Virulence Survey of Puccinia striiformis in Israel Revealed Considerable Changes in the Pathogen Population During the Period 2001 to 2019.

A total of 353 urediniospore isolates of Puccinia striiformis f. sp. tritici (Pst) collected in Israel during 2001 to 2019 were analyzed. Pst pathogenicity was studied with a set of 20 differentials (17 Avocet and 3 other lines). Three periods were compared: 2001 to 2007, 2009 to 2016, and 2017 to 2019. No virulence to Yr5 or Yr15 was detected. Virulence frequencies on Yr4, Yr10, Yr24, and YrSp genes rose to the moderate level (0.28 to 0.44) in 2017 to 2019. Virulence frequencies to Yr2 and Yr9 decreased. One Pst phenotype was identified in all three periods, but its frequency drastically decreased from 0.74 in 2001 to 2016 to 0.21 in 2017 to 2019. The most probable scenario of emergence of wheat yellow rust in Israel is wind dissemination of Pst urediniospores from the Horn of Africa. Variability of the Pst population increased amid considerable evolution with two major transformations in 2009 and 2017. The first modification can be attributed to changes in wheat genetic background in Israel due to deployment of new cultivars resistant to yellow rust since 2004. The second shift in 2017 can be primarily explained by intensive deployment of wheat cultivars resistant to the stem rust race Ug99 in the 2010s in the Horn of Africa. This led to changing genetic backgrounds of the cultivated wheats in the donor region and development and long-distance spread of new Pst phenotypes to Israel. Two singular multivirulent Pst phenotypes were identified in 2019, one of them being closely related to the aggressive Warrior race. Such phenotypes may potentially defeat existing resistances.

Comparative Analysis Highlights Variable Genome Content of Wheat Rusts and Divergence of the Mating Loci.

Three members of the Puccinia genus, Pucciniatriticina (Pt), Pstriiformis f.sp. tritici (Pst), and Pgraminis f.sp. tritici (Pgt), cause the most common and often most significant foliar diseases of wheat. While similar in biology and life cycle, each species is uniquely adapted and specialized. The genomes of Pt and Pst were sequenced and compared to that of Pgt to identify common and distinguishing gene content, to determine gene variation among wheat rust pathogens, other rust fungi, and basidiomycetes, and to identify genes of significance for infection. Pt had the largest genome of the three, estimated at 135 Mb with expansion due to mobile elements and repeats encompassing 50.9% of contig bases; in comparison, repeats occupy 31.5% for Pst and 36.5% for Pgt We find all three genomes are highly heterozygous, with Pst [5.97 single nucleotide polymorphisms (SNPs)/kb] nearly twice the level detected in Pt (2.57 SNPs/kb) and that previously reported for Pgt Of 1358 predicted effectors in Pt, 784 were found expressed across diverse life cycle stages including the sexual stage. Comparison to related fungi highlighted the expansion of gene families involved in transcriptional regulation and nucleotide binding, protein modification, and carbohydrate degradation enzymes. Two allelic homeodomain pairs, HD1 and HD2, were identified in each dikaryotic Puccinia species along with three pheromone receptor (STE3) mating-type genes, two of which are likely representing allelic specificities. The HD proteins were active in a heterologous Ustilago maydis mating assay and host-induced gene silencing (HIGS) of the HD and STE3 alleles reduced wheat host infection.

Annotation of a hybrid partial genome of the coffee rust (Hemileia vastatrix) contributes to the gene repertoire catalog of the Pucciniales.

Coffee leaf rust caused by the fungus Hemileia vastatrix is the most damaging disease to coffee worldwide. The pathogen has recently appeared in multiple outbreaks in coffee producing countries resulting in significant yield losses and increases in costs related to its control. New races/isolates are constantly emerging as evidenced by the presence of the fungus in plants that were previously resistant. Genomic studies are opening new avenues for the study of the evolution of pathogens, the detailed description of plant-pathogen interactions and the development of molecular techniques for the identification of individual isolates. For this purpose we sequenced 8 different H. vastatrix isolates using NGS technologies and gathered partial genome assemblies due to the large repetitive content in the coffee rust hybrid genome; 74.4% of the assembled contigs harbor repetitive sequences. A hybrid assembly of 333 Mb was built based on the 8 isolates; this assembly was used for subsequent analyses. Analysis of the conserved gene space showed that the hybrid H. vastatrix genome, though highly fragmented, had a satisfactory level of completion with 91.94% of core protein-coding orthologous genes present. RNA-Seq from urediniospores was used to guide the de novo annotation of the H. vastatrix gene complement. In total, 14,445 genes organized in 3921 families were uncovered; a considerable proportion of the predicted proteins (73.8%) were homologous to other Pucciniales species genomes. Several gene families related to the fungal lifestyle were identified, particularly 483 predicted secreted proteins that represent candidate effector genes and will provide interesting hints to decipher virulence in the coffee rust fungus. The genome sequence of Hva will serve as a template to understand the molecular mechanisms used by this fungus to attack the coffee plant, to study the diversity of this species and for the development of molecular markers to distinguish races/isolates.

Molecular Phylogenetic Relationships of the Brown Leaf Rust Fungi on Wheat, Rye, and Other Grasses.

The classification of brown leaf rust fungi (Puccinia recondita complex and allied species) on wheat (Triticum aestivum), rye (Secale cereale), and other grasses in the family Poaceae has experienced a long history of controversy and uncertainty due to the reduced morphological characteristics available for taxonomy and difficulty of conducting interfertility experiments. However, because these are pathogens on important crops, it is important to clarify the species delimitations reflecting the natural lineages. In this study, phylogenetic analyses were conducted with DNA sequence data from the ribosomal DNA internal transcribed spacer region and elongation factor 1-α to elucidate this species complex. Three phylogenetic lineages were recovered within the complex of rye leaf rust fungi, P. recondita sensu stricto, which is congruent with existing classifications based on DNA content, sexual compatibility, and morphological studies. The brown leaf rust fungus on wheat (P. triticina) grouped with the related species P. persistens on Elymus repens and E. intermedia as a strongly supported clade. Collections on other Elymus spp. were separated into six clades. Based on the phylogenetic affinities of nine type specimens and aecial host associations, potential taxonomic names were evaluated for selected lineages.

Gene discovery in EST sequences from the wheat leaf rust fungus Puccinia triticina sexual spores, asexual spores and haustoria, compared to other rust and corn smut fungi.

BACKGROUND: Rust fungi are biotrophic basidiomycete plant pathogens that cause major diseases on plants and trees world-wide, affecting agriculture and forestry. Their biotrophic nature precludes many established molecular genetic manipulations and lines of research. The generation of genomic resources for these microbes is leading to novel insights into biology such as interactions with the hosts and guiding directions for breakthrough research in plant pathology. RESULTS: To support gene discovery and gene model verification in the genome of the wheat leaf rust fungus, Puccinia triticina (Pt), we have generated Expressed Sequence Tags (ESTs) by sampling several life cycle stages. We focused on several spore stages and isolated haustorial structures from infected wheat, generating 17,684 ESTs. We produced sequences from both the sexual (pycniospores, aeciospores and teliospores) and asexual (germinated urediniospores) stages of the life cycle. From pycniospores and aeciospores, produced by infecting the alternate host, meadow rue (Thalictrum speciosissimum), 4,869 and 1,292 reads were generated, respectively. We generated 3,703 ESTs from teliospores produced on the senescent primary wheat host. Finally, we generated 6,817 reads from haustoria isolated from infected wheat as well as 1,003 sequences from germinated urediniospores. Along with 25,558 previously generated ESTs, we compiled a database of 13,328 non-redundant sequences (4,506 singlets and 8,822 contigs). Fungal genes were predicted using the EST version of the self-training GeneMarkS algorithm. To refine the EST database, we compared EST sequences by BLASTN to a set of 454 pyrosequencing-generated contigs and Sanger BAC-end sequences derived both from the Pt genome, and to ESTs and genome reads from wheat. A collection of 6,308 fungal genes was identified and compared to sequences of the cereal rusts, Puccinia graminis f. sp. tritici (Pgt) and stripe rust, P. striiformis f. sp. tritici (Pst), and poplar leaf rust Melampsora species, and the corn smut fungus, Ustilago maydis (Um). While extensive homologies were found, many genes appeared novel and species-specific; over 40% of genes did not match any known sequence in existing databases. Focusing on spore stages, direct comparison to Um identified potential functional homologs, possibly allowing heterologous functional analysis in that model fungus. Many potentially secreted protein genes were identified by similarity searches against genes and proteins of Pgt and Melampsora spp., revealing apparent orthologs. CONCLUSIONS: The current set of Pt unigenes contributes to gene discovery in this major cereal pathogen and will be invaluable for gene model verification in the genome sequence.

Leaf Rust on Aegilops speltoides Caused by a New Forma Specialis of Puccinia triticina.

ABSTRACT A leaf rust attacking Aegilops speltoides in its natural habitat is reported for the first time. It was found in two locations in northern and central Israel. The two collections from A. speltoides resemble wheat leaf rust, Puccinia triticina, in most spore dimensions, in the morphology of the substomatal vesicle of the urediniospore, and in DNA content in pycniospore nuclei. Similarly to P. triticina isolates from wheat, isolates taken from A. speltoides are compatible with Thalictrum speciosissimum as an aecial host and they are crossed easily with wheat leaf rust isolates. However, isolates from A. speltoides differ from wheat leaf rust in their telial host range. They are avirulent to cultivated wheat cultivars, but attack hundreds of A. speltoides accessions that were immune to wheat leaf rust. This distinct host preference justifies delineation of the newly found leaf rust as a forma specialis (f. sp. speltoides) within P. triticina.