Chris K. Sørensen

Diversity of Puccinia striiformis on Cereals and Grasses

Yellow (stripe) rust is a common fungal disease on cereals and grasses. It is caused by Puccinia striiformis sensu lato, which is biotrophic and heteroecious. The pathogen is specialized on the primary host at both species and cultivar levels, whereas several Berberis spp. may serve as alternate hosts. One lineage infects mainly cereals and at least two lineages are restricted to grasses. P. striiformis on cereals has a typical clonal population structure in many areas, resulting from asexual reproduction, but high diversity, suggesting frequent recombination, has been observed in certain areas in Asia. Yellow rust is spreading by airborne spores potentially across long distances, which may contribute to sudden disease epidemics in new areas. This has been the case since 2000, where large-scale epidemics in warmer wheat-growing areas have been ascribed to the emergence of two closely related yellow rust strains with increased aggressiveness and tolerance to warm temperatures.

Sexual structures and recombination of the wheat rust fungus Puccinia striiformis on Berberis vulgaris

An isolate of the basidiomycete Puccinia striiformis, which causes yellow (stripe) rust on wheat, was selfed on the newly discovered alternate host, Berberis vulgaris. This allowed a study of the segregation of molecular markers and virulence in the progeny isolates, and of the development of fungal sexual structures and spore forms. Pycnia and aecia were obtained after inoculation of B. vulgaris with basidiospores resulting from germinating teliospores from infected wheat leaves. Subsequent inoculation of wheat with aeciospores from bulked aecia resulted in 16 progeny isolates of the S1 generation. Genotyping with 42 simple sequence repeat (SSR) markers confirmed a parental origin of progeny isolates. Of the 42 analyzed loci, 15 were heterozygous in the parental isolate and 14 revealed segregation in the progenies. This resulted in 11 new multilocus genotypes (MLGs), which confirmed segregation following sexual reproduction. Additionally, parental and progeny isolates were phenotyped using a genetic stock of wheat genotypes representing 21 resistance genes. All S1 progeny isolates had virulence for 14 out of 15 loci where the parental isolate was virulent. This was consistent with the hypothesis that virulence in plant pathogens is often recessive to avirulence, i.e., only expressed in a homozygous state. Furthermore, no segregation was observed for five out of six loci, for which the parental isolate had an avirulent phenotype. The results for one of the two segregating virulence/avirulence loci suggested that the parental isolate was heterozygous with Avr alleles resulting in different but clearly avirulent phenotypes. The other locus indicated that additional genes modifying the phenotypic expression of avirulence were involved.

Yellow Rust epidemics worldwide were caused by Pathogen Races from divergent genetic lineages

We investigated whether the recent worldwide epidemics of wheat yellow rust were driven by races of few clonal lineage(s) or populations of divergent races. Race phenotyping of 887 genetically diverse Puccinia striiformis isolates sampled in 35 countries during 2009–2015 revealed that these epidemics were often driven by races from few but highly divergent genetic lineages. PstS1 was predominant in North America; PstS2 in West Asia and North Africa; and both PstS1 and PstS2 in East Africa. PstS4 was prevalent in Northern Europe on triticale; PstS5 and PstS9 were prevalent in Central Asia; whereas PstS6 was prevalent in epidemics in East Africa. PstS7, PstS8 and PstS10 represented three genetic lineages prevalent in Europe. Races from other lineages were in low frequencies. Virulence to Yr9 and Yr27 was common in epidemics in Africa and Asia, while virulence to Yr17 and Yr32 were prevalent in Europe, corresponding to widely deployed resistance genes. The highest diversity was observed in South Asian populations, where frequent recombination has been reported, and no particular race was predominant in this area. The results are discussed in light of the role of invasions in shaping pathogen population across geographical regions. The results emphasized the lack of predictability of emergence of new races with high epidemic potential, which stresses the need for additional investments in population biology and surveillance activities of pathogens on global food crops, and assessments of disease vulnerability of host varieties prior to their deployment at larger scales.

Evaluation of Spray and Point Inoculation Methods for the Phenotyping of Puccinia striiformis on Wheat

The fungus Puccinia striiformis causes yellow (stripe) rust on wheat worldwide. In the present article, new methods utilizing an engineered fluid (Novec 7100) as a carrier of urediniospores were compared with commonly used inoculation methods. In general, Novec 7100 facilitated a faster and more flexible application procedure for spray inoculation and it gave highly reproducible results for virulence phenotyping. Six point inoculation methods were compared to find the most suitable for assessment of pathogen aggressiveness. The use of Novec 7100 and dry dilution with Lycopodium spores gave an inoculation success rate of 100% in two independent trials, which was significantly higher and more consistent than for spore suspension in Soltrol 170, water, water + Tween 20, and Noble agar + Tween 20. Both Soltrol 170 and Novec 7100 allowed precise quantification of inoculum, which is important for the assessment of quantitative epidemiological parameters. New protocols for spray and point inoculation of P. striiformis on wheat are presented, along with the prospect for applying these in rust research and resistance breeding activities.

3-D imaging of temporal and spatial development of Puccinia striiformis haustoria in wheat

Differentiation of haustoria on primary infection hyphae of the fungal pathogen Puccinia striiformis was studied in wheat seedlings with two-photon microscopy in combination with a classical staining technique. Our results showed a significant increase in the average haustorium size 22, 44, 68, 92 and 116 h after inoculation (hai). After 116 hai no significant change was observed until 336 hai. Haustorium morphology also changed significantly during the time of infection. Initially small spherical haustoria were seen, but as they grew the haustoria gradually became apically branched. At 22 hai all observed haustoria were spherical, but at 44 hai most haustoria had an irregular structure, and at 92 hai all observed haustoria appeared branched. Along with the changes of the haustorial body the haustorial neck changed from narrow and slender to having an expanded appearance with a rough and invaginated structure. The structural changes were similar in two susceptible wheat varieties, 514W and Cartago, although the mean haustorium size was larger in 514W than in Cartago at all intervals.

Genetic diversity within and among aecia of the wheat rust fungus Puccinia striiformis on the alternate host Berberis vulgaris

An isolate of the fungus Puccinia striiformis, causing yellow (stripe) rust on cereals and grasses, was selfed on the alternate (sexual) host, Berberis vulgaris. This enabled us to investigate genetic variability of progeny isolates within and among aecia. Nine aecial clusters each consisting of an aecium (single aecial cup) and nine clusters containing multiple aecial cups were selected from 18 B. vulgaris leaves. Aeciospores from each cluster were inoculated on susceptible wheat seedlings and 64 progeny isolates were recovered. Molecular genotyping using 37 simple sequence repeat markers confirmed the parental origin of all progeny isolates. Thirteen molecular markers, which were heterozygous in the parental isolate, were used to analyse genetic diversity within and among aecial cups. The 64 progeny isolates resulted in 22 unique recombinant multilocus genotypes and none of them were resampled in different aecial clusters. Isolates derived from a single cup were always of the same genotype whereas isolates originating from clusters containing up to nine aecial cups revealed one to three genotypes per cluster. These results implied that each aecium was the result of a successful fertilization in a corresponding pycnium and that an aecium consisted of genetically identical aeciospores probably multiplied via repetitive mitotic divisions. Furthermore, the results suggested that aecia within a cluster were the result of independent fertilization events often involving genetically different pycniospores. The application of molecular markers represented a major advance in comparison to previous studies depending on phenotypic responses on host plants. The study allowed significant conclusions about fundamental aspects of the biology and genetics of an important cereal rust fungus.

Cloning of the wheat Yr15 resistance gene sheds light on the plant tandem kinase-pseudokinase family

Yellow rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a devastating fungal disease threatening much of global wheat production. Race-specific resistance (R)-genes are used to control rust diseases, but the rapid emergence of virulent Pst races has prompted the search for a more durable resistance. Here, we report the cloning of Yr15, a broad-spectrum R-gene derived from wild emmer wheat, which encodes a putative kinase-pseudokinase protein, designated as wheat tandem kinase 1, comprising a unique R-gene structure in wheat. The existence of a similar gene architecture in 92 putative proteins across the plant kingdom, including the barley RPG1 and a candidate for Ug8, suggests that they are members of a distinct family of plant proteins, termed here tandem kinase-pseudokinases (TKPs). The presence of kinase-pseudokinase structure in both plant TKPs and the animal Janus kinases sheds light on the molecular evolution of immune responses across these two kingdoms.Yellow rust fungus severely limits global wheat production and breeding of durable resistance is challenging. Here Klymiuk et al. isolate the broad-spectrum Yr15 resistance gene from wild emmer wheat and show that it is a member of a distinct tandem kinase-pseudokinase family of plant proteins.

Assessment of Aggressiveness of Puccinia striiformis on Wheat

A simple point-inoculation method using Novec™ 7100, a volatile engineered fluid, is presented for the assessment of aggressiveness of Puccinia striiformis isolates on seedlings of wheat. The method allows for quantification of the applied inoculum with a minimal risk of cross-contamination of rust from leaves grown side by side. The method is also applicable for the assessment of qualitative differences inferred by compatible and incompatible host-pathogen interactions, and it can be adjusted to other cereal rust and powdery mildew fungi on other host species, and other plant growth stages as appropriate.

Temporal and Spatial Variability of Fungal Structures and Host Responses in an Incompatible Rust–Wheat Interaction

Information about temporal and spatial variability of fungal structures and host responses is scarce in comparison to the vast amount of genetic, biochemical, and physiological studies of host–pathogen interactions. In this study, we used avirulent wild type and virulent mutant isolates of Puccinia striiformis to characterize the interactions in wheat carrying yellow rust Yr2 resistance. Both conventional and advanced microscopic techniques were used for a detailed study of morphology and growth of fungal colonies and associated host cell responses. The growth of the wild type isolates was highly restricted due to hypersensitive response (HR, plant cell death) indicated by autofluorescence and change in the shape of the affected plant cells. The host response appeared post-haustorial, but large variation in the time and stage of arrest was observed for individual fungal colonies, probably due to a delay between detection and response. Some colonies were stopped right after the formation of the primary infection hyphae whereas others formed highly branched mycelia. HR was first observed in host cells in direct contact with fungal structures, after which the defense responses spread to adjacent host cells, and eventually led to encasement of the fungal colony. Several cells with HR contained haustoria, which were small and underdeveloped, but some cells contained normal sized haustoria without signs of hypersensitivity. The growth of the virulent mutants in the resistant plants was similar to the growth in plants without Yr2 resistance, which is a strong indication that the incompatible phenotype was associated with Yr2. The interaction between P. striiformis and wheat with Yr2 resistance was highly variable in time and space, which demonstrate that histological studies are important for a deeper understanding of host–pathogen interactions and plant defense mechanisms in general.