduced into orthologous plant hexose transporters via single amino acid mutation, highlighting the strong possibility of generating disease resistance in other crops, especially with gene editing.

Plant Disease Models and Forecasting: Changes in Principles and Applications over the Last 50 Years.

This review gives a perspective of selected advances made since the middle of the 20th century in plant disease modeling, and the associated increase in the number of models published during that time frame. This progress can be mainly attributed to advances in (i) sensors and automatic environmental data collection technology, (ii) instrumentation and methods for studying botanical epidemiology, and (iii) data analytics and computer science. We review the evolution of techniques for developing data-based (empirical) models and process-based (mechanistic) models using the wheat rusts as a case study. We also describe the increased importance of knowledge about biological processes for plant disease modeling by using apple scab as a second case study. For both wheat rusts and apple scab, we describe how the models have evolved over the last 50 years by considering certain milestones that have been achieved in disease modeling. Finally, we describe how plant disease models are used as part of a multi-modeling approach to develop decision-making tools in the application of integrated pest management.

The NWRD Dataset: An Open-Source Annotated Segmentation Dataset of Diseased Wheat Crop.

Wheat stripe rust disease (WRD) is extremely detrimental to wheat crop health, and it severely affects the crop yield, increasing the risk of food insecurity. Manual inspection by trained personnel is carried out to inspect the disease spread and extent of damage to wheat fields. However, this is quite inefficient, time-consuming, and laborious, owing to the large area of wheat plantations. Artificial intelligence (AI) and deep learning (DL) offer efficient and accurate solutions to such real-world problems. By analyzing large amounts of data, AI algorithms can identify patterns that are difficult for humans to detect, enabling early disease detection and prevention. However, deep learning models are data-driven, and scarcity of data related to specific crop diseases is one major hindrance in developing models. To overcome this limitation, in this work, we introduce an annotated real-world semantic segmentation dataset named the NUST Wheat Rust Disease (NWRD) dataset. Multileaf images from wheat fields under various illumination conditions with complex backgrounds were collected, preprocessed, and manually annotated to construct a segmentation dataset specific to wheat stripe rust disease. Classification of WRD into different types and categories is a task that has been solved in the literature; however, semantic segmentation of wheat crops to identify the specific areas of plants and leaves affected by the disease remains a challenge. For this reason, in this work, we target semantic segmentation of WRD to estimate the extent of disease spread in wheat fields. Sections of fields where the disease is prevalent need to be segmented to ensure that the sick plants are quarantined and remedial actions are taken. This will consequently limit the use of harmful fungicides only on the targeted disease area instead of the majority of wheat fields, promoting environmentally friendly and sustainable farming solutions. Owing to the complexity of the proposed NWRD segmentation dataset, in our experiments, promising results were obtained using the UNet semantic segmentation model and the proposed adaptive patching with feedback (APF) technique, which produced a precision of 0.506, recall of 0.624, and F1 score of 0.557 for the rust class.

Image Classification of Wheat Rust Based on Ensemble Learning.

Rust is a common disease in wheat that significantly impacts its growth and yield. Stem rust and leaf rust of wheat are difficult to distinguish, and manual detection is time-consuming. With the aim of improving this situation, this study proposes a method for identifying wheat rust based on ensemble learning (WR-EL). The WR-EL method extracts and integrates multiple convolutional neural network (CNN) models, namely VGG, ResNet 101, ResNet 152, DenseNet 169, and DenseNet 201, based on bagging, snapshot ensembling, and the stochastic gradient descent with warm restarts (SGDR) algorithm. The identification results of the WR-EL method were compared to those of five individual CNN models. The results show that the identification accuracy increases by 32%, 19%, 15%, 11%, and 8%. Additionally, we proposed the SGDR-S algorithm, which improved the f1 scores of healthy wheat, stem rust wheat and leaf rust wheat by 2%, 3% and 2% compared to the SGDR algorithm, respectively. This method can more accurately identify wheat rust disease and can be implemented as a timely prevention and control measure, which can not only prevent economic losses caused by the disease, but also improve the yield and quality of wheat.

MARPLE, a point-of-care, strain-level disease diagnostics and surveillance tool for complex fungal pathogens.

BACKGROUND: Effective disease management depends on timely and accurate diagnosis to guide control measures. The capacity to distinguish between individuals in a pathogen population with specific properties such as fungicide resistance, toxin production and virulence profiles is often essential to inform disease management approaches. The genomics revolution has led to technologies that can rapidly produce high-resolution genotypic information to define individual variants of a pathogen species. However, their application to complex fungal pathogens has remained limited due to the frequent inability to culture these pathogens in the absence of their host and their large genome sizes. RESULTS: Here, we describe the development of Mobile And Real-time PLant disEase (MARPLE) diagnostics, a portable, genomics-based, point-of-care approach specifically tailored to identify individual strains of complex fungal plant pathogens. We used targeted sequencing to overcome limitations associated with the size of fungal genomes and their often obligately biotrophic nature. Focusing on the wheat yellow rust pathogen, Puccinia striiformis f.sp. tritici (Pst), we demonstrate that our approach can be used to rapidly define individual strains, assign strains to distinct genetic lineages that have been shown to correlate tightly with their virulence profiles and monitor genes of importance. CONCLUSIONS: MARPLE diagnostics enables rapid identification of individual pathogen strains and has the potential to monitor those with specific properties such as fungicide resistance directly from field-collected infected plant tissue in situ. Generating results within 48 h of field sampling, this new strategy has far-reaching implications for tracking plant health threats.

Genome-Wide Mapping of Adult Plant Resistance to Leaf Rust and Stripe Rust in CIMMYT Wheat Line Arableu#1.

Leaf (brown) rust (LR) and stripe (yellow) rust (YR), caused by Puccinia triticina and P. striiformis f. sp. tritici, respectively, significantly reduce wheat production worldwide. Disease-resistant wheat varieties offer farmers one of the most effective ways to manage these diseases. The common wheat (Triticum aestivum L.) Arableu#1, developed by the International Maize and Wheat Improvement Center and released as Deka in Ethiopia, shows susceptibility to both LR and YR at the seedling stage but a high level of adult plant resistance (APR) to the diseases in the field. We used 142 F5 recombinant inbred lines (RILs) derived from Apav#1 × Arableu#1 to identify quantitative trait loci (QTLs) for APR to LR and YR. A total of 4,298 genotyping-by-sequencing markers were used to construct a genetic linkage map. The study identified four LR resistance QTLs and six YR resistance QTLs in the population. Among these, QLr.cim-1BL.1/QYr.cim-1BL.1 was located in the same location as Lr46/Yr29, a known pleiotropic resistance gene. QLr.cim-1BL.2 and QYr.cim-1BL.2 were also located on wheat chromosome 1BL at 37 cM from Lr46/Yr29 and may represent a new segment for pleiotropic resistance to both rusts. QLr.cim-7BL is likely Lr68 given its association with the tightly linked molecular marker cs7BLNLRR. In addition, QLr.cim-3DS, QYr.cim-2AL, QYr.cim-4BL, QYr.cim-5AL, and QYr.cim-7DS are probably new resistance loci based on comparisons with published QTLs for resistance to LR and YR. Our results showed the diversity of minor resistance QTLs in Arableu#1 and their role in conferring near-immune levels of APR to both LR and YR, when combined with the pleiotropic APR gene Lr46/Yr29.

An immunofluorescence assay for the detection of wheat rust species using monoclonal antibody against urediniospores of Puccinia triticina.

AIMS: Wheat (Triticum aestivum) is one of the most important crop species, but yields are often drastically reduced by rust epidemics. In this report, we describe a rapid and sensitive immunofluorescence method for the detection of urediniospores of the fungi Puccinia striiformis f. sp. tritici, Puccinia triticina and Puccinia graminis f. sp. tritici, which are causal agents of wheat rust. METHODS AND RESULTS: The method uses monoclonal antibody LPT-2 against the urediniospores of P. triticina and PE-cy3 goat anti-mouse. Urediniospores of P. triticina or those of two species that are difficult to distinguish from P. triticina, P. striiformis f. sp. tritici or P. graminis f. sp. tritici were immobilized on a glass slide, and the sample was then treated with LPT-2. Thereafter, a second antibody, goat anti-mouse conjugated PE-cy3, was added, and the slide was observed in a fluoroscope. The fluorescent signal was strong with P. triticina urediniospores, weak with P. striiformis f. sp. tritici urediniospores and weak-to-intermediate with P. graminis f. sp. tritici urediniospores. The detection limit of this method was 2 ng ml(-1) of the monoclonal antibody LPT-2. CONCLUSIONS: In this article, we describe the production and diagnostic application of a novel mouse monoclonal antibody specific to urediniospores of P. triticina. SIGNIFICANCE AND IMPACT OF THE STUDY: After further technical development, this method may become a tool for on-site identification of P. triticina urediniospores and will therefore help in the selection and timing of fungicide applications for control of wheat rust outbreaks.

Identification and Functional Analysis of CAP Genes from the Wheat Stripe Rust Fungus Puccinia striiformis f. sp. tritici.

Cysteine-rich secretory proteins (C), antigen 5 (A), and pathogenesis-related 1 proteins (P) comprise widespread CAP superfamily proteins, which have been proven to be novel virulence factors of mammalian pathogenic fungi and some plant pathogens. Despite this, the identification and function of CAP proteins in more species of plant pathogens still need to be studied. This work presents the identification and functional analysis of CAP superfamily proteins from Puccinia striiformis f. sp. tritici (Pst), an important fungal pathogen that causes wheat stripe rust on wheat worldwide. A total of six CAP genes were identified in the Pst genome, designated as PsCAP1-PsCAP6. Five PsCAP proteins, including PsCAP1, PsCAP2, PsCAP3, PsCAP4, and PsCAP5, have N-terminal signal peptides secreted with the yeast signal sequence trap assay. Single-nucleotide polymorphism (SNP) analysis indicated that they showed a low level of intraspecies polymorphism. The expression abundance of PsCAP genes at different Pst infection stages was detected by RT-qPCR, and most of them were highly expressed during Pst infection on wheat and also Pst sexual reproduction on barberry (Berberis shensiana). Noticeably, the silencing of these six PsCAP genes by BSMV-mediated HIGS indicated that PsCAP1, PsCAP4, and PsCAP5 contribute significantly to Pst infection in wheat. These results indicate that PsCAP proteins may act as virulence factors during Pst infection, which also provides insights into Pst pathogenicity.

Genome-wide association mapping of rust resistance in Aegilops longissima.

The rust diseases, including leaf rust caused by Puccinia triticina (Pt), stem rust caused by P. graminis f. sp. tritici (Pgt), and stripe rust caused by P. striiformis f. sp. tritici (Pst), are major limiting factors in wheat production worldwide. Identification of novel sources of rust resistance genes is key to developing cultivars resistant to rapidly evolving pathogen populations. Aegilops longissima is a diploid wild grass native to the Levant and closely related to the modern bread wheat D subgenome. To explore resistance genes in the species, we evaluated a large panel of Ae. longissima for resistance to several races of Pt, Pgt, and Pst, and conducted a genome-wide association study (GWAS) to map rust resistance loci in the species. A panel of 404 Ae. longissima accessions, mostly collected from Israel, were screened for seedling-stage resistance to four races of Pt, four races of Pgt, and three races of Pst. Out of the 404 accessions screened, two were found that were resistant to all 11 races of the three rust pathogens screened. The percentage of all accessions screened that were resistant to a given rust pathogen race ranged from 18.5% to 99.7%. Genotyping-by-sequencing (GBS) was performed on 381 accessions of the Ae. longissima panel, wherein 125,343 single nucleotide polymorphisms (SNPs) were obtained after alignment to the Ae. longissima reference genome assembly and quality control filtering. Genetic diversity analysis revealed the presence of two distinct subpopulations, which followed a geographic pattern of a northern and a southern subpopulation. Association mapping was performed in the genotyped portion of the collection (n = 381) and in each subpopulation (n = 204 and 174) independently via a single-locus mixed-linear model, and two multi-locus models, FarmCPU, and BLINK. A large number (195) of markers were significantly associated with resistance to at least one of 10 rust pathogen races evaluated, nine of which are key candidate markers for further investigation due to their detection via multiple models and/or their association with resistance to more than one pathogen race. The novel resistance loci identified will provide additional diversity available for use in wheat breeding.

Wheat leaf rust fungus effector Pt13024 is avirulent to TcLr30.

Wheat leaf rust, caused by Puccinia triticina Eriks. (Pt), is a global wheat disease threatening wheat production. Dissecting how Pt effector proteins interact with wheat has great significance in understanding the pathogenicity mechanisms of Pt. In the study, the cDNA of Pt 13-5-72 interacting with susceptible cultivar Thatcher was used as template to amplify Pt13024 gene. The expression pattern and structure of Pt13024 were analyzed by qRT-PCR and online softwares. The secretion function of Pt13024 signal peptide was verified by the yeast system. Subcellular localization of Pt13024 was analyzed using transient expression on Nicotiana benthamiana. The verification that Pt13024 inhibited programmed cell death (PCD) was conducted on N. benthamiana and wheat. The deletion mutation of Pt13024 was used to identify the virulence function motif. The transient transformation of wheat mediated by the type III secretion system (TTSS) was used to analyze the activity of regulating the host defense response of Pt13024. Pt13024 gene silencing was performed by host-induced gene silencing (HIGS). The results showed that Pt13024 was identified as an effector and localized in the cytoplasm and nucleus on the N. benthamiana. It can inhibit PCD induced by the Bcl-2-associated X protein (BAX) from mice and infestans 1 (INF1) from Phytophthora infestans on N. benthamiana, and it can also inhibit PCD induced by DC3000 on wheat. The amino acids 22 to 41 at N-terminal of the Pt13024 are essential for the inhibition of programmed cell death (PCD) induced by BAX. The accumulation of reactive oxygen species and deposition of callose in near-isogenic line TcLr30, which is in Thatcher background with Lr30, induced by Pt13024 was higher than that in 41 wheat leaf rust-resistant near-isogenic lines (monogenic lines) with different resistance genes and Thatcher. Silencing of Pt13024 reduced the leaf rust resistance of Lr30 during the interaction between Pt and TcLr30. We can conclude that Pt13024 is avirulent to TcLr30 when Pt interacts with TcLr30. These findings lay the foundation for further investigations into the role of Pt effector proteins in pathogenesis and their regulatory mechanisms.

Harnessing genetic resistance to rusts in wheat and integrated rust management methods to develop more durable resistant cultivars.

Wheat is one of the most important staple foods on earth. Leaf rust, stem rust and stripe rust, caused by Puccini triticina, Puccinia f. sp. graminis and Puccinia f. sp. striiformis, respectively, continue to threaten wheat production worldwide. Utilization of resistant cultivars is the most effective and chemical-free strategy to control rust diseases. Convectional and molecular biology techniques identified more than 200 resistance genes and their associated markers from common wheat and wheat wild relatives, which can be used by breeders in resistance breeding programmes. However, there is continuous emergence of new races of rust pathogens with novel degrees of virulence, thus rendering wheat resistance genes ineffective. An integration of genomic selection, genome editing, molecular breeding and marker-assisted selection, and phenotypic evaluations is required in developing high quality wheat varieties with resistance to multiple pathogens. Although host genotype resistance and application of fungicides are the most generally utilized approaches for controlling wheat rusts, effective agronomic methods are required to reduce disease management costs and increase wheat production sustainability. This review gives a critical overview of the current knowledge of rust resistance, particularly race-specific and non-race specific resistance, the role of pathogenesis-related proteins, non-coding RNAs, and transcription factors in rust resistance, and the molecular basis of interactions between wheat and rust pathogens. It will also discuss the new advances on how integrated rust management methods can assist in developing more durable resistant cultivars in these pathosystems.

How Target-Sequence Enrichment and Sequencing (TEnSeq) Pipelines Have Catalyzed Resistance Gene Cloning in the Wheat-Rust Pathosystem.

The wheat-rust pathosystem has been well-studied among host-pathogen interactions since last century due to its economic importance. Intensified efforts toward cloning of wheat rust resistance genes commenced in the late 1990s with the first successful isolation published in 2003. Currently, a total of 24 genes have been cloned from wheat that provides resistance to stem rust, leaf rust, and stripe rust. Among them, more than half (15) were cloned over the last 4 years. This rapid cloning of resistance genes from wheat can be largely credited to the development of approaches for reducing the genome complexity as 10 out of the 15 genes cloned recently were achieved by approaches that are summarized as TEnSeq (Target-sequence Enrichment and Sequencing) pipelines in this review. The growing repertoire of cloned rust resistance genes provides new tools to support deployment strategies aimed at achieving durable resistance. This will be supported by the identification of genetic variation in corresponding Avr genes from rust pathogens, which has recently begun. Although developed with wheat resistance genes as the primary targets, TEnSeq approaches are also applicable to other classes of genes as well as for other crops with complex genomes.

Marker-Trait Associations for Enhancing Agronomic Performance, Disease Resistance, and Grain Quality in Synthetic and Bread Wheat Accessions in Western Siberia.

Exploiting genetically diverse lines to identify genes for improving crop performance is needed to ensure global food security. A genome-wide association study (GWAS) was conducted using 46,268 SNP markers on a diverse panel of 143 hexaploid bread and synthetic wheat to identify potential genes/genomic regions controlling agronomic performance (yield and 26 yield-related traits), disease resistance, and grain quality traits. From phenotypic evaluation, we found large genetic variation among the 35 traits and recommended five lines having a high yield, better quality, and multiple disease resistance for direct use in a breeding program. From a GWAS, we identified a total of 243 significant marker-trait associations (MTAs) for 35 traits that explained up to 25% of the phenotypic variance. Of these, 120 MTAs have not been reported in the literature and are potentially novel MTAs. In silico gene annotation analysis identified 116 MTAs within genes and of which, 21 MTAs were annotated as a missense variant. Furthermore, we were able to identify 23 co-located multi-trait MTAs that were also phenotypically correlated to each other, showing the possibility of simultaneous improvement of these traits. Additionally, most of the co-located MTAs were within genes. We have provided genomic fingerprinting for significant markers with favorable and unfavorable alleles in the diverse set of lines for developing elite breeding lines from useful trait-integration. The results from this study provided a further understanding of genetically complex traits and would facilitate the use of diverse wheat accessions for improving multiple traits in an elite wheat breeding program.

Biocontrol Agents for Controlling Wheat Rust.

Bacterial endophytes are potential biocontrol agents of wheat rusts. Apart from disease control, these bacterial endophytes have growth-promoting efficacies which differ significantly from one isolate to another. Here, we describe the procedure for isolation, screening, and identification of endophytic bacterial isolates with high capacities to suppress strip rust infection and better ability to enhance wheat yields.

Investigating Gene Function in Cereal Rust Fungi by Plant-Mediated Virus-Induced Gene Silencing.

Cereal rust fungi are destructive pathogens, threatening grain production worldwide. Targeted breeding for resistance utilizing host resistance genes has been effective. However, breakdown of resistance occurs frequently and continued efforts are needed to understand how these fungi overcome resistance and to expand the range of available resistance genes. Whole genome sequencing, transcriptomic and proteomic studies followed by genome-wide computational and comparative analyses have identified large repertoire of genes in rust fungi among which are candidates predicted to code for pathogenicity and virulence factors. Some of these genes represent defence triggering avirulence effectors. However, functions of most genes still needs to be assessed to understand the biology of these obligate biotrophic pathogens. Since genetic manipulations such as gene deletion and genetic transformation are not yet feasible in rust fungi, performing functional gene studies is challenging. Recently, Host-induced gene silencing (HIGS) has emerged as a useful tool to characterize gene function in rust fungi while infecting and growing in host plants. We utilized Barley stripe mosaic virus-mediated virus induced gene silencing (BSMV-VIGS) to induce HIGS of candidate rust fungal genes in the wheat host to determine their role in plant-fungal interactions. Here, we describe the methods for using BSMV-VIGS in wheat for functional genomics study in cereal rust fungi.

Recent trends and perspectives of molecular markers against fungal diseases in wheat.

Wheat accounts for 19% of the total production of major cereal crops in the world. In view of ever increasing population and demand for global food production, there is an imperative need of 40-60% increase in wheat production to meet the requirement of developing world in coming 40 years. However, both biotic and abiotic stresses are major hurdles for attaining the goal. Among the most important diseases in wheat, fungal diseases pose serious threat for widening the gap between actual and attainable yield. Fungal disease management, mainly, depends on the pathogen detection, genetic and pathological variability in population, development of resistant cultivars and deployment of effective resistant genes in different epidemiological regions. Wheat protection and breeding of resistant cultivars using conventional methods are time-consuming, intricate and slow processes. Molecular markers offer an excellent alternative in development of improved disease resistant cultivars that would lead to increase in crop yield. They are employed for tagging the important disease resistance genes and provide valuable assistance in increasing selection efficiency for valuable traits via marker assisted selection (MAS). Plant breeding strategies with known molecular markers for resistance and functional genomics enable a breeder for developing resistant cultivars of wheat against different fungal diseases.

Tansley Review No. 18 Mechanisms of resistance and pathogenic specialization in rust-wheat interactions.

Forms of resistance and pathogenic specialization in interactions between wheat and its stem and leaf rust Fungi have been selected for analysis with two hypotheses in mind. One is that phytoalexin formation is a universal defence mechanism in plants; the other is that resistance is elicited specifically by products of avirulent strains of pathogens. A search for phytoalexins in wheat, using a range of extraction and separation procedures and a number of forms of expression of resistance, has indicated the presence of several inhibitors of fungal development but has yielded very little evidence for post-infectional increases in the amounts of these inhibitors. No evidence for the formation of phytoalexins in wheat has been obtained. Hypersensitivity has been confirmed as being closely associated with the expression of most forms of resistance studied. Recent work with some very rapidly expressed forms of resistance to rust fungi has implicated lignification as a component of active defence in wheat, a finding in agreement with those of other workers. The expression of resistance conferred by the Lr20 gene towards avirulent strains of the leaf rust fungus was the principal subject of explorations for elicitors. Histological observations of the sequence of events in fungal and wheat cells suggested the action of rust strain and Lr20-specific elicitors in bringing about hypersensitivity. Experimental manipulations involving heat treatments or use of epidermis-free segments of leaves incubated on infected mesophylls gave results consistent with this interpretation. Attempts to extract specific elicitors from infected leaves failed but yielded non-specific elicitors. Non-specific elicitors have been obtained in intercellular washing fluids of leaves infected with strains of both the leaf and stem rust fungi. They have also been obtained from extracts of germ tubes of both species of fungus. Although not specific for any known resistance gene, the elicitors are selective for a character determined by a factor on chromosome 5 A in some wheat cultivars. A role for the non-specific elicitors as incompatibility factors between rust fungi and wheat is suggested. A revised hypothesis is presented in order to explain the evolution of pathogenesis in rust fungi towards wheat and it is discussed in relation to concepts of basic compatibility between pathogens and their hosts, as proposed also by others. CONTENTS Summary 233 I. Introduction 234 II. Working Ideas and Hypotheses 234 III. The Search for Phytoalexins 235 IV. Elicitation of Resistance 237 V. Revised Hypothesis 240 Acknowledgements 242 References 242.

The past, present and future of breeding rust resistant wheat.

Two classes of genes are used for breeding rust resistant wheat. The first class, called R (for resistance) genes, are pathogen race specific in their action, effective at all plant growth stages and probably mostly encode immune receptors of the nucleotide binding leucine rich repeat (NB-LRR) class. The second class is called adult plant resistance genes (APR) because resistance is usually functional only in adult plants, and, in contrast to most R genes, the levels of resistance conferred by single APR genes are only partial and allow considerable disease development. Some but not all APR genes provide resistance to all isolates of a rust pathogen species and a subclass of these provides resistance to several fungal pathogen species. Initial indications are that APR genes encode a more heterogeneous range of proteins than R proteins. Two APR genes, Lr34 and Yr36, have been cloned from wheat and their products are an ABC transporter and a protein kinase, respectively. Lr34 and Sr2 have provided long lasting and widely used (durable) partial resistance and are mainly used in conjunction with other R and APR genes to obtain adequate rust resistance. We caution that some APR genes indeed include race specific, weak R genes which may be of the NB-LRR class. A research priority to better inform rust resistance breeding is to characterize further APR genes in wheat and to understand how they function and how they interact when multiple APR and R genes are stacked in a single genotype by conventional and GM breeding. An important message is do not be complacent about the general durability of all APR genes.

Exploring Durable Genetic Resistance against Leaf Rust through Phenotypic Characterization and Lr34Linked STS Marker in Wheat Germplasm / Explorando a resistência genética duradoura contra a ferrugem da folha através da caracterização fenotípica e do STS marker relacionado ao (GENE) LR34 do germoplasma do trigo

Present study was aimed to screening the population of 25 wheat genotypes from Baluchistan region of Pakistan along with five commercial cultivars for leaf rust adult plant resistance (APR) through gene postulation using natural inoculation of Puccinia triticina Erikss local pathotype. Infection severity was recorded on scale in comparison with susceptible control "Morroco" cultivar. On the basis of phenotypic score, seven accessions and four varieties (Zardana-89, Sariab-92, Zarlashta-99 and Raskoh-05) with AUDPC values up to 20% were characterized as resistant genotypes. Coefficient of infection (CI) score ranged from 0-10 for some accessions and cultivars showing high level of adult plant resistance. Furthermore, bi-allelic STS marker csLV34 having close linkage with Lr34 (0.4cM). This marker amplified one gene specific allele of 150bp in 21 genotypes, including 19 accessions and two commercial varieties (Sariab-92 and Zarghoon-79) which confirmed presence of Lr34 gene conferring adult plant resistance against leaf rust. The rust pathogenicity scale varied for accessions from resistant to moderately susceptible. However, beside Lr34, phenotypic gene postulation, in combination with marker assisted selection for leaf rust resistance, has revealed presence of some other unknown resistance genes in local wheat germplasm which signified its use in wheat improvement programs both locally and abroad.

O presente estudo teve como objetivo a triagem da população de 25 genótipos de trigo do Baluchistão, região do Paquistão, juntamente com cinco cultivares comerciais para o estudo da resistência à ferrugem da folha em plantas adultas (leaf rust adult plant resistance, APR, em inglês) através da postulação gênica usando a inoculação natural do patótipo local da Puccinia triticina Erikks. A gravidade da infecção foi registrada na escala em comparação ao cultivar de controle suscetível "Morroco". Com base na pontuação fenotípica, sete acessões e quatro variedades (Zardana-89, Sariab-92, Zarlashta-99 and Raskoh-05) com valores de AUDPC (area under the disease progress curve, em inglês) até 20% foram caracterizados como genótipos resistentes. A pontuação do coeficiente de infecção (CI) variou no intervalo de 0-10 para algumas acessões e cultivares evidenciando uma elevada resistência nas plantas adultas. Além disso, o STS marker para o csLV34 bi-alélico demonstrou uma ligação estreita com o Lr34 (0.4cM). Este marcador amplificou um alelo específico do gene do 150bp em 21 genótipos, incluindo 19 acessões e duas variedades comerciais (Sariab-92 and Zarghoon-79) o que confirmou a presença do gene Lr34 conferindo resistência às plantas adultas contra a ferrugem da folha. A escala de patogenicidade da ferrugem para as acessões de resistente a moderadamente suscetível. Contudo, além do Lr34, a postulação gênica fenotípica, em combinação com a seleção auxiliada (ou assistida) por marcadores para a resistência da ferrugem da folha, revelou a presença de outros genes resistentes desconhecidos no germoplasma do trigo local o que justifica a sua utilização em programas de melhoramento do trigo tanto a nível local quanto a nível internacional.