M. H. Pei

Identity and host alternation of some willow rusts (Melampsora spp.) in England

Six species of Melampsora: M. salicis-albae, M. larici-pentandrae, M. amygdalinae, M. capraearum, M. ribesii-viminalis and M. epitea , were identified among 26 willow leaf samples collected mainly in SW England. Identification of species was based on the morphology of the uredinial and telial stages as well as on the results of inoculations of the aecial hosts. M. epitea occurred alone in 18 leaf samples, and together with M. ribesii-viminalis in one further sample. Basidiospores produced from teliospores of the autoecious M. amygdalinae infected Salix triandra (a species of tree willows). All other species were heteroecious, with M. larici-pentandrae, M. capraearum and M. epitea producing spermogonia and aecia on Larix decidua (European larch), and less readily on L. kaempferi (Japanese larch). M. ribesii-viminalis and a form of M. epitea from S. purpurea , occasionally formed spermogonia but not aecia on Ribes spp. M. epitea, M. capraearum , and M. ribesii-viminalis occurred on cultivars of shrub willows which are favoured for short-rotation, coppiced energy cropping in the U.K.

Occurrence of Melampsora rusts in biomass willow plantations for renewable energy in the United Kingdom.

The natural occurrence of Melampsora rusts in biomass willow plantations for renewable energy was investigated at four sites in the UK during 1992–1994. Three sites, located at Long Ashton, south west England, Loughgall, Northern Ireland, and Craibstone, Scotland, were rust/clone trials that contained standard 24 willow clones and the fourth site at Markington, northern England, had a similar range of willows. Larch-alternating M. epitea (M. larici-epitea) was found to be most prevalent. Melampsora capraearum was predominant on S. caprea hybrids and the stem-infecting form occurred mainly on S. viminalis ‘Bowles Hybrid’. To identify different pathotypes in larch-alternating M. epitea, field rust samples were tested for pathogenicity in the laboratory using eight willow differentials. A total of 12 pathotypes were identified. More pathotypes were found at Long Ashton, Loughgall and Markington, where extensive, long-term willow plantings were grown, than at Craibstone, where the planting was more isolated. A major breakdown of rust resistance in S. ×mollissima ‘Q83’ in the UK in 1992 was caused by the emergence of a new pathotype. Our results also suggest that the larch-alternating M. epitea found in UK biomass willow plantations may consist of genetically separate populations. This would provide an opportunity to plant willow clones that harbour different mating populations, in mixed plantations in order to enhance the property of mixtures to suppress rust.

AFLP evidence of the distinctive patterns of life-cycle in two forms of Melampsora rust on Salix viminalis

Amplified fragment length polymorphism (AFLP) was used to examine genetic variation in two forms of Melampsora rust on Salix viminalis , the ‘stem-infecting form’ (SIF) and the f. sp. larici-epitea typica (LET) of M. epitea. A simple two-tube method was used to obtain genomic DNA suitable for AFLP. Eleven SIF and 26 LET isolates from the UK were tested using two primer combinations. Of the 215 AFLP markers scored, 93% were polymorphic. AFLP profiles were distinct between SIF and LET (Nei & Lis similarity coefficients between SIF and LET isolates = 22-35%). Within SIF, AFLP patterns were very similar (similarity > 98.9%), indicating that SIF is an asexual population and may have a clonal lineage. Within LET, similarities were > 69%. LET isolates collected from a site at Long Ashton in 1991-1993 were closely related and, therefore, may have come from the same local source. Similarity data from AFLP were in good agreement (Spearmans rank correlation = 0.85) with that of RAPD when both SIF and LET isolates were included, but the correlation was less obvious (= 0.49) when only LET isolates were included. Identical banding patterns were obtained when AFLP was performed using either a Perkin-Elmer 480 or a Perkin-Elmer 9700 Thermocyclers, each programmed with a different PCR profile.

Distinction between stem- and leaf-infecting forms of Melampsora rust on Salix viminalis using RAPD markers

The RAPD technique was used to distinguish two forms of Melampsora rust on Salix viminalis . Fifteen isolates of the stem-infecting form and 19 of the leaf-infecting form collected from the U.K. were tested with 10 arbitrary primers. All the primers generated polymorphic bands and 46 putative loci were examined. Seven primers gave a total of eight bands specific for the stem-infecting form and four primers produced five bands characteristic of the leaf-infecting form. Cluster analysis using Unweighted Pair-Group Method with Averaging (UPGMA) revealed 62% disagreement between the two forms. The RAPD band patterns of the stem- infecting form isolates were almost identical, while those of the leaf-infecting isolates showed up to 29% variation.

A comparative study of stem- and leaf-infecting forms of Melampsora rust on Salix viminalis in the U.K.

A form of Melampsora rust was found to cause stem cankers on Salix viminalis clones in south-west England. Comparative studies suggested that the rust differed from the leaf-infecting form (M. epitea var. epitea f. sp. larici-epitea typica) by causing infection of both young stems and shoot tips, having distinct host specificity, and existing predominantly as the uredinial stage. After incubation for a period at low temperature the rust produced only poorly developed hypophyllous telia similar to those of the leaf-infecting form. In the field, this form of rust overwintered in buds or cankered stems of infected willows. Survey results of willow plantations in the U.K. in 1991 showed that the stem-infecting form occurred in most study sites including Northern Ireland and Scotland. The leaf-infecting form was present in all locations, whereas M. ribesii-viminalis was occasionally found only in south-west England.

Pathogenicity to willow rust, Melampsora epitea, of the mycoparasite Sphaerellopsis filum from different sources

Using leaf disc techniques, nine isolates of the mycoparasite Sphaerellopsis filum were examined for pathogenicity to Melampsora epitea, the most important species of rust in renewable energy willow plantations. Five isolates were derived from Melampsora spp. on willows and poplar, one from Puccinia coronata on couch grass, two from Phragmidium violaceum on blackberry and one from Triphragmiopsis laricinum on larch. Two inoculation experiments were carried out. In the first, S. Filum and rust were applied simultaneously on to leaf discs of Salix burjatica cv. Korso, in the second, leaf discs were inoculated with rust initially, then the resulting rust uredinial pustules were inoculated with S. filum. In both experiments, all S. filum isolates from Melampsora spp., and that from P. coronata, developed pycnidia on willow rust. No pycnidia were produced from the other three isolates. Among isolates which produced pycnidia, the frequency of rust pustules colonized by S. filum and suppression of rust spore production differed markedly. In the first experiment, 55-99% pustules were colonized and rust spore production was suppressed by 64-98%. In the second, the proportions were 33-97% and 53-73%, respectively. Suppression of rust sporulation was closely correlated with the frequency of rust pustules bearing S. filum pycnidia. The results suggest that S. filum is composed of pathogenically specialized populations differing widely in their virulence.

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).

Heterogeneous Nature of a ‘new’ Pathotype of Melampsora Rust on Salix Revealed by AFLP

An outbreak of rust on Salix × mollissima (S. triandra × S. viminalis) ‘Q83’, an important biomass willow, was first observed at several locations in the UK in 1992. Rust collections obtained from ‘Q83’ in 1992 at Long Ashton (south west England), Markington (Northern England) and Loughgall (Northern Ireland), were tested for pathogenicity and examined using amplified fragment length polymorphism (AFLP). All collections showed the same pathogenicity patterns on the eight willow differentials and were assigned to f.sp. larici-epitea typica of Melampsora epitea. A total of 304 AFLP markers was scored for 54 rust isolates, 20 from Long Ashton, 20 from Markington and 14 from Loughgall. Cluster analysis placed the isolates into three distinct groups according to the collection sites. Within each site, Markington isolates were least variable, Nei & Lis similarity coefficients averaging 0.996. Average similarities within isolates from Long Ashton and Loughgall were 0.899 and 0.883, respectively. Average per-locus diversity within site (Hj′), calculated using Shannon information index, was 0.014 in Markington, 0.24 in Long Ashton and 0.23 in Loughgall population. Most diversity (69.1%) was partitioned between populations. An analysis of molecular variance (AMOVA) attributed 85.8% of variance to between populations and 14.2% to the individuals within populations. The results suggest that, in 1992, this previously unknown pathotype was not spread from a common source but from separate sources. The AFLP analysis and early records on the host range of M. epitea indicate that the rust virulent to S. × mollissima may have existed in nature before 1992.

Genetic structure and population dynamics of a heteroecious plant pathogen Melampsora larici‐epitea in short‐rotation coppice willow plantations

Complex life strategies are common among plant pathogens belonging to rust fungi (Uredinales). The heteroecious willow rust Melampsora larici‐epitea produces five spore stages and alternates on larch (Larix). To shed light on the epidemiology of this pathogen, amplified fragment length polymorphisms (AFLPs) were used to determine the genetic diversity and genetic structure of rust samples collected from coppice willow (Salix) plantations at three UK sites (LA, CA and MC) over three sampling dates (September 2000, July 2001 and September 2001). Of the total of 819 isolates, 465 were unique AFLP phenotypes and there was a shift in genotype diversity between the two seasons (0.67 in 2000 and 0.87–0.89 in 2001). No phenotypes were common between the two seasons within a site, suggesting that the rust did not overwinter as an asexual stage within plantations. A temporal analysis detected large amounts of genetic drift (FS = 0.15–0.26) between the two seasons and very small effective population sizes (Ne = 2–3) within sites. These results all point to a new colonization of the plantations by the rust in the second season (2001). The FST‐analogue values were ΦCT = 0.121, Weir and Cockerham’s θ = 0.086 and the Bayesian estimate θB = 0.087–0.096. The results suggest that the sources of inoculum were somewhat localized and the same sources were mainly responsible for disease epidemics in LA and CA over the two seasons. The relatively low FST‐values among sites (0.055–0.13) suggest the existence of significant gene flow among the three sampled sites.

Eudarluca caricis, the teleomorph of the mycoparasite Sphaerellopsis filum, on blackberry rust Phragmidium violaceum

Eudarluca caricis, the teleomorph of Sphaerellopsis filum, was found on blackberry rust (Phragmidium violaceum) in south west England and its morphology is described. Cultures derived from ascospores produced pycnidial conidiomata and conidia typical of S. filum, confirming the connection between the meiotic and mitotic stages of the fungus. Microconidia were also found in the cultures derived from ascospores. Conidiomata developed on the majority of uredinial pustules of P. violaceum 10 d after inoculating detached, uredinia-bearing blackbery leaves with the conidia from the cultures derived from ascospores.