Complex life strategies are common among plant pathogens belonging to rust fungi (Uredinales). Th... more 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 (F(S) = 0.15-0.26) between the two seasons and very small effective population sizes (N(e) = 2-3) within sites. These results all point to a new colonization of the plantations by the rust in the second season (2001). The F(ST)-analogue values were Phi(CT) = 0.121, Weir and Cockerham's theta = 0.086 and the Bayesian estimate theta(B) = 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 F(ST)-values among sites (0.055-0.13) suggest the existence of significant gene flow among the three sampled sites.
The 5' end of the large subunit (... more The 5' end of the large subunit (LSU) region and the entire internal transcribed spacer (ITS) region of ribosomal DNA were sequenced from 11 species or special forms of Melampsora on Salix, and three species on Populus. For all the species, except for M. larici-epitea and M. coleosporioides, the sequences in both the examined regions were identical within a species. Within M. larici-epitea, f. sp. larici-epitea typica and f. sp. larici-retusae shared the same sequences which slightly differed from that of f. sp. larici-daphnoides. In the LSU region, M. larici-epitea, M. capraearum and the stem-infecting form on S. viminalis shared the same sequence and the Far-Eastern species M. epiphylla differed from them only slightly (p distance 0.006), indicating that they may share a common ancestral lineage. M. amygdalinae and M. coleosporioides formed a distict group (bootstrap value 100% for combined ITS and LSU data). M. larici-epitea and M. ribesii-purpurea, both belonging to the M. epitea complex, appeared to be distinct. The molecular data also suggest that the differences in certain characteristics, such as the thickness of teliospore walls and host specificity, may have evolved relatively recently.
Genetic diversity among 51 isolates of the mycoparasite Sphaerellopsis filum from Melampsora rust... more Genetic diversity among 51 isolates of the mycoparasite Sphaerellopsis filum from Melampsora rusts on willow and poplar was examined using AFLP. Genetic variation was relatively low among the isolates (Nei & Li's similarities > or =90). Genetic diversity calculated using Shannon index was 0.119 at Loughgall, Northern Ireland, 0.109 at Markington, northern England, 0.039 at Craibstone, Scotland, and 0.015 at Long Ashton, southwest England. At Long Ashton, 14 out of 16 isolates shared the same AFLP bands. Two genotypes were found at both Markington and Loughgall. The low genetic diversity and a high rate of clonality suggested that asexual reproduction plays a major role in S. filum epidemics. Sequence information was also obtained from the ITS-5.8S region of the ribosomal DNA from the S. filum isolates derived from willow and poplar rusts and six isolates derived from other sources. ITS sequences were identical among all the 51 isolates from willow and poplar rusts. ITS analysis placed S. filum isolates from Melampsora spp. on willow and poplar, Puccinia coronata on grass and Melampsora sp. on Euphorbia sp. into one clade and the isolates from blackberry rust Phragmidium violaceum and larch rust Triphragmiopsis laricinum into another. Nucleotide sequence differences between the two groups were 8.4-10.4 %. The ITS-5.8S sequences obtained in this study were compared with those deposited in the GenBank database.
The mycoparasite Sphaerellopsis filum (teleomorph Eudarluca caricis) was applied simultaneously w... more The mycoparasite Sphaerellopsis filum (teleomorph Eudarluca caricis) was applied simultaneously with Melampsora larici-epitea on to willow leaf discs using eight concentrations of conidia. Inoculum densities were quantified and the numbers of uredinia of the rust, pycnidia and conidia of S. filum and rust spores produced per leaf disc were measured 13 d after inoculation (first assessment). Higher S. filum inoculum densities resulted in more rust uredinia being infected, but did not reduce the number of uredinia produced. The ratios of infected rust pustules: S. filum conidia applied were in a range of 0.25-0.31 when less than 20 S. filum spores were inoculated on to a leaf disc (0.95 cm2). Suppressive effects of S. filum on rust spore production were more obvious in the second assessment, carried out 23 d after inoculation. Inoculum densities of S. filum were significantly (P < 0.001) correlated with the frequency of uredinia infected (% variance accounted for [VAF] = 85.8), the number of S. filum pycnidia (% VAF = 81.4), S. filum spores produced (% VAF = 72.3) and rust spore production (% VAF = 48.6). Rust spore production was significantly (P < 0.001) negatively correlated with the frequency of uredinia infected (% VAF = 51.1), the number of S. filum pycnidia (% VAF = 42.0) and the number of S. filum spores produced (% VAF = 40.6). The best correlation was found between the number of pycnidia and the number of S. filum spores produced (% VAF = 88.8).
Using a leaf disc method, 19 isolates of the poplar rust, Melampsoralarici-populina , and one iso... more Using a leaf disc method, 19 isolates of the poplar rust, Melampsoralarici-populina , and one isolate of M.?populnea from England were inoculated on to 25 poplar clones belonging to Populus nigra and P.?trichocarpa, and hybrids between P. deltoides and P. nigra, P. deltoidesand P. trichocarpa, P.?tacamahaca and P.?trichocarpa, and P. alba and P. tremula. Disease was scored based on the pustule area and inoculum density. In terms of whether sporulating uredinia formed, the 19 isolates showed seven different patterns to the tested poplar clones. The majority of the rust isolates infected P. nigra ‘P3090’ and ‘Vereecken’, P.?nigra?×?P. deltoides ‘Casale’ and ‘Tasman’, P. tacamahaca?×?trichocarpa ‘36’ and ‘Balsam Spire’, and P.?trichocarpa ‘Blom’. Populus trichocarpa?×?P. deltoides ‘69039/4’ was infected by only three isolates collected from southern England. No visible symptoms appeared on P. alba ?×?P. tremula‘Tower’ and P.?trichcarpa?×?P. deltoides?×?P. deltoides‘76028/5’ in inoculations with M. larici-populina isolates. Populus alba?×?P.?tremula ‘Tower’ was infected only by M. populnea. When M.larici-populina isolates were tested using AFLP, no differences were found either between isolates from different geographical regions or between those having ‘narrow’ spectrum of virulence and those showing ‘wide’ spectrum of virulence on the tested clones. The results suggest that the UK rust populations possess virulences which were found in races E1, E2, E3 and E4 in continental Europe and that rust having virulence patterns similar to race E4 has occurred in UK poplar plantations since 1996.
Rust resistance of 23 commonly grown biomass willow varieties and 71 genotypes produced from a UK... more Rust resistance of 23 commonly grown biomass willow varieties and 71 genotypes produced from a UK-based willow-breeding programme was tested in laboratory inoculation experiments. Leaf discs were inoculated with six pathotypes of Melampsora larici-epitea and disease was scored based on uredinial pustule area and inoculum density. In the field, rust severity of the same varieties and genotypes was scored using an assessment key. Of the varieties and commonly grown willows tested, several North American willows and Salix schwerinii×Salix viminalis ‘Bjorn’ and ‘Tora’ were highly resistant. Overall, the great majority of the willows produced through breeding showed high levels of resistance to rust. There was a significant correlation between infection-type scores obtained from the laboratory testing and field rust severity (Spearman's rank correlation coefficient=0.624, P<1×10−6). There was also a good agreement in field disease severity scores between the years (Kendall's coefficient of concordance=0.669, P<0.001).
Complex life strategies are common among plant pathogens belonging to rust fungi (Uredinales). Th... more 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 (F(S) = 0.15-0.26) between the two seasons and very small effective population sizes (N(e) = 2-3) within sites. These results all point to a new colonization of the plantations by the rust in the second season (2001). The F(ST)-analogue values were Phi(CT) = 0.121, Weir and Cockerham&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s theta = 0.086 and the Bayesian estimate theta(B) = 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 F(ST)-values among sites (0.055-0.13) suggest the existence of significant gene flow among the three sampled sites.
The 5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; end of the large subunit (... more The 5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; end of the large subunit (LSU) region and the entire internal transcribed spacer (ITS) region of ribosomal DNA were sequenced from 11 species or special forms of Melampsora on Salix, and three species on Populus. For all the species, except for M. larici-epitea and M. coleosporioides, the sequences in both the examined regions were identical within a species. Within M. larici-epitea, f. sp. larici-epitea typica and f. sp. larici-retusae shared the same sequences which slightly differed from that of f. sp. larici-daphnoides. In the LSU region, M. larici-epitea, M. capraearum and the stem-infecting form on S. viminalis shared the same sequence and the Far-Eastern species M. epiphylla differed from them only slightly (p distance 0.006), indicating that they may share a common ancestral lineage. M. amygdalinae and M. coleosporioides formed a distict group (bootstrap value 100% for combined ITS and LSU data). M. larici-epitea and M. ribesii-purpurea, both belonging to the M. epitea complex, appeared to be distinct. The molecular data also suggest that the differences in certain characteristics, such as the thickness of teliospore walls and host specificity, may have evolved relatively recently.
Genetic diversity among 51 isolates of the mycoparasite Sphaerellopsis filum from Melampsora rust... more Genetic diversity among 51 isolates of the mycoparasite Sphaerellopsis filum from Melampsora rusts on willow and poplar was examined using AFLP. Genetic variation was relatively low among the isolates (Nei &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp; Li&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s similarities &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; or =90). Genetic diversity calculated using Shannon index was 0.119 at Loughgall, Northern Ireland, 0.109 at Markington, northern England, 0.039 at Craibstone, Scotland, and 0.015 at Long Ashton, southwest England. At Long Ashton, 14 out of 16 isolates shared the same AFLP bands. Two genotypes were found at both Markington and Loughgall. The low genetic diversity and a high rate of clonality suggested that asexual reproduction plays a major role in S. filum epidemics. Sequence information was also obtained from the ITS-5.8S region of the ribosomal DNA from the S. filum isolates derived from willow and poplar rusts and six isolates derived from other sources. ITS sequences were identical among all the 51 isolates from willow and poplar rusts. ITS analysis placed S. filum isolates from Melampsora spp. on willow and poplar, Puccinia coronata on grass and Melampsora sp. on Euphorbia sp. into one clade and the isolates from blackberry rust Phragmidium violaceum and larch rust Triphragmiopsis laricinum into another. Nucleotide sequence differences between the two groups were 8.4-10.4 %. The ITS-5.8S sequences obtained in this study were compared with those deposited in the GenBank database.
The mycoparasite Sphaerellopsis filum (teleomorph Eudarluca caricis) was applied simultaneously w... more The mycoparasite Sphaerellopsis filum (teleomorph Eudarluca caricis) was applied simultaneously with Melampsora larici-epitea on to willow leaf discs using eight concentrations of conidia. Inoculum densities were quantified and the numbers of uredinia of the rust, pycnidia and conidia of S. filum and rust spores produced per leaf disc were measured 13 d after inoculation (first assessment). Higher S. filum inoculum densities resulted in more rust uredinia being infected, but did not reduce the number of uredinia produced. The ratios of infected rust pustules: S. filum conidia applied were in a range of 0.25-0.31 when less than 20 S. filum spores were inoculated on to a leaf disc (0.95 cm2). Suppressive effects of S. filum on rust spore production were more obvious in the second assessment, carried out 23 d after inoculation. Inoculum densities of S. filum were significantly (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.001) correlated with the frequency of uredinia infected (% variance accounted for [VAF] = 85.8), the number of S. filum pycnidia (% VAF = 81.4), S. filum spores produced (% VAF = 72.3) and rust spore production (% VAF = 48.6). Rust spore production was significantly (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.001) negatively correlated with the frequency of uredinia infected (% VAF = 51.1), the number of S. filum pycnidia (% VAF = 42.0) and the number of S. filum spores produced (% VAF = 40.6). The best correlation was found between the number of pycnidia and the number of S. filum spores produced (% VAF = 88.8).
Using a leaf disc method, 19 isolates of the poplar rust, Melampsoralarici-populina , and one iso... more Using a leaf disc method, 19 isolates of the poplar rust, Melampsoralarici-populina , and one isolate of M.?populnea from England were inoculated on to 25 poplar clones belonging to Populus nigra and P.?trichocarpa, and hybrids between P. deltoides and P. nigra, P. deltoidesand P. trichocarpa, P.?tacamahaca and P.?trichocarpa, and P. alba and P. tremula. Disease was scored based on the pustule area and inoculum density. In terms of whether sporulating uredinia formed, the 19 isolates showed seven different patterns to the tested poplar clones. The majority of the rust isolates infected P. nigra ‘P3090’ and ‘Vereecken’, P.?nigra?×?P. deltoides ‘Casale’ and ‘Tasman’, P. tacamahaca?×?trichocarpa ‘36’ and ‘Balsam Spire’, and P.?trichocarpa ‘Blom’. Populus trichocarpa?×?P. deltoides ‘69039/4’ was infected by only three isolates collected from southern England. No visible symptoms appeared on P. alba ?×?P. tremula‘Tower’ and P.?trichcarpa?×?P. deltoides?×?P. deltoides‘76028/5’ in inoculations with M. larici-populina isolates. Populus alba?×?P.?tremula ‘Tower’ was infected only by M. populnea. When M.larici-populina isolates were tested using AFLP, no differences were found either between isolates from different geographical regions or between those having ‘narrow’ spectrum of virulence and those showing ‘wide’ spectrum of virulence on the tested clones. The results suggest that the UK rust populations possess virulences which were found in races E1, E2, E3 and E4 in continental Europe and that rust having virulence patterns similar to race E4 has occurred in UK poplar plantations since 1996.
Rust resistance of 23 commonly grown biomass willow varieties and 71 genotypes produced from a UK... more Rust resistance of 23 commonly grown biomass willow varieties and 71 genotypes produced from a UK-based willow-breeding programme was tested in laboratory inoculation experiments. Leaf discs were inoculated with six pathotypes of Melampsora larici-epitea and disease was scored based on uredinial pustule area and inoculum density. In the field, rust severity of the same varieties and genotypes was scored using an assessment key. Of the varieties and commonly grown willows tested, several North American willows and Salix schwerinii×Salix viminalis ‘Bjorn’ and ‘Tora’ were highly resistant. Overall, the great majority of the willows produced through breeding showed high levels of resistance to rust. There was a significant correlation between infection-type scores obtained from the laboratory testing and field rust severity (Spearman's rank correlation coefficient=0.624, P<1×10−6). There was also a good agreement in field disease severity scores between the years (Kendall's coefficient of concordance=0.669, P<0.001).
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