Development and characterization of novel, polymorphic microsatellite markers for oat crown rust, Puccinia coronata

We report the development of 37 novel and polymorphic microsatellite markers for oat crown rust, Puccinia coronata f.sp. avenae. The allelic diversity ranged from two to 16 alleles per locus. Observed heterozygosity ranged from 0.000 to 0.971, and expected heterozygosity from 0.057 to 0.848. Thirteen of the loci were not in Hardy-Weinberg equilibrium, due to either the presence of null alleles, small sample size, or the effects of population subdivision (Wahlund's effect). All 37 primer pairs were tested with P. graminis and P. triticina showing that they are specific to P. coronata.     

Spore dimensions of Puccinia species of cereal hosts as determined by image analysis

Digital image analysis was used to measure dimensions of spores produced by Puccinia coronata, P. graminis, P. hordei, P. recondita, P. striiformis and P. triticina. Included were teliospores, basidiospores, urediniospores and, except for P. striiformis, pycniospores and aeciospores. Length, width and projection area of spores were measured with NIH Image or Scion software. By using limits on size, spores were automatically selected and measured, except for teliospores, which required manual elimination of the pedicel and separation of images of adhering spores. Length and width were determined as the major and minor axes of the best fitting ellipse for each spore. This procedure gave values for length and width close to results obtained with an ocular micrometer. Projection area was determined as the number of pixels within spore boundaries multiplied by the area represented by each pixel, giving values that are not feasible to obtain accurately with an ocular micrometer. Of the species studied, spores of P. recondita had the largest dimensions, P. triticina had the smallest. The rank of the six species based on increasing width, length or projection area was almost the same, using each spore type except pycniospores. Generally, differences of 5% in a given spore dimension between two species were significant. Differences between species were greater with basidiospores and aeciospores than with other spore types. Teliospores were unique in that length and width were negatively correlated, resulting in less variation in area than in length or width. The results indicate that image analysis is useful for measuring spore dimensions, that projection area of spores is a useful added parameter for characterizing rust species and that dimensions of teliospores, basidiospores, aeciospores and urediniospores each are potentially useful for differentiating species.     

Rds and Rih mediate hypersensitive cell death independent of gene-for-gene resistance to the oat crown rust pathogen Puccinia coronata f. sp. avenae.

The Pca crown rust resistance cluster in the diploid Avena genus confers gene-for-gene specificity to numerous isolates of Puccinia coronata f. sp. avenae. Recombination breakpoint analysis indicates that specificities conferred by the Pca cluster are controlled by at least five distinct genes, designated Pc81, Pc82, Pc83, Pc84, and Pc85. Avena plants with the appropriate genotype frequently respond to P. coronata by undergoing hypersensitive cell death at the sites of fungal infection. Autofluorescence of host cells in response to P. coronata occurs in plants that develop visible necrotic lesions but not in plants that lack this phenotype. Two newly described, non-Pc loci were shown to control hypersensitive cell death. Rds (resistance-dependent suppressor of cell death) suppresses the hypersensitive response (HR), but not the resistance, mediated by the Pc82 resistance gene. In contrast, Rih (resistance-independent hypersensitive cell death) confers HR in both resistant and susceptible plants. Linkage analysis indicates that Rds is unlinked to the Pca cluster, whereas Rih is tightly linked to it. These results indicate that multiple synchronous pathways affect the development of hypersensitive cell death and that HR is not essential for resistance to crown rust. Further characterization of these genes will clarify the relationship between plant disease resistance and localized hypersensitive cell death.     

Aphanocladiutn album, a fungus inducing teliospore production in rusts

The hyphomycetous fungus Aphanocladiutn album can grow over and around uredia of the rusts Puccinia coronata, P. hordei, P. graminis f.sp. avenae and P. recondita f.sp. triticina when host plants are kept under very humid conditions, but not on such plants not infected with rusts; uredia are adversely affected and telia develop in their vicinity. Plants inoculated with these rusts and with five isolates of A. album (one from a dead insect) showed: (1) much earlier development of telia on detached and non-detached rusted leaves inoculated with A. album than on corresponding leaves not thus inoculated; (2) telial induction by A. album in some isolates of rust species which hitherto had rarely or never produced telia; (3) precocious telial formation, in comparison with controls, when A. album spores were sprayed on leaves as much as 3 days before and 9 days after rust inoculation, and occasionally after uredia had already matured. As affected leaves remained green until the whole leaf became moribund, senescence is apparently not the factor inducing telia formation. The normal-appearing teliospores of some isolates were induced to germinate, whereas others did not. Rhamnus palaestina inoculated with basidiospores of one isolate of A. album-treated P. coronata f.sp. avenae produced pycnia and fertile aecia. The importance of A. album as a working tool in rust research and as a possible means for biological control of rust epiphytotics is discussed.     

Inheritance of resistance in smooth bromegrass to the crown rust fungus

Common smooth bromegrass (Bromus inermis Leyss.) is octoploid, 2n = 8x = 56, with a genome structure of AAAAB1B1B2B2. Tetrasomic inheritance patterns have been observed in smooth bromegrass, but disomic inheritance is also expected from cytologic observations. Smooth bromegrass is susceptible to the crown rust fungus (Puccinia coronata Corda.). The objective of this study was to determine the inheritance of smooth bromegrass resistance to P. coronata. Seven smooth bromegrass clones, three susceptible and four resistant, were selfed and crossed in a diallel with bulked reciprocals. Inoculations were made with a population of P. coronata from PL-BDR1 smooth bromegrass. Resistance of smooth bromegrass to this population of P. coronata is complex. At least three genes appear to be involved in this host-pathogen interaction, one tetrasomic dominant gene which determines susceptibility (S) and two dominant genes (R1 and R2) that may be complementary and could be inherited either tetrasomically or disomically. Other genes may be involved in the smooth bromegrass-P. coronata interaction, possibly accounting for the lack of fit to expected ratios of some progeny. Heterogeneity for avirulence phenotype in the pathogen population may also have contributed to lack of fit of some progeny. Multiple resistance genes were detected because a pathogen population, likely consisting of genotypes with different genes for virulence, was used to challenge the host.     

禾顶囊壳4个变种对禾本科牧草的致病性研究

采用人工接种法测定了禾顶囊壳小麦变种、燕麦变种、玉米变种和禾谷变种对4种禾本科牧草鸭茅、披碱草、苇状羊茅和无芒雀麦的致病性。结果表明,除禾顶囊壳玉米变种Ggm01菌株对披碱草没有致病性外,4个变种对供试的其它牧草都有致病性,且能产生全蚀病的典型症状。其中小麦变种和燕麦变种对供试牧草的致病性强于禾谷变种,玉米变种致病性最弱。各变种的菌株存在着致病性分化。小麦变种Ggt9813菌株对苇状羊茅和披碱草的致病性强于燕麦变种,发病严重度均达到50％以上。目前在我国尚未发现燕麦变种,小麦变种主要出现在北方地区,在牧草上的发生亦不广泛,因此二者具有检疫重要性。     

Antifungal activity of three spermidine conjugates

Three tri-substituted spermidines, di-p-coumaroyl-caffeoylspermidine, tri-caffeoylspermidine and tri-p-coumaroylspermidine, isolated from pollen of Quercus alba, were examined for antifungal activity. Both di-p-coumaroyl-caffeoylspermidine and tri-p-coumaroylspermidine reduced mycelial growth of the oat leaf stripe pathogen, Pyrenophora avenae and reduced powdery mildew (Blumeria graminis f. sp. hordei) infection of barley seedlings when applied as a post-inoculation treatment. When used as a pre-inoculation treatment, only di-p-coumaroyl-caffeoylspermidine reduced powdery mildew infection significantly. Growth of P. avenae in the presence of 100 microM di-p-coumaroyl-caffeoylspermidine reduced activity of S-adenosylmethionine decarboxylase (AdoMetDC), and led to a reduction in the incorporation of labelled ornithine into spermidine. The other two spermidine conjugates increased AdoMetDC activity and the flux label from ornithine into spermine in P. avenae significantly.     

Relatedness Among Agronomically Important Rusts Based on Mitochondrial Cytochrome b Gene and Ribosomal ITS Sequences

A fragment of the mitochondrial cytochrome b (cyt b) gene of 13 agronomically important plant pathogenic Basidiomycetes was sequenced, including several Puccinia spp., Uromyces appendiculatus, Phakopsora pachyrhizi, Hemileia vastatrix and Rhizoctonia solani. The deduced amino acid sequences (residues 142–266) were used to study the relatedness of these pathogens as compared to other species of the Basidiomycetes, Ascomycetes and Oomycetes. The relatedness was also studied at nuclear level using the Internal Transcribed Spacers (ITS) in the ribosomal DNA. Phylogenic trees were constructed with the maximum parsimony (MP) and the neighbour‐joining (NJ) methods. On the basis of both cytochrome b and ITS sequences, the Puccinia species pathogenic to graminaceous crop plants, such as Puccinia recondita f. sp. tritici, Puccinia graminis f. sp. tritici, Puccinia striiformis f. sp. tritici, Puccinia coronata f. sp. avenae, Puccinia hordei, P. recondita f. sp. secalis and Puccinia sorghi, together with Puccinia horiana from Chrysanthemum, were very closely related to each other, whereas Puccinia arachidis (from peanut) was closely related to U. appendiculatus (from beans) but more distant from the other Puccinia species. Both rusts on soybean (P. pachyrhizi) and coffee (H. vastatrix) were outside the Puccinia cluster. All rusts were separated from other Basidiomycetes such as R. solani and the strobilurin producing species Strobilurus tenacellus and Mycena galopoda. Our results demonstrate that the amino acid sequence of the mitochondrial cytochrome b is a valid tool to study phylogenic relatedness among plant pathogenic Basidiomycetes and supports taxonomic grouping based on morphological structures and host specificity. Because of their high variability, ITS sequences were able to discriminate Puccinia species, which were identical on the basis of the cytochrome b amino acid sequence. Thus, ITS sequences could better show differences among species or within a species, whereas cytochrome b is more suitable than ITS for phyologenic inference at family or genus level. In addition, the sequence data obtained during this study represent essential information for easy isolation of the cyt b gene and detection of point mutations conferring resistance to Qo Inhibitor fungicides that eventually may evolve.     

Genetic and physical mapping of Lrk10-like receptor kinase sequences in hexaploid oat (Avena sativa L.).

Oat receptor-like kinase gene sequences, homologous to the Lrk10 gene from wheat (Triticum aestivum L.), were mapped in oat (Avena sativa L.). PCR primers designed from the wheat Lrk10 were used to produce ALrk10 from oat. Two DNA sequences, ALrk1A1 and ALrk4A5, were produced from primers designed from coding and noncoding regions of ALrk10. Their use as RFLP probes indicated that the kinase genes mapped to four loci on different hexaploid oat 'Kanota' x 'Ogle' linkage groups (4_12, 5, 6, and 13) and to a fifth locus unlinked to other markers. Three of these linkage groups contain a region homologous to the short arm of chromosome I of wheat and the fourth contains a region homologous to chromosome 3 of wheat. Analysis with several nullisomics of oat indicated that two of the map locations are on satellite chromosomes. RFLP mapping in a 'Dumont' x 'OT328' population indicated that one map location is closely linked to Pg9, a resistance gene to oat stem rust (Puccinia graminis subsp. avenae). Comparative mapping indicates this to be the region of a presumed cluster of crown rust (Puccinia coronata subsp. avenae) and stem rust resistance genes (Pg3, Pg9, Pc44, Pc46, Pc50, Pc68, Pc95, and PcX). The map position of several RGAs located on KO6 and KO3_38 with respect to Lrk10 and storage protein genes are also reported.     

Induction to Resistance to Crown Rust in Oat

Induction of resistance to crown rust, caused by Puccinia coronata Cda. var, avenae Frazer et Led., occurred in seedlings of three genetic lines of oats (Avena sativa L.) inoculated sequentially with one of three inducer-challenger combinations of the pathogen. There was significant reduction in both the number of pustules per leaf and the weight of urediospores harvested in all three host genotypes tested. There were highly significant differences between individual spore harvests correlated with age of the pustules. Development of telia by the challenger was found to occur considerably later, and was less intense, when the challenger was inoculated alone in contrast to treatments in which the challenger followed pre-inoculation with the inducer. There was no correlation between the numbers of resistant and susceptible lesions within the range of inoculum density used in these experiments.     

Apoptotic cell death is a common response to pathogen attack in oats

We have examined the characteristics of cell death induced by pathogen infection in oats with respect to following hallmark apoptotic features: DNA laddering, chromatin condensation, and electron microscopic-terminal deoxynucleotidyl transferase-mediated UTP end labeling positive response. A wide range of plant pathogens representing different levels of parasitism in susceptible and resistant interactions were used for the inocula, which include (i) an obligate parasite, Puccinia coronata f. sp. avenae (the crown rust fungus); (ii) a facultative biotroph parasite, Magnaporthe grisea (the blast fungus); (iii) pathogenic bacteria, Pseudomonas syringae pv. atropurpurea and P. syringae pv. coronafaciens (the halo or stripe blights of oats); and (iv) Ryegrass mottle virus. Surprisingly, any of the pathogens used induced most of the apoptotic features in oat cells at and around the infection sites, indicating that apoptotic cell death is a common phenomenon in oats during pathogen attack. The localization and the timing of apoptotic cell death during a course of infection were, however, quite different depending on the interactions (compatible or incompatible) and the pathogens (fungi, bacteria, or viruses). Possible roles of apoptotic cell death in the susceptible and resistant interactions are discussed.     

Host and environmental effects on the penetration of oats by Puccinia graminis avenae and Puccinia coronata avenae

The frequency of penetration from appressoria of Puccinia graminis avenae and P. coronata avenae varied among Avena species and between oat cultivars, although both rusts produced susceptible infection type pustules on the cultivars tested. Penetration on cv. Garry was significantly less than that on the Avena species (A. barbata, A.fatua and A. sterilis) studied and penetration of these Avena species was significantly less than on the cvs Algerian and Fulmark. When the rusts were allowed to develop into pustules on seedlings which had been inoculated with fixed amounts of inoculum, there was a direct relationship between number of pustules produced and penetration frequency. The effects of temperature, light and dew period on penetration from appressoria of ‘single race’ and ‘mixed race’ inocula was also studied on these cultivars and species. Penetration by P. graminis avenae was greatest at 30–35 °C and at light intensities of 5625 lux and above, whereas that by P. coronata avenae was greatest at 20 °C and was unaffected by artificial light intensities up to n 250 lux. Maximal penetration by P. graminis avenae and P. coronata avenae was observed after inoculated plants had been exposed to dew periods of 16 and 12 h respectively. Some penetration was observed after a dew period of 8 h. The time taken for each rust to attain maximum penetration varied from 36 to 52 h after inoculation, depending on the environment, and was usually less for P. coronata avenae than for P. graminis avenae.     

Resistance gene analogs within an introgressed chromosomal segment derived from Triticum ventricosum that confers resistance to nematode and rust pathogens in wheat

A resistance (R) gene-rich 2S chromosomal segment from Triticum ventricosum contains a cereal cyst nematode (CCN; Heterodera avenae) R gene locus CreX and a closely linked group of genes (Sr38, Yr17, and Lr37) that confer resistance to stem rust (Puccinia graminis f. sp. tritici), stripe rust (P. striiformis f. sp. tritici), and leaf rust (P. recondita f. sp. tritici) when introgressed into wheat. The 2S chromosomal segment from T. ventricosum is further delineated in translocations onto chromosome 2A of bread wheat, where the rust genes are retained but not the CreX gene. Using these critical genetic stocks, we have isolated family members of R gene analogs that are associated with either the 2S segment from T. ventricosum carrying the CreX locus or the rust genes. Derivatives of the Cre3 candidate R gene sequence and a rice (Oryza sativa) R gene analog that mapped to the 2S homologous chromosome groups in wheat were used to isolate related gene sequences from T. ventricosum that contain a nucleotide binding site-leucine rich repeat domain. The potential of these gene sequences as entry points for isolating candidate genes or gene family members of the CreX or rust genes and their further applications to plant breeding are discussed.     

Development and characterization of EST-SSR markers for the crown rust pathogen of ryegrass (Puccinia coronata f.sp. lolii).

The causative organism of crown rust in ryegrasses (Puccinia coronata f.sp. lolii) is an obligate biotroph that causes significant economic losses within the temperate grazing industries of dairy, meat, and wool production. This study reports on the development, transferability, and utility of gene-associated simple sequence repeat (SSR) molecular markers for crown rust. Analysis of 1,100 expressed sequence tag (EST) sequences from a urediniospore-derived cDNA library detected 55 SSR loci. The majority of EST-SSR arrays contained perfect trinucleotide repeats with consistently low repeat numbers, and the motifs (ACC)n and (CAT)n were most commonly represented. DNA extraction from single pustules, in conjunction with multiple displacement amplification, provided the basis for PCR-based screening to evaluate genetic marker performance. An example of the identification of intraspecific genetic diversity was obtained from the analysis of 16 P. coronata isolates originating from the United Kingdom, Australia, New Zealand, and Japan. A subset of 12 robust EST-SSR markers was informative for determination of pathogen diversity within and between these localities. It was also demonstrated that crown rust EST-SSR markers were capable of cross-amplification in closely related fungal taxa (Puccinia spp.) and filamentous fungi within the Ascomycota.     

Incidence of crown rust and virulence of puccinia coronata cda. f.sp. avenae eriks. and the effectiveness of pc genes for resistance in europe,middle east and north africa: Inzidenz von kronenrost und virulenz von puccinia coronata cda. f.sp. avenae eriks. und die wirksamkeit von pc-genen für resistenz in europa,nahost und nordafrika

Crown rust on oat incited by Puccinia coronata Cda f.sp. avenae is a wide-spread disease in Europe, Middle East (Israel) and North Africa (Morocco). High natural levels of the disease were recorded in Austria, Bulgaria, Czech Republic, Estonia, Israel, Italy, Poland, Russia and the former Yugoslavia. The severity of the disease in the European and Mediterranean Oat Disease Nursery (EMODN) between 1995 and 1999 showed that it occurred at damaging levels in a number of countries. Considerable differences in a disease resistance index (DRI) to crown rust among 67 oat lines in the period 1995?–?2000 were found. The values of the DRI ranged from18 (KR 8122) to over 290 (Pc 59, Pc 68). Detailed studies of the virulence combinations of P.coronata f.sp. avenae in Europe, Middle East and North Africa were carried out between 1995?–?2001. There were considerable differences in the average number of virulence among countries. Virulence to Pc 39 and 68 was found for the first time and is a significant finding. Nevertheless the major genes Pc 68, Pc 39, Pc 50-2, Pc Pc 59, Pc 60, Pc 61 proved to be highly effective.     

Quantitative trait loci for partial resistance to crown rust, Puccinia coronata, in cultivated oat, Avena sativa L.

To facilitate the detection of quantitative trait loci (QTLs) for partial resistance to oat crown rust, Puccinia coronata f. sp. avenae Eriks., a genetic map was generated in a population of 158 F(6)-derived oat recombinant inbred lines from a cross of a partial resistance line MN841801-1 by a susceptible cultivar selection 'Noble-2'. The map, developed using 230 marker loci, mostly restriction fragment length polymorphism and amplified fragment length polymorphism markers, spanned 1,509 cM (Haldane) arranged into 30 linkage groups of 2-18 markers each. Four consistently detected major QTLs for partial rust resistance, Prq1a, Prq1b, Prq2, and Prq7, and three minor QTLs, Prq3, Prq5, and Prq6, were found in tests involving three field and two greenhouse environments. In addition, two major QTLs for flowering time, Ftq1 and Ftq7, and five weaker QTLs, Ftq2, Ftq3, Ftq4, Ftq5, and Ftq6, were revealed. Overlapping of the map segments of Ftq1 and Prq1 and of Ftq7 and Prq7 suggested either linkage between the flowering time QTLs and resistance QTLs or a pleiotropic effect of the Ftq QTLs on rust resistance. Relatively low heritability estimates (0.30) obtained for partial resistance to crown rust in the field indicate a potential value for marker-assisted selection.     

Phytoecdysteroids: biological effects,application in agriculture and complementary medicine (as presented at the 14-th Ecdysone Workshop,July, 2000, Rapperswil,Switzerland)

Serratula coronata and Serratula inermis (fam. Asteraceae) have been chosen as the most perspective sources for ecdysteroids from the flora of Ukraine. Ecdysterone was the major component in both species (approximately 2%). The minor components have been identified as alpha-ecdysone, polypodine B, integristerone, pterosterone, monoacetonide and diacetonide of ecdysterone, monoacetonide of ajugasterone C (S. coronata); and viticosterone E, 2-desoxyecdysterone, polypodine B, integristerone, 26-hydroxyecdysterone (S. inermis). Antioxidant activity, antiradical and immunomodulators effects, anabolic, adaptogenic, stimulating, hypocholesterolemic, hepatoprotective actions have been found on the variety of model systems in vitro and in vivo (D-hypovitaminosis, hypercholesterolemia, liver intoxication, increased physical loading). Biochemical mechanisms of these findings have been discussed. A number of preparations-biostumulators have been elaborated on the basis of ecdysteroids from Serratula sp.--for poultry-keeping, agricultural entomology and as food supplements for sick or healthy people.     

Isozyme Variation and Genetic Distances of Erysiphe graminis DC. Formae Speciales

Abstract Isozymes of ten different enzymes and unspecific stained proteins were used as biochemical genetic markers to study genetic variation within and between E. graminis ff. sp. hordei, avenae, secalis and tritici. In addition, grainproteins of the corresponding host species were examined. In each forma specialis, one genotype proved to be predominant. 131 distinct isozyme and 93 protein bands were distinguishable in these genotypes. However, divergent banding patterns differed only in 8 bands from the predominant banding patterns found within the formae speciales avena, secalis and tritici. The genetic relationships between powdery mildew formae speciales and host species were computed by cluster analysis from similarity (F) and dissimilarity (D) coefficients and illustrated by phylogenetic trees. Marked correspondence was found between E. graminis ff. sp. secalis and tritici (F: 82–90%). Lower homologies were obtained from the comparison ofthese formae speciales respectively with E. graminis ff. sp. hordei (F: 28–34%) and avenae (F: 24–32%). All phylogenetic trees constructed revealed the same arrangement classification of the formae speciales with similar graduation. The comparison of the host species revealed the highest similarity between S. cereale and T. aestivum (F: 74%). Regression analysis confirmed significant correlation between the genetic relationships within host species and powdery mildew formae speciales (r²= 0.81).     

Hypersensitive lignification response as the mechanism of non-host resistance of wheat against oat crown rust

Seven-day-old seedlings of the near-isogenic wheat ( Triticum aestivum L.) lines Prelude and Prelude-Sr5, susceptible and resistant to wheat stem rust, respectively, were inoculated with uredospores of the oat crown rust fungus Puccinia coronata Cda. f. sp. avenae Fraser & Led. Fluorescence microscopy revealed that the majority of colonies developed intercellular infection structures including haustorial mother cells and haustoria after penetration of wheat mesophyll cells. All penetrated cells became necrotic, and exhibited bright yellow autofluorescence. This autofluorescence was not extractable with alkali, and fluorescent cells stained positively with phloroglucinol/HCI, suggesting that hypersensitive cell death was correlated with cellular lignification. Accordingly, the lignin biosynthetic enzymes phenylalanine ammonia-lyase (EC4.3.1.5). 4-coumarate:CoA ligase (EC6.2.1.12), cinnamyl-alcohol dehydrogenase (EC1.1.1.149), and peroxidases (EC1.11.1.7) increased in activity during the expression of resistance. The induced pattern of peroxidase iso/ymes closely resembled that observed for highly incompatible wheat/wheat stem rust interactions. Furthermore, an elieitor was extracted from oat crown rust germlings. which induces lignification when injected into the intercellular space of wheat leaves. This elieitor appears to be functionally similar to that isolated from wheat stem rust germlings. The results suggest that the non-host resistance of wheat to the xenopara-site oat crown rust closely resembles the race/cullivar-speeific resistant mechanism of highly resistant wheat varieties to wheat stem rust.     

中国西部柄锈菌新资料

本文报告了我国西部的四种柄锈菌。黑果茜草Rubia cordifolia var. pratensis上的西北柄锈菌Puccinia boreo-occidentalis被描述为新种。垂花报春Primula nutans上的北极柄锈菌P. arctica、车叶草Asperula aparine上的车叶草柄锈菌P. asperulae-aparines和黄水枝Tiarella polyphylla上的黄水枝生柄锈菌P. tiarellicola为中国新记录。根据我国的标本对每个种作了描述和讨论并附有孢子线条图。所有标本保藏在中国科学院菌物标本馆(HMAS)。