Transient transformation of the rust fungus Puccinia graminis f. sp. tritici

The biotrophic rust fungus Puccinia graminis f. sp. tritici (Pgt) was transformed by particle bombardment. The promoter from the Pgt translation elongation factor 1alpha (EF-1alpha) gene was fused to the bacterial marker genes hygromycin B phosphotransferase (hpt) and beta-glucuronidase (GUS). Transformation constructs were introduced into uredospores of Pgt, an obligate pathogen of wheat, by biolistic bombardment. Uredospores transformed with the construct containing the hpt gene germinated and initiated branching on selective medium, indicating that they had acquired resistance to hygromycin B. However, transformants stopped growing 5 days after bombardment. GUS activity in uredospores and germlings was histochemically detected 4-16 h after bombardment. GUS expression was also obtained using the INF24 promoter from the bean rust fungus Uromyces appendiculatus, demonstrating that heterologous genes can be expressed in P. graminis under the control of regulatory sequences from closely related organisms.     

Modifications ultrastructurales provoquees par Puccinia graminis f. sp. tritici chez le ble

Susceptible varieties of wheat infected withPuccinia graminis (Pers.) f. sp.tritici Eriks. &E. Henn. were studied using chlorotic samples fixed with glutaraldehyde and postfixed in potassium permanganate or osmium tetroxide. Intercellular mycelium and haustoria are surrounded with a thick wall covered with dense material. Many kinds of organelles or structures were found in the electron-dense cytoplasm of fungi: nucleus, mitochondria, endoplasmic reticulum and lomasomes. Golgi apparatus was not recognized. Haustorium is characterized by pinocytosis figures lined with fungal plasmalemma and by numerous mitochondria. The haustorium is enclosed in an encapsulation of electron-dense, fibrillar or granular material. The thickness of the encapsulation increases with age. A membrane, which seems to be continuous with the host plasmalemma, forms the interface between the encapsulation and the host cytoplasm. This membrane is invaginated by lomasomes and small vesicles occur in his vicinity. The origin of the encapsulation and of the encapsulation membrane remains unknown.Many host organelles (mitochondria, endoplasmic reticulum, nucleus, chloroplasts) are closely appressed to the encapsulation membrane. The host nucleus appears to encase the haustorium; the chloroplasts develop excrescences and are devoid of starch, while photosynthesis is reduced. A large number of mitochondria encircles the haustorium and may be related with respiratory increase in the host. Rough-surfaced endoplasmic reticulum develops into long threads. Lipid droplets were observed in the host infected cytoplasm.

---

Résumé L'étude de l'ultrastructure de variétés de Blé sensibles parasitées parPuccinia graminis (Pers.) f. sp.tritici Eriks. &E. Henn a été conduite sur des échantillons chlorotiques fixés à la glutaraldéhyde et postfixés par le permanganate de potassium ou l'acide osmique. Une paroi peu dense et recouverte d'exsudat entoure le mycélium intercellulaire et les haustorias. Le cytoplasme, dense aux électrons, renferme diverses structures ou organites: lomasomes, mitochondries, noyau, réticulum endoplasmique. Aucun dictyosome n'a été relevé. L'haustoria se caractérise par des figures de pinocytose qui tapissent la membrane plasmique et par la présence de nombreuses mitochondries. Une encapsulation de densité variable selon la fixation, encapsulation qui s'épaissit en vieillissant, sépare l'haustoria du protoplaste de l'hôte. Elle contient des produits fibrillaires ou granuleux, plus opaques aux électrons. La membrane qui recouvre l'encapsulation au contact du cytoplasme de l'hôte semble en continuité avec le plasmalemme de ce dernier. Elle est interrompue par des lomasomes et de nombreuses vésicules la cotoient. L'origine de l'encapsulation et de cette membrane demeure hypothétique.De nombreux organites de la plante (mitochondries, réticulum endoplasmique, noyau, chloroplastes) établissent des liens étroits avec le parasite. Le noyau devient lobé et les chloroplastes présentent parfois des excroissances. L'absence d'amidon indique un ralentissement de la photosynthèse. L'élévation du rythme respiratoire se traduit par la présence de nombreuses mitochondries qui encerclent l'haustoria. Le réticulum endoplasmique, souvent long et flexueux est riche en ribosomes. Le cytoplasme des cellules infectées contient des globules lipidiques.

---

---

Contribution No. 105 de la Faculté d'Agriculture, Université Laval, Québec 10e.     

Performance and survival of different races of Puccinia graminis f.sp. tritici on detached leaves of wheat

The detached leaf culture of Puccinia graminis f.sp. tritici was tested with eight races and two biotypes of the pathogen. The inherent characteristics of the races were similar to those in intact plants. Continuous maintenance of races in pure culture on detached leaves showed the usefulness of the technique for multiplication of inoculum as well as for maintenance of races in other basic studies.     

Establishment of wheat-Thinopyrum ponticum translocation lines with resistance to Puccinia graminis f. sp. tritici Ug99

<正>Stem rust, caused by Puccinia graminis Pers.:Pers. f. sp. tritici Eriks. E. Henn. (Pgt), is a destructive wheat disease and has the potential to cause significant yield losses (Olivera et al., 2015;Soko et al., 2018). The emergence of Pgt race TTKSK, virulent to the widely deployed stem rust resistance gene Sr31 and originally named as isolate Pgt-Ug99 (Pretorius et al., 2000), emphasized     

MicroRNA regulated defense responses in Triticum aestivum L. during Puccinia graminis f.sp. tritici infection

Plants have evolved diverse mechanism to recognize pathogen attack and triggers defense responses. These defense responses alter host cellular function regulated by endogenous, small, non-coding miRNAs. To understand the mechanism of miRNAs regulated cellular functions during stem rust infection in wheat, we investigated eight different miRNAs viz. miR159, miR164, miR167, miR171, miR444, miR408, miR1129 and miR1138, involved in three different independent cellular defense response to infection. The investigation reveals that at the initiation of disease, accumulation of miRNAs might be playing a key role in hypersensitive response (HR) from host, which diminishes at the maturation stage. This suggests a possible host-fungal synergistic relation leading to susceptibility. Differential expression of these miRNAs in presence and absence of R gene provides a probable explanation of miRNA regulated R gene mediated independent pathways.     

Genetic comparison of Ug99 with selected South African races of Puccinia graminis f.sp. tritici

Using simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) marker analyses, the genetic structure of selected South African wheat stem rust races was compared with Ug99. SSR analysis divided the population into two distinct groups with 24.5% similarity between them. A local race, UVPgt55 (North American race notation TTKSF), grouped with Ug99 (TTKSK) with a 100% similarity. When AFLP data were included, the same groups were found, but with an increased similarity of 66.7%. Although the SSR data were unable to distinguish between all individual isolates, the AFLP data alone and in combination with the SSR data discriminated between the isolates. The grouping of individual isolates resembled the pathogenicity profile of the different races. On the basis of its similarity with Ug99, it was concluded that UVPgt55 was most probably an exotic introduction into South Africa, whereas the other races specialized locally through mutational adaptation.     

An EST library from Puccinia graminis f. sp. tritici reveals genes potentially involved in fungal differentiation

The rust fungus Puccinia graminis f. sp. tritici is an obligately biotrophic pathogen on wheat plants and thus difficult to investigate. Hence, little is known about this fungus at the molecular level. We constructed a differential suppression subtractive hybridization cDNA-library from rust-infected vs. healthy wheat plants. The majority of expressed sequence tags (ESTs) showed similarities to fungal sequences. Semiquantitative RT-PCR using mRNA from rust-infected leaves, and from axenically grown, differentiating and nondifferentiating young rust colonies as well as sporulating and nonsporulating mature mycelia revealed rather diverse expression patterns for different ESTs, shedding new light on their potential involvement in differentiation and host-pathogen interaction.     

Production of teliospores by some races of Puccinia graminis f. sp. tritici in detached wheat and barley leaves

Five races of Puccinia graminis f. sp. tritici cultured on wheat and barley leaves in two nutrient solutions were studied for teliospore formation by subjecting them to varying treatments of temperature and light. The early appearance as well as a high percentage of teliospore formation occurred in 100 ppm benzimidazole solution on wheat or barley leaves kept at 30°C and 500 footcandles of light. The feasibility of maintaining and multiplying races of Puccinia graminis f. sp. tritici in continuous cultures on detached leaves depends on various factors, the most important being the onset of the teliostage of the fungus. The appearance of the teliospore denotes the culmination of the sporophytic or repeating stage of the wheat rusts. In this paper, some factors that influence the production of teliospores by certain races of Puccinia graminis tritici in detached leaves are discussed.     

2012–2013年中国禾柄锈菌小麦专化型小种动态及新毒力谱分析

监测具有潜在威胁的小麦秆锈病病原菌小种动态并分析其毒力谱变化,是培育抗病品种不可或缺的基础工作。在小麦秆锈病鲜有发生的情况下,于2012–2013年获得小麦田间秆锈病标样11份和经有性生殖过程的禾柄锈菌小麦专化型菌样22份。上述菌样经过分离、纯化,得到了53个单孢子堆菌株。利用国内最新采用的禾柄锈菌小麦专化型小种与毒力鉴别寄主体系进行分析,鉴定出13个生理小种,其中34C3RTGQM和34Oro II-MRGQM为优势小种,出现频率均为13.2%,首次发现了对Sr5+Sr11具有联合毒力的6个新小种,其出现频率处在1.9%–13.2%范围内。同时测定出Sr9e、Sr26、Sr31、Sr33、Sr37、Sr38、Sr47和Sr Tt3等8个抗秆锈病单基因对全部供试菌株表现抗病,余下40个单基因系则分别表现对1个以上至全部菌株感病。结论:(1)首次报道了6个对Sr5+Sr11具有危险性的联合毒力谱的新小种类型,这类毒力类型应当被密切关注;(2)明确了当前完全有效的8个抗秆锈病基因和其他部分有效的抗病基因;(3)初步说明了有性循环菌株与无性循环菌株在毒力结构上存在差异。     

小麦秆锈菌生理小种21C3CPH和Ug99 SSR标记的筛选

小麦秆锈病是一种专化性很强的大区远距气传病害,曾造成多个小麦种植国家和地区的毁灭性损失,新的强毒力小种Ug99含有对Sr31等多个重要抗秆锈基因的联合毒性,对我国的小麦生产有巨大潜在威胁,因此,加强小麦秆锈菌生理小种的监测和鉴定是有效防治该病害的基础性研究工作和关键环节。现代分子生物学的迅猛发展,为许多研究提供了新的方法和手段,分子标记技术在区分小麦秆锈菌生理小种方面显示了充分的可行性。本研究利用25对SSR引物对7个小麦秆锈菌主要生理小种进行DNA多态性分析,结果显示,所有特异引物对秆锈菌的扩增结果均呈现出丰富的多态性,秆锈菌的不同生理小种之间存在遗传差异。其中引物SSR180在21C3CPH中扩增出205bp的特异性条带;引物SSR6在Ug99中扩增出170bp的特异性条带,经过多次的重复试验,这些特异性条带均能够比较稳定地重复出现,说明引物SSR180和SSR6可用于小种21C3CPH和Ug99的特异性检测。     

Race analysis of Puccinia striiformis f.sp. tritici in Iran

The wheat stripe (yellow) rust is one of the most important diseases in Iran. In this study, 41 races out of 104 isolates in greenhouse were determined from 2008 to 2010. Races 6E6A+, 6E10A+ and 6E0A+ were more common. Races 0E0A+ was less aggressive than races 166E158A+ and 134E158A+ with virulence on 11 known genes. Virulence on plant/s with gene/s Yr1, Yr2, Yr4, Yr6, Yr7, Yr8, Yr9, Yr10, Yr25, Yr27, YrSU, YrSD, YrND, Yr3, Yr2+, Yr6+, Yr9+, Yr7+, YrCV and YrA was detected. The majority of isolates with high frequency (more than 70%) showed virulence on plant/s with Yr2, Yr7, Yr9 and YrA genes. No virulence was detected on plant/s with Yr3, Yr5 and YrSP. In greenhouse test, frequency of virulence to wheat genotypes with Yr1, Yr4, Yr10, YrCV (32+) and YrSD gene was less than 7%. Frequency of virulence to other wheat genotypes was between 8 and 100%.     

Isolation of haustoria ofPuccinia graminis f.sp.tritici from wheat leaves

Haustoria of the wheat stem rust fungus,Puccinia graminis f.sp.tritici, race 32, were isolated from two different infected wheat cultivars. Yield of haustoria from the highly susceptible wheat cv. “Little Club” was 10 times higher than that of the resistant wheat line “Prelude × Eagle” carrying the Sr26 gene for resistance toP. graminis f.sp.tritici. Tests for the integrity of haustoria using the stain methylene blue showed that up to 91% of the haustoria were undamaged.