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Diseases of Theobroma cacao L. (Malvaceae) disrupt cocoa bean supply and economically impact growers. Vascular streak dieback (VSD), caused by Ceratobasidium theobromae, is a new encounter disease of cacao currently contained to southeast Asia and Melanesia. Resistance to VSD has been tested with large progeny trials in Sulawesi, Indonesia, and in Papua New Guinea with the identification of informative quantitative trait loci (QTLs). Using a VSD susceptible progeny tree (clone 26), derived from a resistant and susceptible parental cross, we assembled the genome to chromosome-level and discriminated alleles inherited from either resistant or susceptible parents. The parentally phased genomes were annotated for all predicted genes and then specifically for resistance genes of the nucleotide-binding site leucine-rich repeat class (NLR). On investigation, we determined the presence of NLR clusters and other potential disease response gene candidates in proximity to informative QTLs. We identified structural variants within NLRs inherited from parentals. We present the first diploid, fully scaffolded, and parentally phased genome resource for T. cacao L. and provide insights into the genetics underlying resistance and susceptibility to VSD., (© 2024 The Author(s). The Plant Genome published by Wiley Periodicals LLC on behalf of Crop Science Society of America.)

Bread wheat (Triticum aestivum) is a globally dominant crop and major source of calories and proteins for the human diet. Compared with its wild ancestors, modern bread wheat shows lower genetic diversity, caused by polyploidisation, domestication and breeding bottlenecks 1,2 . Wild wheat relatives represent genetic reservoirs, and harbour diversity and beneficial alleles that have not been incorporated into bread wheat. Here we establish and analyse extensive genome resources for Tausch's goatgrass (Aegilops tauschii), the donor of the bread wheat D genome. Our analysis of 46 Ae. tauschii genomes enabled us to clone a disease resistance gene and perform haplotype analysis across a complex disease resistance locus, allowing us to discern alleles from paralogous gene copies. We also reveal the complex genetic composition and history of the bread wheat D genome, which involves contributions from genetically and geographically discrete Ae. tauschii subpopulations. Together, our results reveal the complex history of the bread wheat D genome and demonstrate the potential of wild relatives in crop improvement., (© 2024. The Author(s).)

Barley stripe or yellow rust (BYR) caused by Puccinia striiformis f. sp. hordei ( Psh ) is a significant constraint to barley production. The disease is best controlled by genetic resistance, which is considered the most economical and sustainable component of integrated disease management. In this study, we assessed the diversity of resistance to Psh in a panel of international barley genotypes (n = 266) under multiple disease environments (Ecuador, India, and Mexico) using genome-wide association studies (GWASs). Four quantitative trait loci (QTLs) (three on chromosome 1H and one on 7H) associated with resistance to Psh were identified. The QTLs were validated by mapping resistance to Psh in five biparental populations, which detected key genomic regions on chromosomes 1H (populations Pompadour/Zhoungdamei, Pompadour/Zug161, and CI9214/Baudin), 3H (Ricardo/Gus), and 7H (Fumai8/Baronesse). The QTL RpshQ.GWA.1H.1 detected by GWAS and RpshQ.Bau.1H detected using biparental mapping populations co-located were the most consistent and stable across environments and are likely the same resistance region. RpshQ.Bau.1H was saturated using population CI9214/Baudin by enriching the target region, which placed the resistance locus between 7.9 and 8.1 Mbp (flanked by markers sun_B1H_03 , 0.7 cM proximal to Rpsh_1H and sun_B1H_KASP_02 , 3.2 cM distal on 1HS) in the Morex reference genome v.2. A Kompetitive Allele Specific PCR (KASP) marker sun_B1H_KASP_01 that co-segregated for RpshQ.Bau.1H was developed. The marker was validated on 50 Australian barley cultivars, showing well-defined allelic discrimination and presence in six genotypes (Baudin, Fathom, Flagship, Grout, Sakurastar, and Shepherd). This marker can be used for reliable marker-assisted selection and pyramiding of resistance to Psh and in diversifying the genetic base of resistance to stripe rust., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Singh, Ziems, Chettri, Dracatos, Forrest, Bhavani, Singh, Barnes, Zapata, Gangwar, Kumar, Bhardwaj and Park.)

Leaf rust caused by the pathogen Puccinia triticina ( Pt ) is a destructive fungal disease of wheat that occurs in almost all wheat-growing areas across the globe. Genetic resistance has proven to be the best solution to mitigate the disease. Wheat breeders are continuously seeking new diversified and durable sources of resistance to use in developing new varieties. We developed recombinant inbred line (RIL) populations from two leaf rust-resistant genotypes (Kenya Kudu and AUS12568) introduced from Kenya to identify and characterize resistance to Pt and to develop markers linked closely to the resistance that was found. Our studies detected four QTL conferring adult plant resistance (APR) to leaf rust. Two of these loci are associated with known genes, Lr46 and Lr68 , residing on chromosomes 1B and 7B, respectively. The remaining two, QLrKK_2B and QLrAus12568_5A , contributed by Kenya Kudu and AUS12568 respectively, are putatively new loci for Pt resistance. Both QLrKK_2B and QLrAus12568_5A were found to interact additively with Lr46 in significantly reducing the disease severity at adult plant growth stages in the field. We further developed a suite of six closely linked markers within the QLrAus12568_5A locus and four within the QLrKK_2B region. Among these, markers sunKASP_522 and sunKASP_524 , flanking QLrAus12568_5A , and sunKASP_536 , distal to QLrKK_2B , were identified as the most closely linked and reliable for marker-assisted selection. The markers were validated on a selection of 64 Australian wheat varieties and found to be polymorphic and robust, allowing for clear allelic discrimination. The identified new loci and linked molecular markers will enable rapid adoption by breeders in developing wheat varieties carrying diversified and durable resistance to leaf rust.

Stem rust, caused by Puccinia graminis f. sp. tritici ( Pgt ), is an important disease of wheat in South Africa (SA) and is primarily controlled using resistant cultivars. Understanding virulence diversity of Pgt is essential for successful breeding of resistant cultivars. Samples of infected wheat stems were collected across the major wheat-growing regions of SA from 2016 to 2020 to determine the pathogenic variability of Pgt isolates. Seven races were identified from 517 isolates pathotyped. The most frequently found races were 2SA104 (BPGSC + Sr9h , 27 , Kw ) (35% frequency) and 2SA88 (TTKSF + Sr8b ) (33%). Race 2SA42 (PTKSK + Sr8b ), which was found in 2017, and 2SA5 (BFGSF + Sr9h ), identified in 2017, are new races. The Ug99 variant race 2SA42 is similar in its virulence to 2SA107 (PTKST + Sr8b ) except for avirulence to Sr24 and virulence to Sr8155B1 . Race 2SA5 is closely related in its virulence to existing races that commonly infect triticale. Certain races showed limited geographical distribution. Races 2SA5, 2SA105, and 2SA108 were found only in the Western Cape, whereas 2SA107 and 2SA42 were detected only in the Free State province. The new and existing races were compared using microsatellite (SSR) marker analysis and their virulence on commercial cultivars was also determined. Seedling response of 113 wheat entries against the new races, using 2SA88, 2SA88+9h, 2SA106, and 2SA107 as controls, revealed 2SA107 as the most virulent (67 entries susceptible), followed by 2SA42 (64), 2SA106 (60), 2SA88+9h (59), 2SA88 (25), and 2SA5 (17). Thus, 2SA5 may not pose a significant threat to local wheat production. SSR genotyping revealed that 2SA5 is genetically distinct from all other SA Pgt races., Competing Interests: The author(s) declare no conflict of interest.

Leaf rust, caused by Puccinia hordei, is one of the most widespread and damaging foliar diseases affecting barley. The barley leaf rust resistance locus Rph7 has been shown to have unusually high sequence and haplotype divergence. In this study, we isolate the Rph7 gene using a fine mapping and RNA-Seq approach that is confirmed by mutational analysis and transgenic complementation. Rph7 is a pathogen-induced, non-canonical resistance gene encoding a protein that is distinct from other known plant disease resistance proteins in the Triticeae. Structural analysis using an AlphaFold2 protein model suggests that Rph7 encodes a putative NAC transcription factor with a zinc-finger BED domain with structural similarity to the N-terminal DNA-binding domain of the NAC transcription factor (ANAC019) from Arabidopsis. A global gene expression analysis suggests Rph7 mediates the activation and strength of the basal defence response. The isolation of Rph7 highlights the diversification of resistance mechanisms available for engineering disease control in crops., (© 2023. Springer Nature Limited.)

Austropuccinia psidii is a biotrophic fungus that causes myrtle rust. First described in Brazil, it has since spread to become a globally important pathogen that infects more than 480 myrtaceous species. One of the most important commercial crops affected by A. psidii is eucalypt, a widely grown forestry tree. The A. psidii-Eucalyptus spp. interaction is poorly understood, but pathogenesis is likely driven by pathogen-secreted effector molecules. Here, we identified and characterized a total of 255 virulence effector candidates using a genome assembly of A. psidii strain MF-1, which was recovered from Eucalyptus grandis in Brazil. We show that the expression of seven effector candidate genes is modulated by cell wax from leaves sourced from resistant and susceptible hosts. Two effector candidates with different subcellular localization predictions, and with specific gene expression profiles, were transiently expressed with GFP-fusions in Nicotiana benthamiana leaves. Interestingly, we observed the accumulation of an effector candidate, Ap28303, which was upregulated under cell wax from rust susceptible E. grandis and described as a peptidase inhibitor I9 domain-containing protein in the nucleus. This was in accordance with in silico analyses. Few studies have characterized nuclear effectors. Our findings open new perspectives on the study of A. psidii-Eucalyptus interactions by providing a potential entry point to understand how the pathogen manipulates its hosts in modulating physiology, structure, or function with effector proteins.

Global barley production is threatened by plant pathogens, especially the rusts. In this study we used a targeted genotype-by-sequencing (GBS) assisted GWAS approach to identify rust resistance alleles in a collection of 287 genetically distinct diverse barley landraces and historical cultivars available in the Australian Grains Genebank (AGG) and originally sourced from Eastern Europe. The accessions were challenged with seven US-derived cereal rust pathogen races including Puccinia hordei ( Ph -leaf rust) race 17VA12C, P. coronata var. hordei ( Pch -crown rust) race 91NE9305 and five pathogenically diverse races of P. striiformis f. sp. hordei ( Psh -stripe rust) (PSH-33, PSH-48, PSH-54, PSH-72 and PSH-100) and phenotyped quantitatively at the seedling stage. Novel resistance factors were identified on chromosomes 1H, 2H, 4H and 5H in response to Pch , whereas a race-specific QTL on 7HS was identified that was effective only to Psh isolates PSH-72 and PSH-100. A major effect QTL on chromosome 5HL conferred resistance to all Psh races including PSH-72, which is virulent on all 12 stripe rust differential tester lines. The same major effect QTL was also identified in response to leaf rust (17VA12C) suggesting this locus contains several pathogen specific rust resistance genes or the same gene is responsible for both leaf rust and stripe rust resistance. Twelve accessions were highly resistant to both leaf and stripe rust diseases and also carried the 5HL QTL. We subsequently surveyed the physical region at the 5HL locus for across the barley pan genome variation in the presence of known resistance gene candidates and identified a rich source of high confidence protein kinase and antifungal genes in the QTL region.

A panel of 114 genetically diverse barley lines were assessed in the greenhouse and field for resistance to the pathogen Puccinia hordei , the causal agent of barley leaf rust. Multi-pathotype tests revealed that 16.6% of the lines carried the all-stage resistance (ASR) gene Rph3 , followed by Rph2 (4.4%), Rph1 (1.7%), Rph12 (1.7%) or Rph19 (1.7%). Five lines (4.4%) were postulated to carry the gene combinations Rph2+9.am , Rph2+19 and Rph8+19 . Three lines (2.6%) were postulated to carry Rph15 based on seedling rust tests and genotyping with a marker linked closely to this gene. Based on greenhouse seedling tests and adult-plant field tests, 84 genotypes (73.7%) were identified as carrying APR, and genotyping with molecular markers linked closely to three known APR genes ( Rph20 , Rph23 and Rph24 ) revealed that 48 of the 84 genotypes (57.1%) likely carry novel (uncharacterized) sources of APR. Seven lines were found to carry known APR gene combinations ( Rph20+Rph23 , Rph23+Rph24 and Rph20+Rph24 ), and these lines had higher levels of field resistance compared to those carrying each of these three APR genes singly. GWAS identified 12 putative QTLs; strongly associated markers located on chromosomes 1H, 2H, 3H, 5H and 7H. Of these, the QTL on chromosome 7H had the largest effect on resistance response to P. hordei . Overall, these studies detected several potentially novel genomic regions associated with resistance. The findings provide useful information for breeders to support the utilization of these sources of resistance to diversify resistance to leaf rust in barley and increase resistance durability.

Crown rust, caused by Puccinia coronata f. sp. avenae (Pca), is a significant impediment to global oat production. Some 98 alleles at 92 loci conferring resistance to Pca in Avena have been designated; however, allelic relationships and chromosomal locations of many of these are unknown. Long-term monitoring of Pca in Australia, North America and elsewhere has shown that it is highly variable even in the absence of sexual recombination, likely due to large pathogen populations that cycle between wild oat communities and oat crops. Efforts to develop cultivars with genetic resistance to Pca began in the 1950s. Based almost solely on all all-stage resistance, this has had temporary benefits but very limited success. The inability to eradicate wild oats, and their common occurrence in many oat growing regions, means that future strategies to control Pca must be based on the assumption of a large and variable prevailing pathogen population with high evolutionary potential, even if cultivars with durable resistance are deployed and grown widely. The presence of minor gene, additive APR to Pca in hexaploid oat germplasm opens the possibility of pyramiding several such genes to give high levels of resistance. The recent availability of reference genomes for diploid and hexaploid oat will undoubtedly accelerate efforts to discover, characterise and develop high throughput diagnostic markers to introgress and pyramid resistance to Pca in high yielding adapted oat germplasm., (© 2022. Crown.)

Barley leaf rust (BLR), caused by Puccinia hordei, is best controlled through genetic resistance. An efficient resistance breeding program prioritizes the need to identify, characterize, and map new sources of resistance as well as understanding the effectiveness, structure, and function of resistance genes. In this study, three mapping populations were developed by crossing Israelian barley lines "AGG-396," "AGG-397," and "AGG-403" (carrying unknown leaf rust resistance) with a susceptible variety "Gus" to characterize and map resistance. Genetic analysis of phenotypic data from rust testing F 3 s with a P. hordei pathotype 5457 P+ revealed monogenic inheritance in all three populations. Targeted genotyping-by-sequencing of the three populations detected marker trait associations in the same genomic region on the short arm of chromosome 2H between 39 and 57 Mb (AGG-396/Gus), 44 and 64 Mb (AGG-397/Gus), and 31 and 58 Mb (AGG-403/Gus), suggesting that the resistance in all three lines is likely conferred by the same locus (tentatively designated RphAGG396 ). Two Kompetitive allele-specific PCR (KASP) markers, HvGBSv2-902 and HvGBSv2-932, defined a genetic distance of 3.8 cM proximal and 7.1 cM distal to RphAGG396 , respectively. To increase the marker density at the RphAGG396 locus, 75 CAPS markers were designed between two flanking markers. Integration of marker data resulted in the identification of two critical recombinants and mapping RphAGG396 between markers- Mloc-28 (40.75 Mb) and Mloc-41 (41.92 Mb) narrowing the physical window to 1.17 Mb based on the Morex v2.0 reference genome assembly. To enhance map resolution, 600 F 2 s were genotyped with markers- Mloc-28 and Mloc-41 and nine recombinants were identified, placing the gene at a genetic distance of 0.5 and 0.2 cM between the two markers, respectively. Two annotated NLR (nucleotide-binding domain leucine-rich repeat) genes (r2.2HG0093020 and r2.2HG0093030) were identified as the best candidates for RphAGG396 . A closely linked marker was developed for RphAGG396 that can be used for marker-assisted selection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mehnaz, Dracatos, Dinh, Forrest, Rouse, Park and Singh.)

Leaf rust of barley causes significant losses in crops of susceptible cultivars. Deploying host resistance is the most cost-effective and eco-sustainable strategy to protect the harvest. However, most known leaf rust resistance genes have been overcome by the pathogen due to the pathogen's evolution and adaptation. The discovery of novel sources of genetic resistance is vital to keep fighting against pathogen evolution. In this study, we investigated the genetic basis of resistance in barley breeding line GID 5779743 (GID) from ICARDA, found to carry high levels of seedling resistance to prevalent Australian pathotypes of Puccinia hordei . Multipathotype tests, genotyping, and marker-trait associations revealed that the resistance in GID is conferred by two independent genes. The first gene, Rph3 , was detected using a linked CAPS marker and QTL analysis. The second gene was detected by QTL analysis and mapped to the same location as that of the Rph5 locus on the telomeric region of chromosome 3HS. The segregating ratio in F 2 (conforming to 9 resistant: 7 susceptible genetic ratio; p > 0.8) and F 3 (1 resistant: 8 segregating: 7 susceptible; p > 0.19) generations of the GID × Gus population, when challenged with pathotype 5477 P- (virulent on Rph3 and Rph5 ) suggested the interaction of two genes in a complementary fashion. This study demonstrated that Rph3 interacts with Rph5 or an additional locus closely linked to Rph5 (tentatively designated RphGID ) in GID to produce an incompatible response when challenged with a pathotype virulent on Rph3+Rph5 ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Dinh, Pourkheirandish, Park and Singh.)

Breeding for leaf rust resistance has been successful worldwide and is underpinned by the discovery and characterisation of genetically diverse sources of resistance. An English scientist, Arthur Watkins, collected pre-Green Revolution wheat genotypes from 33 locations worldwide in the early part of the 20th Century and this collection is now referred to as the 'Watkins Collection'. A common wheat genotype, Aus27352 from Yugoslavia, showed resistance to currently predominating Australian pathotypes of the wheat leaf rust pathogen. We crossed Aus27352 with a leaf rust susceptible wheat selection Avocet S and a recombinant inbred line (RIL) F 6 population of 200 lines was generated. Initial screening at F 3 generation showed monogenic segregation for seedling response to leaf rust in Aus27352. These results were confirmed by screening the Aus27352/Avocet S RIL population. The underlying locus was temporarily named LrAW2 . Bulked segregant analysis using the 90K Infinium SNP array located LrAW2 in the long arm of chromosome 2B. Tests with molecular markers linked to two leaf rust resistance genes, Lr50 and Lr58 , previously located in chromosome 2B, indicated the uniqueness of LrAW2 and it was formally designated Lr82 . Kompetitive allele-specific polymerase chain reaction assays were developed for Lr82 -linked SNPs. KASP_22131 mapped 0.8 cM proximal to Lr82 and KASP_11333 was placed 1.2 cM distal to this locus. KASP_22131 showed 91% polymorphism among a set of 89 Australian wheat cultivars. We recommend the use of KASP_22131 for marker assisted pyramiding of Lr82 in breeding programs following polymorphism check on parents.

Sexual reproduction, mutation, and reassortment of nuclei increase genotypic diversity in rust fungi. Sexual reproduction is inherent to rust fungi, coupled with their coevolved plant hosts in native pathosystems. Rust fungi are hypothesised to exchange nuclei by somatic hybridisation with an outcome of increased genotypic diversity, independent of sexual reproduction. We provide criteria to demonstrate whether somatic exchange has occurred, including knowledge of parental haplotypes and rejection of fertilisation in normal rust life cycles., Competing Interests: The authors have declared that no competing interests exist.

Leaf rust, caused by Puccinia hordei, is an economically significant disease of barley, but only a few major resistance genes to P. hordei (Rph) have been cloned. In this study, gene Rph3 was isolated by positional cloning and confirmed by mutational analysis and transgenic complementation. The Rph3 gene, which originated from wild barley and was first introgressed into cultivated Egyptian germplasm, encodes a unique predicted transmembrane resistance protein that differs from all known plant disease resistance proteins at the amino acid sequence level. Genetic profiles of diverse accessions indicated limited genetic diversity in Rph3 in domesticated germplasm, and higher diversity in wild barley from the Eastern Mediterranean region. The Rph3 gene was expressed only in interactions with Rph3-avirulent P. hordei isolates, a phenomenon also observed for transcription activator-like effector-dependent genes known as executors conferring resistance to Xanthomonas spp. Like known transmembrane executors such as Bs3 and Xa7, heterologous expression of Rph3 in N. benthamiana induced a cell death response. The isolation of Rph3 highlights convergent evolutionary processes in diverse plant-pathogen interaction systems, where similar defence mechanisms evolved independently in monocots and dicots., (© 2022. The Author(s).)

Austropuccinia psidii is a fungal plant pathogen that infects species within the Myrtaceae, causing the disease myrtle rust. Myrtle rust is causing declines in populations within natural and managed ecosystems and is expected to result in species extinctions. Despite this, variation in response to A. psidii exist within some species, from complete susceptibility to resistance that prevents or limits infection by the pathogen. Untargeted metabolomics using Ultra Performance Liquid Chromatography with Ion Mobility followed by analysis using MetaboAnalyst 3.0, was used to explore the chemical defence profiles of resistant, hypersensitive and susceptible phenotypes within Melaleuca quinquenervia during the early stages of A. psidii infection. We were able to identify three separate pools of secondary metabolites: (i) metabolites classified structurally as flavonoids that were naturally higher in the leaves of resistant individuals prior to infection, (ii) organoheterocyclic and carbohydrate-related metabolites that varied with the level of host resistance post-infection, and (iii) metabolites from the terpenoid pathways that were responsive to disease progression regardless of resistance phenotype suggesting that these play a minimal role in disease resistance during the early stages of colonization of this species. Based on the classes of these secondary metabolites, our results provide an improved understanding of key pathways that could be linked more generally to rust resistance with particular application within Melaleuca .

Long-distance migration and host adaptation by transboundary plant pathogens often brings detrimental effects to important agroecosystems. Efficient surveillance as a basis for responding to the dynamics of such pathogens is often hampered by a lack of information on incursion origin, evolutionary pathways and the genetic basis of rapidly evolving virulence across larger timescales. Here, we studied these genetic features by using historical isolates of the obligate biotrophic pathogen Puccinia striiformis f. sp. tritici (Pst), which causes one of the most widespread and devastating diseases, stripe (yellow) rust, of wheat. Through a combination of genotypic, phenotypic and genomic analyses, we assigned eight Pst isolates representing putative exotic Pst incursions into Australia to four previously defined genetic groups, PstS0, PstS1, PstS10 and PstS13. We showed that isolates of an additional incursion of P. striiformis, known locally as P. striiformis f. sp. pseudo-hordei, had a new and unique multilocus SSR genotype (MLG). We provide results of overall genomic variation of representative Pst isolates from each genetic group by comparative genomic analyses. We showed that isolates within the PstS1 and PstS13 genetic groups are most distinct at the whole-genome variant level from isolates belonging to genetic group PstS0, whereas the isolate from the PstS10 genetic group is intermediate. We further explored variable gene content, including putative effectors, representing both shared but also unique genetic changes that have occurred following introduction, some of which may additionally account for local adaptation of these isolates to triticale. Our genotypic and genomic data revealed new genetic insights into the evolution of diverse phenotypes of rust pathogens following incursion into a geographically isolated continental region., (© 2021 John Wiley & Sons Ltd.)

Plant root-produced constitutive and inducible defences inhibit pathogenic microorganisms within roots and in the rhizosphere. However, regulatory mechanisms underlying host responses during root-pathogen interactions are largely unexplored. Using the model species Brachypodium distachyon (Bd), we studied transcriptional and metabolic responses altered in Bd roots following challenge with Fusarium graminearum (Fg), a fungal pathogen that causes diseases in diverse organs of cereal crops. Shared gene expression patterns were found between Bd roots and spikes during Fg infection associated with the mycotoxin deoxynivalenol (DON). Overexpression of BdMYB78, an up-regulated transcription factor, significantly increased root resistance during Fg infection. We show that Bd roots recognize encroaching Fg prior to physical contact by altering transcription of genes associated with multiple cellular processes such as reactive oxygen species and cell development. These changes coincide with altered levels of secreted host metabolites detected by an untargeted metabolomic approach. The secretion of Bd metabolites was suppressed by Fg as enhanced levels of defence-associated metabolites were found in roots during pre-contact with a Fg mutant defective in host perception and the ability to cause disease. Our results help to understand root defence strategies employed by plants, with potential implications for improving the resistance of cereal crops to soil pathogens., (© 2021 John Wiley & Sons Ltd.)

The causal agent of maize common rust (CR), Puccinia sorghi , has increased in incidence and severity in Australia in recent years, prompting the assessment of sources of resistance and a preliminary survey of the diversity of P. sorghi populations. The maize commercial hybrids tested carried no resistance to 14 isolates of P. sorghi and had infection types comparable with that of a susceptible check. The resistance gene Rp1_D that remained effective in the United States for 35 years was ineffective against 7 of the 14 isolates. Maize lines carrying known "resistance to Puccinia " ( Rp ) genes were inoculated with the five isolates considered most diverse based on year of collection (2018 or 2019), location (Queensland or Victoria), and host from which they were isolated (maize or sweet corn). Lines carrying the resistance genes RpG, Rp5, Rp1_E, Rp1_I, Rp1_L, RpGDJ, RpGJF , and Rp5GCJ were resistant to all five isolates and to isolates collected in many agroecological regions. These lines were recommended as donors of effective resistance for maize breeding programs in Australia. Lines carrying no known resistance or resistance genes Rp8_A, Rp8_B, Rp1_J, Rp1_M, Rp7 , and Rpp9 (conferring resistance to P. polysora ) were susceptible to all five isolates. Differential lines carrying resistance genes Rp1_B, Rp1_C, Rp1_D, Rp1_F, Rp1_K, Rp3_D , or Rp4_A were either resistant or susceptible depending upon the isolate used, showing that the isolates varied in virulence for these genes. Urediniospore production was reduced on adult compared with juvenile plants, presumably due to changes in plant physiology associated with age or the presence of adult plant resistance.

Despite the global economic importance of the wheat leaf rust pathogen Puccinia triticina ( Pt ), genomic resources for Pt are limited and chromosome-level assemblies of Pt are lacking. Here, we present a complete haplotype-resolved genome assembly at a chromosome-scale for Pt using the Australian pathotype 64-(6),(7),(10),11 (Pt64; North American race LBBQB) built upon the newly developed technologies of PacBio and Hi-C sequencing. PacBio reads with ∼200-fold coverage (29.8 Gb data) were assembled by Falcon and Falcon-unzip and subsequently scaffolded with Hi-C data using Falcon-phase and Proximo. This approach allowed us to construct 18 chromosome pseudomolecules ranging from 3.5 to 12.3 Mb in size for each haplotype of the dikaryotic genome of Pt64. Each haplotype had a total length of ∼147 Mb, scaffold N 50 of ∼9.4 Mb, and was ∼93% complete for BUSCOs. Each haplotype had ∼29,800 predicted genes, of which ∼2,000 were predicted as secreted proteins (SPs). The investigation of structural variants (SVs) between haplotypes A and B revealed that 10% of the total genome was spanned by SVs, highlighting variations previously undetected by short-read based assemblies. For the first time, the mating type (MAT) genes on each haplotype of Pt64 were identified, which showed that MAT loci a and b are located on two chromosomes (chromosomes 7 and 14), representing a tetrapolar type. Furthermore, the Pt64 assembly enabled haplotype-based evolutionary analyses for 21 Australian Pt isolates, which highlighted the importance of a haplotype resolved reference when inferring genetic relationships using whole genome SNPs. This Pt64 assembly at chromosome-scale with full phase information provides an invaluable resource for genomic and evolutionary research, which will accelerate the understanding of molecular mechanisms underlying Pt -wheat interactions and facilitate the development of durable resistance to leaf rust in wheat and sustainable control of rust disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wu, Song, Ding, Dong, Hasan and Park.)

Key Message: A new gene Rph28 conferring resistance to barley leaf rust was discovered and fine-mapped on chromosome 5H from wild barley. Leaf rust is a highly destructive disease of barley caused by the fungal pathogen Puccinia hordei. Genetic resistance is considered to be the most effective, economical and eco-friendly approach to minimize losses caused by this disease. A study was undertaken to characterize and fine map a seedling resistance gene identified in a Hordeum vulgare ssp. spontaneum-derived barley line, HEB-04-101, that is broadly effective against a diverse set of Australian P. hordei pathotypes. Genetic analysis of an F 3 population derived from a cross between HEB-04-101 and the H. vulgare cultivar Flagship (seedling susceptible) confirmed the presence of a single dominant gene for resistance in HEB-04-101. Selective genotyping was performed on representative plants from non-segregating homozygous resistant and homozygous susceptible F 3 families using the targeted genotyping-by-sequencing (tGBS) assay. Putatively linked SNP markers with complete fixation were identified on the long arm of chromosome 5H spanning a physical interval between 622 and 669 Mb based on the 2017 Morex barley reference genome assembly. Several CAPS (cleaved amplified polymorphic sequences) markers were designed from the pseudomolecule sequence of the Morex assembly (v1.0 and v2.0), and 16 polymorphic markers were able to delineate the RphHEB locus to a 0.05 cM genetic interval spanning 98.6 kb. Based on its effectiveness and wild origin, RphHEB is distinct from all other designated Rph genes located on chromosome 5H and therefore the new locus symbol Rph28 is recommended for RphHEB in accordance with the rules and cataloguing system of barley gene nomenclature.

The re-emergence of stem rust on wheat in Europe and Africa is reinforcing the ongoing need for durable resistance gene deployment. Here, we isolate from wheat, Sr26 and Sr61, with both genes independently introduced as alien chromosome introgressions from tall wheat grass (Thinopyrum ponticum). Mutational genomics and targeted exome capture identify Sr26 and Sr61 as separate single genes that encode unrelated (34.8%) nucleotide binding site leucine rich repeat proteins. Sr26 and Sr61 are each validated by transgenic complementation using endogenous and/or heterologous promoter sequences. Sr61 orthologs are absent from current Thinopyrum elongatum and wheat pan genome sequences, contrasting with Sr26 where homologues are present. Using gene-specific markers, we validate the presence of both genes on a single recombinant alien segment developed in wheat. The co-location of these genes on a small non-recombinogenic segment simplifies their deployment as a gene stack and potentially enhances their resistance durability.

Leaf rust, caused by Puccinia hordei, is a devastating fungal disease affecting barley (Hordeum vulgare subsp. vulgare) production globally. Despite the effectiveness of genetic resistance, the deployment of single genes often compromises durability due to the emergence of virulent P. hordei races, prompting the search for new sources of resistance. Here we report on the cloning of Rph15, a resistance gene derived from barley's wild progenitor H. vulgare subsp. spontaneum. We demonstrate using introgression mapping, mutation and complementation that the Rph15 gene from the near-isogenic line (NIL) Bowman + Rph15 (referred to as BW719) encodes a coiled-coil nucleotide-binding leucine-rich repeat (NLR) protein with an integrated Zinc finger BED (ZF-BED) domain. A predicted KASP marker was developed and validated across a collection of Australian cultivars and a series of introgression lines in the Bowman background known to carry the Rph15 resistance. Rph16 from HS-680, another wild barley derived leaf rust resistance gene, was previously mapped to the same genomic region on chromosome 2H and was assumed to be allelic with Rph15 based on genetic studies. Both sequence analysis, race specificity and the identification of a knockout mutant in the HS-680 background suggest that Rph15- and Rph16-mediated resistances are in fact the same and not allelic as previously thought. The cloning of Rph15 now permits efficient gene deployment and the production of resistance gene cassettes for sustained leaf rust control., (© 2021 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Diseases caused by rust fungi pose a significant threat to global plant production. Although carotenoid pigments are produced in spores of nearly all rust species, the corresponding biosynthesis pathway(s) have not been investigated. Here, candidate genes for carotenoid biosynthesis in Puccinia graminis f. sp. tritici (Pgt) were identified, cloned and functionally complemented using specifically engineered strains of Escherichia coli. A part of the carotenoid biosynthesis pathway in rust fungi was elucidated, with only two genes, CrtYB and CrtI, catalysing the reactions from geranyl-geranyl diphosphate (GGPP) to γ-carotene. The CrtYB gene encodes a bi-functional lycopene cyclase/phytoene synthase, which catalyses the condensation of two GGPP into phytoene, as well as the cyclisation of the ψ-end of lycopene to form γ-carotene. The CrtI gene encodes a phytoene desaturase that carries out four successive desaturations of phytoene, through the intermediates phytofluene and neurosporene to lycopene. The evolution of carotenoid pigmentation in rust fungi, including Pgt, P. graminis avenae, P. graminis secalis (Pgs), P. graminis lolli, P. striiformis f. sp. tritici, P. striiformis f. sp. pseudohordei, P. striiformis f. sp. hordei, the "scabrum" rust (putative hybrids between Pgt and Pgs), P. triticina, and P. hordei, was investigated by phylogenetic analysis. Both CrtYB and CrtI were found to be closely related among rust fungi, other pathogenic fungi, and some aphids. Our results provide a springboard to increase the understanding of the physiological role(s) of carotenoid pigments in rust fungi, to better understand evolution within the Pucciniales, and to develop robust molecular diagnostics for rust fungi., (Crown Copyright © 2020. Published by Elsevier Ltd. All rights reserved.)

Austropuccinia psidii, originating in South America, is a globally invasive fungal plant pathogen that causes rust disease on Myrtaceae. Several biotypes are recognized, with the most widely distributed pandemic biotype spreading throughout the Asia-Pacific and Oceania regions over the last decade. Austropuccinia psidii has a broad host range with more than 480 myrtaceous species. Since first detected in Australia in 2010, the pathogen has caused the near extinction of at least three species and negatively affected commercial production of several Myrtaceae. To enable molecular and evolutionary studies into A. psidii pathogenicity, we assembled a highly contiguous genome for the pandemic biotype. With an estimated haploid genome size of just over 1 Gb (gigabases), it is the largest assembled fungal genome to date. The genome has undergone massive expansion via distinct transposable element (TE) bursts. Over 90% of the genome is covered by TEs predominantly belonging to the Gypsy superfamily. These TE bursts have likely been followed by deamination events of methylated cytosines to silence the repetitive elements. This in turn led to the depletion of CpG sites in TEs and a very low overall GC content of 33.8%. Compared to other Pucciniales, the intergenic distances are increased by an order of magnitude indicating a general insertion of TEs between genes. Overall, we show how TEs shaped the genome evolution of A. psidii and provide a greatly needed resource for strategic approaches to combat disease spread., (© The Author(s) 2020. Published by Oxford University Press on behalf of Genetics Society of America.)

Barley is an intermediate or near nonhost to many cereal rust pathogens that infect grasses, making it a highly suitable model to understand the evolution and genetic basis of nonhost resistance (NHR) in plants. To characterize the genetic architecture of NHR in barley, we used the Oregon Wolfe Barley doubled haploid and Morex × SusPtrit recombinant inbred line mapping populations. To elicit a wide array of NHR responses, we tested 492 barley accessions and both mapping populations with pathogenically diverse cereal rust isolates representing distinct formae speciales adapted to Avena , Hordeum , Triticum , and Lolium spp.: P. coronata f. sp. avenae (oat crown rust pathogen) and P. coronata f. sp. lolii (ryegrass crown rust pathogen), P. graminis f. sp. avenae (oat stem rust pathogen) and P. graminis f. sp. lolii (the ryegrass stem rust pathogen), and P. striiformis f. sp. tritici (wheat stripe rust pathogen) and P. striiformis f. sp. pseudo-hordei (barley grass stripe rust pathogen). With the exception of P. coronata f. sp. lolii and P. coronata f. sp. avenae , susceptibility and segregation for NHR was observed in the barley accessions and both mapping populations. Quantitative trait loci (QTLs) for NHR were mapped on all seven chromosomes. NHR in barley to the heterologous rusts tested was attributable to a combination of QTLs with either or both overlapping and distinct specificities. Across both mapping populations, broadly effective NHR loci were also identified that likely play a role in host specialization.

Improving resistance to barley leaf rust (caused by Puccinia hordei ) is an important breeding objective in most barley growing regions worldwide. The development and subsequent utilization of high-throughput PCR-based codominant molecular markers remains an effective approach to select genotypes with multiple effective resistance genes, permitting efficient gene deployment and stewardship. The genes Rph20 and Rph24 confer widely effective adult plant resistance (APR) to leaf rust, are common in European and Australian barley germplasm (often in combination), and act interactively to confer high levels of resistance. Here we report on the development and validation of codominant insertion-deletion (indel) based PCR markers that are highly predictive for the resistance alleles Rph20.ai and Rph24.an (both referred to as Rph20 and Rph24 ).

NAC (NAM, AFAT1/2, and CUC2) transcription factors play important roles in plant growth and in resistance to abiotic and biotic stresses. Here, we show that the TaNAC35 gene negatively regulates leaf rust resistance in the wheat line Thatcher + Lr14b (TcLr14b) when challenged with a virulent isolate of Puccinia triticina (Pt). The TaNAC35 gene was cloned from this line, and blastp results showed that its open reading frame (ORF) was 96.16% identical to the NAC35-like sequence reported from Aegilops tauschii, and that it encoded a protein with 387 amino acids (aa) including a conserved NAM domain with 145 aa at the N-terminal alongside the transcriptional activation domain with 220 aa in the C-terminal. Yeast-one-hybrid analysis proved that the C-terminal of the TaNAC35 protein was responsible for transcriptional activation. A 250-bp fragment from the 3'-end of this target gene was introduced to a BSMV-VIGS vector and used to infect the wheat line Thatcher + Lr14b (TcLr14b). The BSMV-VIGS/TaNAC35-infected plant material showed enhanced resistance (infection type "1") to Pt pathotype THTT, which was fully virulent (infection type "4") on BSMV-VIGS only infected TcLr14b plants. Histological studies showed that inhibition of TaNAC35 reduced the formation of haustorial mother cells (HMC) and mycelial growth, implying that the TaNAC35 gene plays a negative role in the response of TcLr14b to Pt pathotype THTT. These results provide molecular insight into the interaction between Pt and its wheat host, and identify a potential target for engineering resistance in wheat to this damaging pathogen.

The leaf rust pathogen, Puccinia triticina ( Pt ), threatens global wheat production. The deployment of leaf rust ( Lr ) resistance (R) genes in wheat varieties is often followed by the development of matching virulence in Pt due to presumed changes in avirulence (Avr) genes in Pt . Identifying such Avr genes is a crucial step to understand the mechanisms of wheat-rust interactions. This study is the first to develop and apply an integrated framework of gene expression, single nucleotide polymorphism (SNP), insertion/deletion (InDel), and copy number variation (CNV) analysis in a rust fungus and identify candidate avirulence genes. Using a long-read based de novo genome assembly of an isolate of Pt ('Pt104') as the reference, whole-genome resequencing data of 12 Pt pathotypes derived from three lineages Pt104, Pt53, and Pt76 were analyzed. Candidate avirulence genes were identified by correlating virulence profiles with small variants (SNP and InDel) and CNV, and RNA-seq data of an additional three Pt isolates to validate expression of genes encoding secreted proteins (SPs). Out of the annotated 29,043 genes, 2392 genes were selected as SP genes with detectable expression levels. Small variant comparisons between the isolates identified 27-40 candidates and CNV analysis identified 14-31 candidates for each Avr gene, which when combined, yielded the final 40, 64, and 69 candidates for AvrLr1 , AvrLr15, and AvrLr24 , respectively. Taken together, our results will facilitate future work on experimental validation and cloning of Avr genes. In addition, the integrated framework of data analysis that we have developed and reported provides a more comprehensive approach for Avr gene mining than is currently available.

Myrtle rust, caused by the pathogen Austropuccinia psidii , affects species of the Myrtaceae, many of which are endemic to Australia and New Zealand. Originating from South America, A. psidii is now present in both countries, necessitating effective chemical control for disease management. Using an artificial inoculation protocol, the efficacy of eight fungicides (tebuconazole/trifloxystrobin, cyproconazole/azoxystrobin, fosetyl aluminum, triforine, triadimenol, oxycarboxin, copper, and tebuconazole) applied as curative or protectant treatments was tested on two native New Zealand species ( Lophomyrtus × ralphii and Metrosideros excelsa ). The impacts of rate (×2), frequency (single or double), and timing (pre- or postinfection) of fungicide application were investigated. Overall, the most effective fungicides tested across both species were those that included a demethylation inhibitor and strobilurin mix, notably tebuconazole/trifloxystrobin (Scorpio) and cyproconazole/azoxystrobin (Amistar Xtra). These fungicides significantly reduced infection of host plants relative to the water control. Timing of application significantly affected bioefficacy, with applications made 7 days before inoculation or 7 days after inoculation being generally the most effective. The rate of fungicide application was not significant for both host species, with few interaction terms showing overall significance. Key findings from this study will set the foundation for further fungicide bioefficacy research conducted to evaluate formulations and adjuvant mixtures, determine suitable application methods for enhanced retention and coverage, and derive optimum application time for effective protection of native and exotic Myrtaceae species in New Zealand.

The demand for cereal grains as a main source of energy continues to increase due to the rapid increase in world population. The leaf rust diseases of cereals cause significant yield losses, posing challenges for global food security. The deployment of resistance genes has long been considered as the most effective and sustainable way to control cereal leaf rust diseases. While genetic resistance has reduced the impact of these diseases in agriculture, losses still occur due to the ability of the respective rust pathogens to change and render resistance genes ineffective plus the slow pace at which resistance genes are discovered and characterized. This article highlights novel recently developed strategies based on advances in genome sequencing that have accelerated gene isolation by overcoming the complexity of cereal genomes. The leaf rust resistance genes cloned so far from wheat and barley belong to various protein families, including nucleotide binding site/leucine-rich repeat receptors and transporters. We review recent studies that are beginning to reveal the defense mechanisms conferred by the leaf rust resistance genes identified to date in cereals and their roles in either pattern-triggered immunity or effector-triggered immunity.