The population genetic structure of the rust fungus Melampsora lini as revealed by pathogenicity, isozyme and RFLP markers

Isozyme and RFLP markers were used to investigate genetic variation underlying the racial structure of populations of Melampsora lini occurring on the Australian endemic host species, Linum marginale . Previous studies of the pathogenicity of isolates occurring in a series of discrete but interconnected populations detected an overall structure characterized by linkage disequilibrium—indicative of a pathogen population reproducing asexually as a number of clonal lines (Burdon & Jarosz, 1992), Using samples from that study, we detected two isozyme multi-locus phenotypes. One of these was restricted to two closely related races which appear to be examples of recent migrants into the area. Variation was also detected among and between races of the pathogen using two RFLP markers. Differences detected by one of these markers suggested the occurrence of a previous migrational event. Variation in the other marker detected two phenotypes within three common, widespread races. However, not all possible phenotypes were recovered, suggesting that the variation detected was not generated by recent sexual recombination.     

Rust resistance in Salix to Melampsora larici-epitea

A total of 174 Salix (willow) clones belonging to 57 species and 14 interspecific hybrids were inoculated with seven pathotypes of Melampsora larici-epitea using the leaf disc method. Infection types were scored based on the uredinial pustule area data and the inoculum density. A close correlation ( R ² = 0·82) was found between the average pustule area and the average number of spores produced. Most of the willows were also assessed in the field for rust in 1999. Most willow clones belonging to the species native to western Europe were infected by the rust. In inoculation experiments, uredinia developed on 46 S. viminalis clones, out of a total of 47. In the field, all the S. viminalis clones were infected by rust. Within the subgenus Vetrix , eight out of the 17 willow species that originated from North and South America produced rust pustules in inoculation experiments. Of these, S. pellita was most susceptible. Salix irrorata and S. lasiolepsis var. bracelinae produced well developed pustules after inoculation but no rust infections were detected in the field. In both leaf disc tests and field assessments, no rust infections were found on S. candida , S. cordata , S. drummondiana , S. eriocephala , S. hookeriana , S. houghtonii , S. humilis , S. rigida var. mackenziana and S. syrticola . Of 12 species of subgenus Vetrix native to northeast Asia and Japan, only S. kochiana was susceptible both in inoculation tests and in the field. Salix rossica produced no symptoms in leaf disc tests but showed low levels of infection in the field. The maximum infection type scores in leaf disc tests were highly significantly correlated with field disease severity scores (Spearman rank correlation coefficient was 0·76, P  < 1 × 10⁻¹⁰).     

Quantitative Inoculations of Poplars with Melampsora Larici-populina

Four poplar clones were inoculated with four isolates of Melampsora larici-populina at seven spore concentrations (inoculum densities up to 680 spores cm^–2 using a leaf-disc method. Disease reactions were recorded using a digital camera. The number and size of uredinia were examined using image analysis software and the number of spores produced per leaf disc was counted. The infection efficiency was estimated in a range of 0.008–0.167 and the pustule diameter measured 0.75–0.94 mm. Rust resistance/susceptibility was expressed by the differences in both the number and the size of uredinia. Within a clone/isolate combination, pustule diameter and the number of spores produced per pustule did not differ significantly between different levels of inoculum density. There was a close correlation between the pustule area and spore yield. When Spearman rank correlation was tested between the disease variables, a close correlation was found between pustule number and pustule area per leaf disc (0.98) and between the number of spores produced and the pustule area/number per leaf disc (0.94 and 0.92, respectively). There was significant correlation between the number and the diameter of pustules (0.54, P < 0.001).     

Pathotypes of Melampsora rust on Salix in short-rotation forestry plantations

Melampsora epitea , the causal agent of leaf rust on willow ( Salix spp.) in short-rotation forestry, was sampled over four consecutive years (1993–96) in Sweden. The pathotype patterns of 332 single-spore isolates were examined for virulence characteristics using a defined set of willow hosts. Thirty-seven pathotypes of M. epitea were identified and grouped into three formae speciales . No race or morph subdivision was observed among the isolates, other than the three formae speciales . For monitoring and studying the ecological and evolutionary dynamics of virulence for M. epitea , an internationally useful naming system for pathotypes is proposed, with a three-digit code referring to virulence on a specified set of standard test clones.     

Occurrence of Melampsora euphorbiae on Euphorbia lagascae in south-east Spain

Melampsora euphorbiae (Schub.) Castagne is reported for the first time on Euphorbia lagascae , a spurge which is a native of south-east Spain and has seed oil rich in vernolic acid. In Spanish trials, rust caused by M. euphorbiae was the major disease of this potential crop.     

Population structure of poplar rust Melampsora larici-populina in the UK inferred from AFLP

AFLP profiles were generated from poplar leaf rust ( Melampsora larici-populina ) collected from Populus trichocarpa cv. Trichobel at six sites in the UK in 2001. Of a total of 145 isolates, 121 were unique AFLP phenotypes. On one occasion, the same AFLP phenotype was found at two sites. The analysis indicated that 81·88% of the total variation was attributable to differences among individuals within replicate plots, 3·47% to differences among plots within sites and 14·65% to differences among sites. Weir & Cockerham's θ was calculated as 0·138 and θ ^B , the Bayesian analogue of F _ST, as 0·146. Overall, there was sufficient gene flow among different sites to prevent genetic drift, as Nm was calculated as 1·562 using θ and as 1·462 using θ ^B . Nei's genetic distance and population-assignment test indicated that the rust from a site in north-east England was differentiated from those from other sites. There was no significant correlation between genetic and geographical distances. The results indicate that sexual reproduction has a major influence on the relative lack of population structure of M. larici-populina in the UK.     

中国落叶松-杨栅锈菌(Melampsora laricipopulina Kleb.)生理小种分化

 90年代末,作者首次对我国秦岭林区落叶松一杨栅锈菌的致病性及生理小种分化进行了研究,并初步将其划分为3个生理小种^[1]。2001~2003年,作者进一步对我国西北、华北、东北和西南的广大杨树种植地区的落叶松一杨栅锈菌致病性分化进行深入研究,证明该锈菌在全国范围内存在明显不同的5个致病类型,并将其划分为5个生理小种。     

Pathogenic specialization in Melampsora epitea var. epitea on Salix

Eighteen isolates of the rust fungus Melampsora from different locations in England were tested for pathogenicity to a large range of willow clones ( Salix spp.) in experiments involving inoculation of leaf discs. Seventeen of the isolates were of leaf-infecting M. epitea var. epitea , 16 of which represented forms which alternated on Larix and one of which alternated on Ribes . The remaining isolate was of uncertain identity. Two experiments were carried out. In the first experiment, 24 willow clones consisting of 20 Salix species and interspecific hybrids were inoculated with eight isolates from clones of S. viminalis . In the other experiment, 77 clones from 57 species or hybrids were inoculated with 10 isolates from several Salix spp. The M. epitea var. epitea isolates from S. viminalis clones were all similarly pathogenic, whilst most of the other isolates expressed distinct host specificity. Eight distinct pathotypes were recognized within M. epitea var. epitea . All except one of these alternated on Larix and could be assigned to three formae speciales which had been reported previously in Europe: four pathotypes to f.sp. larici-epitea typica , two to f.sp. larici-retusae , and one to f.sp. larici-daphnoides . The Ribes -alternating pathotype of M. epitea var. epitea infected only S. purpurea . Nine differential willow hosts are proposed as reference clones to distinguish between the larch-alternating pathotypes, the Ribes -alternating rust and the 'stem-infecting' form. The extent of pathogenic variation encountered amongst sexually reproducing rusts suggests that more pathotypes probably exist and will arise in future in response to selection given by long-term clonal willow plantings.     

Histological Aspects of a Hypersensitive Response in Poplar to Melampsora larici-populina

ABSTRACT The course of the infection and development of the biotrophic fungus Melampsora larici-populina on leaf tissue from the hybrid poplar Populus deltoides x P. nigra 'Ogy' was monitored at the histological level. Leaf disks were inoculated with one of two rust physiological races (E1 and E2), resulting in interactions that were either incompatible (race E1) or compatible (race E2). In the compatible interaction, the fungus rapidly colonized the leaf without inducing any apparent host response. Symptoms appeared on the leaf several days after inoculation just prior to spore dissemination. The incompatible interaction was characterized by the early collapse and disorganization of cytoplasm of infected cells 17 h after inoculation and within 2 h after the appearance of the first haustoria. Resistance to M. larici-populina was mediated through a hypersensitive response, since it was extremely localized and involved only the few cells that were in the immediate vicinity of each infected cell.     

Genetic Structure of Melampsora Epitea Populations in Swedish Salix Viminalis Plantations

Amplified fragment length polymorphism (AFLP) was used to study the genetic structure of populations of the willow leaf rust, Melampsora epitea, in Swedish willow plantations. In total, 197 isolates collected from Salix viminalis clones in three locations in Sweden were analysed. AFLP profiles based on 83 markers were used to compute genetic distances between pairs of individuals. High levels of gene and genotypic diversity were detected in all populations, with 96% of the AFLP loci being polymorphic and with normalized Shannon's diversity indices ranging from 0.977 to 1.0. Analysis of molecular variance (AMOVA) showed small significant differences among locations, although most of the molecular variability was found within locations (97.5%). Five isolates from one willow clone in one location differed markedly from the common pattern. When these five exceptional isolates were excluded, no significant differences among willow clones were found with AMOVA. Sexual reproduction and spore migration appear to be important factors for the population genetic structure of this pathogen.     

Abundant Pathogenic Variation in the New Hybrid Rust Melampsora xcolumbiana on Hybrid Poplar

ABSTRACT The recently described rust hybrid Melampsora xcolumbiana was discovered as a result of its novel pathogenic variation on Populus trichocarpa x P. deltoides (TxD) hybrid poplar. To characterize this pathogenic variation, 10 commercial TxD clones, all F(1) clones, were chosen as host differentials. Fourteen mononuredinial isolates of Pacific Northwestern field collections of M. xcolumbiana, from 1996 to 1998 inclusive, were determined to be 13 distinct pathotypes. In contrast, four Southeastern isolates of M. medusae could not be distinguished on the same TxD host differentials, although they can be distinguished as pathotypes using P. deltoides differentials. The first three pathotypes of M. xcolumbiana (Mxc1, Mxc2, and Mxc3) and a Mississippi isolate of M. medusae were inoculated onto a three-generation TxD pedigree, formerly used to characterize the Mmd1 gene for resistance to M. medusae. Resistance to the Mxc3 pathotype mapped to the same linkage group (group Q) as the Mmd1 gene. In contrast, linked genes for resistance to Mxc1 and to Mxc2 were located on linkage group O, and were unlike Mmd1 and Mxc3 in that they were inherited from P. deltoides. The latter two genes resembled Mmd1 and Mxc3 in that infection type was correlated with quantitative traits such as uredinial density and latent period. Pathogenic variation in M. xcolumbiana matches resistance genes from both P. trichocarpa and P. deltoides and reveals the vulnerability to hybrid rust of commercial TxD hybrid poplar clones.     

Genetic Variation in Melampsora Larici-epitea on Biomass willows Assessed using AFLP

Five pathotypes belonging to formae speciales larici-epitea typica (LET), larici-retusae (LR) and larici-daphnoides (LD) of Melampsora larici-epitea were examined using amplified fragment length polymorphism (AFLP). Of 213 AFLP markers scored, several were found to be exclusive to different formae speciales. The dendrogram placed the five pathotypes into distinct groups. Within pathotypes, average Nei & Li's similarity coefficients were calculated as 0.71–0.85. The similarities were 0.66–0.72 among the three pathotypes within LET and 0.34–0.44 between pathotypes belonging to different formae speciales. When assessed using the Shannon index, the diversity within locations was estimated as 0.55–0.59, greater than that found within pathotypes (0.24–0.42). The average per-locus diversity was 0.37 among the pathotypes and 0.11 among the locations. When the data from both LET and LR isolates were examined using AMOVA, the majority of the variation (70.85%) was attributed to among pathotypes within location. When only LET types were included, approximately half of the variation was partitioned to among pathotypes within location and the other half to among the isolates within collection. It appears that the degree of differentiation of LET4 on S. × mollissima between Loughgall and Long Ashton sites has decreased markedly since 1992, when it was first detected.     

Structure of Melampsora larici-populina populations on wild and cultivated poplar

Populations of Melampsora larici-populina, causal agent of poplar leaf rust, have been collected during 1992–1994 in three types of sites: natural stands of Populus nigra in the south-east of France; cultivated poplar stands in the northern half of France, including mainly interspecific and exotic hybrids of poplar; and two overseas populations collected from Northern Ireland and Western USA. The pathotype of each rust isolate was determined by inoculating a differential set of poplar clones. The number of virulences per isolate and the frequency of each virulence in each population were compared, according to the sites and to the clones from which the isolates were collected.Several diversity indices were applied to the populations. The and Simpson indices were retained since they described best the richness and the evenness, respectively, of the populations, and they were the least sensitive to sample size. The complexity of races was lower in the natural stands of P. nigra than in the cultivated poplar stands. Populations of M. larici-populina collected on Populus × euramericana Robusta and P. nigra italica (Lombardy poplar) presented a high richness and evenness, confirming the value of these clones for describing race populations. Populations from Western USA presented a very low diversity, which is in accordance with the recent introduction of the pathogen in North America. These results suggest that the race populations of M. larici-populina are mainly influenced by the structure of the host populations.     

Genetic Diversity in Relation to Sexual and Asexual Reproduction in Populations of Melampsora larici-epitea

Genetic diversity of Melampsora larici-epitea leaf rust from three cultivated stands of the willow Salix viminalis was studied using AFLP polymorphisms at 60 loci. One population was located in Northern Ireland and two in Sweden. Analysis of molecular variance (AMOVA) showed that most of the genetic variation was distributed on a fine scale within the field in all populations. Both Swedish populations displayed a very high genotypic diversity (normalized Shannon's indices of 0.95 and 1.00) and random association among loci. These results suggested that sexual reproduction had an important influence on the Swedish populations. The occurence of the alternate host (larch) adjacent to one of the Swedish rust populations did not affect the genetic diversity. However, severe rust attacks started earlier in the season in this population. The M. larici-epitea population in Northern Ireland was characterized by a low genotypic diversity (normalized Shannon's index = 0.54) and non-random association among loci was indicated by test of multilocus association and by pairwise tests among loci. These results suggested that asexual reproduction had a major effect on the genetic structure of this population, probably because of the overwintering of asexual spores and/or a population bottleneck associated with the annual sexual phase.     

Quantitative relationships between inoculum of Melampsora larici-epitea and corresponding disease on Salix

Six Salix clones were inoculated with urediniospores of four isolates of Melampsora larici-epitea at five inoculum levels using a leaf-disc method. Disease reactions were recorded using a digital camera; the number and size of uredinia were examined using image analysis software; and spore yield per leaf disc was measured. In three Salix / Melampsora combinations, S.  ×  mollissima 'Q83'/Q1 (LET4); S. viminalis '78183'/V1 (LET1); and S.  ×  stipularis /V1, pustule numbers increased as inoculum density became higher. In the remainder, S. viminalis 'Mullatin'/V1; S.  ×  calodendron /DB (LET3); and S. burjatica 'Korso'/K (LR1), pustule numbers initially increased, then decreased as inoculum densities exceeded 140–360 spores per disc. Calculated infection efficiency ranged from 0·11 to 0·20 on the three willows inoculated with V1: 0·16–0·68 for S.  ×  calodendron /DB; 0·20–0·55 for 'Q83'/Q1; and 0·07–0·48 for Korso/K. In single-spore inoculations, up to 10% of spores produced single uredinia. Infection efficiency increased sharply between inoculum densities of 1–40 spores per leaf disc. Spore yield was more closely correlated to pustule area (accounting for 61·2% variance for the combined data) than to the number of pustules (42·7% variance). For spore yields in relation to pustule numbers, clone-specific individual lines having different intercepts and slopes fitted significantly better than either a single line for all the tested willows, or parallel lines fitted to each clone ( P  < 0·001). For spore yields in relation to pustule area, clone-specific individual parallel lines were significantly better than a single line ( P  < 0·001).     

Reassessment of rust fungi on weeping willows in the Americas and description of Melampsora ferrinii sp. nov.

Rust fungi in the genus Melampsora usually cause disease on hosts in the Salicaceae. Identification of Melampsora species is often complicated due to few differences in spore morphology and little publicly available comparative sequence data. Weeping willow trees (primarily Salix babylonica and its hybrids) have been reported to be infected by 11 Melampsora species; however, most of these records are based on morphological characterization. New collections of rust fungi on weeping willows from the central USA were analysed using a combination of morphology, ITS and LSU rDNA sequencing, and host data to determine that they represent an undescribed rust fungus, Melampsora ferrinii sp. nov. Additional studies of herbarium material revealed that M. ferrinii has occasionally been collected but identified as M. epitea. In addition to North America, M. ferrinii is also present in South America and has been infecting weeping willows there since at least the 1990s.     

Simultaneous monitoring and quantification of Melampsora allii‐populina and Melampsora larici‐populina on infected poplar leaves using a duplex real‐time PCR assay

The rust fungi Melampsora larici‐populina (Mlp) and Melampsora allii‐populina (Map) are the main phytosanitary constraints for commercial poplar cultivation in Europe. Although Mlp is more aggressive and prevalent than Map, the two species may co‐infect the same poplar tree or even the same poplar leaf, making the epidemiological surveys of each species difficult to achieve. In this study, a new duplex real‐time PCR assay targeting each species was developed, based on single‐copy genes. This test proved to be specific, inclusive and was successfully used to detect and quantify each species, starting from urediniospore samples or directly from infected poplar leaves, with or without visible symptoms. This new molecular tool was also assessed for comparative studies of time‐course infection experiments on artificially inoculated poplar leaf discs. These studies showed that the growth dynamics of Map were significantly slower when the two species were co‐inoculated on the same leaf disc, confirming that Map is less aggressive than Mlp.     

Bulked Segregant Analysis Identifies Molecular Markers Linked to Melampsora medusae Resistance in Populus deltoides

ABSTRACT In the north central United States, leaf rust caused by Melampsora medusae is a major disease problem on Populus deltoides. In this study we identified molecular markers linked to a M. medusae resistance locus (Lrd1) that was segregating 1:1 within an intraspecific P. deltoides family (C9425DD). Previous field results were confirmed in the controlled environment of a growth chamber through an excised whole-leaf inoculation method. Using bulked segregant analysis we identified two random amplified polymorphic DNA (RAPD) markers (OPG10(340) and OPZ19(1800)) that are linked to Lrd1. Based on segregation in a total of 116 progeny, the genetic distances between OPG10(340) and OPZ19(1800) and the resistance locus were estimated as 2.6 and 7.4 Haldane centimorgans (cM), respectively. Multipoint linkage analyses strongly suggest the most likely order for these loci is Lrd1, OPG10(340), and OPZ19(1800). These markers may prove to be instrumental in the eventual cloning of Lrd1, as well as for marker-assisted selection of leaf-rust resistant genotypes.     

胡杨和灰叶胡杨锈菌夏孢子形态和侵染结构观察

 借助直接观察、显微和超微结构观测等手段,对胡杨和灰叶胡杨锈病显症叶片、锈菌夏孢子的形态、孢子萌发和侵染结构进行了观测。结果表明,胡杨和灰叶胡杨的锈菌夏孢子堆、夏孢子形态结构相似;夏孢子堆散生于叶片正反两面,近半球形或半椭球形,呈橙黄色或金黄色;夏孢子散落分布于叶片正反两面,圆形或卵圆形,透明、橙黄色,厚壁,单胞,表面密生刺瘤;胡杨和灰叶胡杨锈菌夏孢子的长轴和短轴长度、刺瘤高度及刺瘤间距四项指标均无显著性差异;夏孢子萌发产生芽管后形成菌丝,通过叶表气孔直接侵染叶片,以菌丝的形式在叶片组织内扩展。