Development of Melampsora ciliata rust on nursery‐grown poplars in north‐western Himalayas

Development of leaf rust (Melampsora ciliata) in different species, hybrids and cultivars of poplar (Populus spp.) was studied in nursery‐grown plants. Five different criteria were used to assess the disease development. The mean disease index on 10 August was 9.4% which increased to 70.0% on 10 October. The lowest disease index (5.5%) was recorded in P. yunnanensis whereas the maximum was recorded in P. × euramericana‘Rubra‐Poiret’ (59.5%). The apparent infection rate per unit per day was highest in P. nigra × P. trichocarpa (9.7 × 10^–2) whereas the minimum occurred in P. maximowiczii × P. berolinensis‘Oxford’ (3.3 × 10^–2). The area under the disease progress curve was a maximum in P. × euramericana‘Rubra‐Poiret’ (26.7) and a minimum in P. yunnanensis (1.6). Complete defoliation by the first half of October occurred in P. nigra × P. trichocarpa, which also had the maximum apparent infection rate and area under the disease progress curve. Inoculum production was highest in P. nigra × P. trichocarpa and lowest in P. maximowiczii × P. berolinensis‘Oxford’.     

Variation in leaf phenolics of field‐cultivated willow (Salix myrsinifolia) clones in relation to occurrence of Melampsora rust

Concentrations of potential antifungal phenolics in field‐cultivated willow (Salix myrsinifolia) clones were analysed during three growing seasons, and correlated to the occurrence of Melampsora rust. Consistent relationships between phenolics and rust were not found across the experimental years. There was significant clonal and temporal variation in phenolic content and rust frequency. Levels of some phenolics varied considerably within a sequence of four full‐grown leaves, but the variation in rust occurrence within the same leaf sequence was nonsignificant. The results suggest that the possible association between willow phenolics and rusts is not straightforward, and emphasize the importance of long‐term studies to investigate the chemical basis of willow rust interactions.     

Effect of Melampsora rust on the growth and development of Salix burjatica‘Korso’in Northern Ireland

The impact of Melampsora rust on the growth of Salix burjatica‘Korso’was assessed. Comparisons were made between untreated plants and those where the disease had been partially controlled using fungicides. Fungicides delayed the onset of the disease and its effects on growth. Leaf loss was reduced whilst shoot growth and dry matter yields increased. The effect of secondary organisms was also reduced resulting in lower levels of shoot death in the following season.     

Direct genotyping of the poplar leaf rust fungus,Melampsora medusae f. sp. deltoidae,using codominant PCR‐SSCP markers

Two anonymous DNA markers that are revealed by single‐strand conformational polymorphism (SSCP) analysis were developed for detection of polymorphisms in Melampsora medusae f. sp. deltoidae (Mmd). Mono‐uredinial isolates of Mmd were first obtained, DNA was extracted from urediniospores and random amplified polymorphic DNA (RAPD) products of eight mono‐uredinial isolates were separated on a SSCP gel to identify differences among them. Bands representing putative polymorphic loci among the eight isolates tested were excised from the SSCP gel and re‐amplified by polymerase chain reaction (PCR), and then cloned and sequenced. A primer pair was designed to amplify a DNA fragment of a size suitable for SSCP analysis (<600 bp) for two out of three DNA fragments sequenced. Each set of primers amplified a PCR product for all eight isolates that were initially used to generate them and the resulting PCR products were analysed by SSCP. Polymorphisms among isolates were identified for both putative loci. The two primer pairs amplified a PCR product of the expected size on an additional 32 mono‐uredinial isolates of Mmd tested. From the overall 40 mono‐uredinial isolates tested, 5 and 11 alleles were detected, and 12 and 34 isolates showed to be heterozygous, as indicated by the presence of more than two bands on the SSCP gel, at loci A and B, respectively. The primer pairs were tested for specificity against 106 fungal isolates belonging to various taxa, including other rusts, and against DNA extracted from greenhouse‐grown healthy poplar leaves. DNA amplification products of the expected size were obtained only when Mmd DNA was present. Optimization of PCR conditions with these two primer pairs allowed genotyping directly from single uredinia extracted from infected leaves, thus alleviating the need to culture the fungus to characterize individuals, hence making it possible to process large numbers of samples for population studies.     

The influence of pine twist rust (Melampsora pinitorqua) on growth and development of Scots pine (Pinus sylvestris)

The influence of pine twist must (Alelampsora pinitorqsat) on growth and development of Scots pine was studied during three consecutive years. The experimental material included three provenances from natural stands and one open pollinated seed orchrd progeny. Trees severely affected by pine twist must reached an average of 38% lower height than did not-affected trees. while mildly affected trees grew somewhat better, on the average, than did non-affected trees. Pine twist must caused tha greatest loss of height increment in the seed orchard progeny.     

Rust resistance in Salix induced by inoculations with avirulent and virulent isolates of Melampsora larici‐epitea

Six Salix clones and four Melampsora larici‐epitea isolates were used in two leaf‐disc inoculation experiments to determine whether disease severity was affected by the presence of both virulent and avirulent rust genotypes. In the first experiment, an equal amount of urediniospores of a virulent isolate and an avirulent isolate was applied simultaneously using four levels of spore suspension. In the second, the willows were inoculated with one isolate first and then, after 3 days, with another using two spore concentrations. In the first experiment, overall rust spore production was reduced by 48.6% in inoculations with mixed inocula compared with that in the inoculations with single virulent isolates. In the second experiment, 20 of the 36 combinations involving pre‐inoculations with avirulent isolates showed significant reduction in spore production. The suppressive effects on rust sporulation were more obvious at the higher inoculum densities and on the clones S. × calodendron and S. × mollissima‘Q83’.     

Genetic relationships among genetically distinct forms of Melampsora larici‐epitea and related species based on AFLP data

Amplified Fragment Length Polymorphism (AFLP) was used to study genetic relationship among leaf rust fungi (Melampsora spp.) occurring on Salix species in short‐rotation coppice. Special interest was paid to a newly identified rust fungus found on S. viminalis in southern Sweden, morphologically similar to M. larici‐epitea but with a distinct DNA profile. Genetic distances among 40 Melampsora isolates collected from S. viminalis, S. dasyclados, S. viminalis x dasyclados, S. daphnoides, S. acutifolia and M. larici‐populina were calculated based on 101 AFLP markers. Neighbour‐joining analysis revealed the presence of six clusters, which corresponded exactly to predefined groups, namely three formae speciales of M. larici‐epitea, a stem‐infecting form of Melampsora on S. viminalis, the newly identified Melampsora on S. viminalis and a group consisting of two isolates from the poplar rust M. larici‐populina. All six clusters were well supported by bootstrap analysis (84 to 100% support). The newly identified Melampsora on S. viminalis was indicated to be genetically separated from M. larici‐epitea as well as from the stem‐infecting form of Melampsora.     

Experimental testing of rust fungus‐mediated herbivory resistance in Betula pendula

Correlative evidence suggests that pathogenic leaf rust fungus, Melampsoridium betulinum, in late summer may negatively affect folivorous insects in the following summer. Correlative association does not necessarily, however, reveal causality. Alternatively, other interconnected plant characters may determine rust densities and herbivore performance. In this study, we used birch clones and rust fungus inoculations to manipulatively test the effects of birch rust and birch genotypes on the growth performance of folivorous moth larvae of Epirrita autumnata (Lep. Geometridae) in the subsequent year. The inoculation treatment increased rust densities (three‐ to 60‐fold) compared with natural infection levels. E. autumnata performance varied among birch clones and showed 4% lower growth performance on rust‐inoculated trees. However, the larval performance did not differ between rust‐treated shoots and untreated control shoots and the use of tree‐specific rust densities as a covariant in statistical analyses failed to reveal any negative association between rust fungus and larval performance. As the slight difference in larval growth performance also levelled until pupation, we propose that rust infection has biologically insignificant importance to the performance of E. autumnata.     

Short rotation coppice willow in Northern Ireland since 1973: development of the use of mixtures in the control of foliar rust (Melampsora spp.)

A review is given of the use of clonal mixtures in the development of disease control strategies in short rotation coppice willow in Northern Ireland. Salix burjatica‘Korso’ had been grown successfully for over 10 years when, in 1985, Melampsora epitea var. epitea caused serious problems. Although fungicides were effective, their use was not practical for environmental and economic reasons. Therefore, in 1987 large scale field experiments were initiated to investigate the use of clonal mixtures as a disease control strategy. Increased yields were consistently recorded from mixed stands when compared to either the mean yield of component clones or the individual yields of any of the component clones grown in monoculture. Part of this increased yield was due to a reduction in the impact of rust disease. Investigations are currently being conducted on the effect of number of clones within a mixture, the best clones to use and the optimum planting density. It is essential that there is a range of susceptibilities to M. epitea var. epitea pathotypes within the components of the mixture.     

Reliability of field,greenhouse and cut‐shoot screening procedures for evaluating susceptibility of Scots pine to Melampsora pinitorqua

The reliability of field, greenhouse and cut‐shoot screening procedures for the assessment of the susceptibility of Scots pine (Pinus sylvestris) genotypes to Melampsora pinitorqua, the causal agent of twisting rust, was evaluated. Fourteen genotypes evaluated on half‐sib progenies after natural infection of 2‐year‐old plants showed comparable rank for rust susceptibility with those evaluated after controlled inoculation of 1‐year‐old seedlings in a greenhouse. The dynamic of pycnia production was assessed at 2‐day intervals in cut‐shoot assays under controlled conditions, giving reliable rankings of Scots pine genotypes between years and being significantly correlated with rust susceptibility assessed after natural infection of 2‐year‐old progenies. These results underlined the importance of some critical factors such as inoculum load and host phenology in the reliability of susceptibility evaluations assessed under different experimental conditions. Artificial inoculation of 1‐year‐old seedlings in greenhouse experiments could provide a useful early test for the management of Scots pine breeding programmes and study of inheritance of twisting rust susceptibility. However, in specific investigations the cut‐shoot assay would constitute a reliable laboratory test for studying host–pathogen interactions and the variability in pathogenicity of Melampsora pinitorqua populations.     

Incidence and distribution of white pine blister rust in the high‐elevation forests of California

In 2004–2006, a California‐wide survey was conducted to evaluate the incidence and distribution of Cronartium ribicola, cause of white pine blister rust (WPBR), in the high‐elevation white pine forests. WPBR occurrence varied considerably within and between regions, and little to no disease was found in the Basin and Range, eastern Sierra Nevada and Transverse and Peninsular mountain ranges. Field surveys revealed no evidence of rust on Great Basin bristlecone, limber or the southern subspecies of foxtail pine. Rust incidence for western white pine was highest in the North Coast region (42% of trees surveyed), followed by the Klamath (18%) and northern Sierra Nevada (14%). For whitebark pine, WPBR incidence averaged 24% in the northern Sierra Nevada; this was considerably greater than other regions where whitebark pine was found infected. Latitudinal trends in WPBR incidence (i.e. greater in the north) may correspond with earlier introductions of C. ribicola into these regions, reflecting the southward spread of the pathogen since its introduction from the north in the early 1900s and environmental conditions favourable for infection. Distance from inoculum sources and potentially host phenology may also be important factors influencing the distribution of C. ribicola.     

Tree‐ring isotope analysis of Norway spruce suffering from long‐term infection by the pathogenic white‐rot fungus Heterobasidion parviporum

We report for the first time a tree‐ring isotopic analysis on host trees infected with Heterobasidion parviporum. By measuring carbon and oxygen stable isotope ratios in tree rings over ca. 150 years of forest growth, we obtained evidence that stomatal conductance increases in Picea abies affected by H. parviporum. We put forward this approach as a novel way of providing an insight into plant–pathogen relationships during tree life.     

Effect of plantation design on stem‐infecting form of rust in willow biomass coppice

Planting of genotype mixtures is a major strategy for the non‐chemical control of willow rust in short‐rotation coppice willows grown for biomass. In 1997 and 1998, the relative severity and spatiotemporal distribution of stem canker damage by Melampsora spp. on Salix viminalis `Bowles Hybrid' in a field trial containing monocultures and two design mixtures (random or regular) of three or five genotypes were assessed. Sphaerellopsis filum, a hyperparasite of Melampsora spp. was detected in all plots during the summer (1998) between the time of the two canker assessments. The percentage of rust pustules with this hyperparasite was initially greater in monoculture plantings than in mixtures in June, but by July, more than 92% of pustules were parasitized, irrespective of plantation design. The stem rust assessments showed that, in general, there were fewer cankers per stool in plots containing mixtures of willow genotypes compared with monoculture plantings. The distribution of rust cankers was different between monocultures and willow mixture designs, with differences between years for monocultures.     

Temperature sensitivity of qualitative race-cultivar interactions in Melampsora medusae Thüm. and Populus species

Six mono-uredospore isolates (races) of Melampsora medusae Thiim. produced qualitatively distinct reactions, in vitro, when incubated at 15 °C on leaf disks of certain cultivars of Populus deltoides Marsh. The infection types in some race-cultivar combinations were very temperature sensitive with less distinct reactions when incubated at 20 or 25°C. The significance of differential race-cultivar-temperature interaction in the epidemiology of M. medusae is discussed.     

Co‐occurrence of the ascomycete Lophodermium piceae and the rust fungus Chrysomyxa abietis in Norway spruce needles

The frequency of needles and the proportion of needle segments infected by Lophodermium piceae were compared in symptomless and Chrysomyxa abietis‐infected, 1‐year‐old needles of Picea abies. In late spring, symptomless needles from both rust‐infected and healthy saplings were sampled. In addition, rust‐infected, totally chlorotic needles and needles with chlorosis along about half their length from the diseased trees were examined. In all three stands, the proportion of segments infected by L. piceae was larger in the rust‐infected half of the needle than in the symptomless half; but the difference was statistically significant in only one of the stands. The proportion of L. piceae‐infected segments among the nonrust‐infected needles was the same as that found for the uninfected half of rust‐infected needles (after correction for size differences). No differences in the proportion of L. piceae‐infected segments were found between the totally chlorotic, rust‐infected needles and the green needles of diseased or healthy trees.     

The long‐term survival of Phytophthora cinnamomi in mature Banksia grandis killed by the pathogen

The ability of Phytophthora cinnamomi to survive long dry periods is the key to its persistence in the south‐west of Western Australia. It has been proposed that dead Banksia grandis are a significant long‐term reservoir for P. cinnamomi inoculum. To test this, 36 healthy B. grandis trees were inoculated in April 1999, and the presence of viable propagules in planta was determined between 2 and 34 months after tree death. By 10 months after inoculation, 75% of the trees had died, with the remaining seven trees dying by 22 months. The pathogen was more commonly recovered from bark than from wood, except from those trees that died at 22 months, and more commonly from above‐ground trunks than below‐ground trunks and roots until 8 months after plant death. In trees that died 12 months after inoculation, P. cinnamomi was recovered from 60% of trunk and root core samples at 3 months, declining to 33% at 10 months, 5.5% at 12 months and 0.1% at 34 months after tree death. In trees that died at 22 months, P. cinnamomi was recovered from 87% of trunk and root samples 2 months after tree death, decreasing to 0.5% by 33 months. This study suggests that the pathogen does not have a saprotrophic phase within dead B. grandis tissue, and B. grandis is unlikely to be a long‐term reservoir for P. cinnamomi. However, the manipulation of the density of B. grandis and the use of fire to facilitate the breakdown of dead Banksia trunks in the Eucalyptus marginata (jarrah) forest may reduce the spread and impact of P. cinnamomi.     

Varietal mixture of poplar clones: Effects on infection by Melampsora larici-populina and on plant growth

The effects of poplar varietal mixture on infection levels caused by Melampsora larici-populina and on plant growth were studied during two consecutive growing seasons. The varietal mixture consisted of an intimate mixture of three poplar clones, Populus x interamericana‘Boelare’, ‘Rap’ and Populus candicans‘NNT’, expressing different race-specific resistances. Infection levels of M. larici-populina were assessed in monoclonal and polyclonal plots. Plant heights were determined at the end of each growing season. Infection levels in the growing season of the first year were not significantly different between monoclonal and polyclonal plots at the end of the epidemic. During the second growing season, a naturally occurring complex pathotype, able to infect clones ‘Boelare’ and ‘Rap’, invaded some plots prematurely and changed the composition of pathogen population. During this season, four infection assessments were conducted. Infection reduction in polyclonal plots compared with monoclonal plots was observed on ‘Boelare’ and P. candicans plants. Nevertheless, no significant difference was observed for growth between monoclonal and polyclonal plots. Biological factors which may be involved in the reduction of infection levels in polyclonal plots are dilution of susceptible individuals, physical barriers caused by resistant plants and cross-protection. A high auto-infection ratio may have partly hidden the positive effects of the clonal mixture.     

Genetic diversity of European populations of the oak fine‐root pathogen Phytophthora quercina

The recently discovered oak‐specific fine root plant pathogen Phytophthora quercina is a significant factor in the current phase of European oak decline but its origins and ecology are poorly understood. A genome‐wide analysis of 260 amplified fragment length polymorphism (AFLP) markers was used to examine the genetic diversity of 72 isolates from five oak species at 28 sites in Germany (particularly Bavaria), Italy, France, Hungary and the UK. Within‐site diversity was examined at 16 sites. The limited genetic diversity (within and between sites) and lack of genetic substructuring according to geographic origin or host species suggest the rapid spread of a relatively recently introduced species. Two subgroups were distinguished and these may reflect an initial introduction of isolates of two different genetic backgrounds. The relatively low genetic diversity is probably because of the predominantly inbreeding (homothallic) nature of P. quercina. However, evidence of limited intra‐site diversity, temporal variation and the lack of clonality within the European population suggest that some diversity is being maintained by occasional outcrossing and turnover of a reservoir of long‐lived soil‐borne oospore (sexually derived) inoculum.     

PCR‐RFLP markers identify three lineages of the North American and European populations of Phytophthora ramorum

Phytophthora ramorum, the cause of sudden oak death and ramorum blight, has three major clonal lineages and two mating types. Molecular tests currently available for detecting P. ramorum do not distinguish between clonal lineages and mating type is determined by cultural methods on a limited number of samples. In some molecular diagnostic tests, cross‐reaction with other closely related species such as P. hibernalis, P. foliorum or P. lateralis can occur. Regions in the mitochondrial gene Cox1 are different among P. ramorum lineages and mitochondrial genotyping of the North American and European populations seems to be sufficient to differentiate between mating types, because the EU1 lineage is mostly A1 and both NA1 and NA2 lineages are A2. In our study, we were able to identify P. ramorum isolates according to lineage using polymerase chain reaction‐restriction fragment‐length polymorphism (PCR‐RFLP) of the Cox1 gene, first by using ApoI to separate P. ramorum from other species and EU1 from North American populations, and then AvaI to distinguish between NA1 and NA2 genotypes. However, P. foliorum had the same restriction profile as P. ramorum NA1 isolates.     

Gene flow and inter‐sterility between allopatric and sympatric populations of Heterobasidion abietinum and H. parviporum in Europe

The tree pathogenic fungi Heterobasidion parviporum and H. abietinum are recently described sibling species that show marked host preference for Picea abies and Abies alba, respectively. H. parviporum shows a higher reproductive character displacement (i.e. higher inter‐sterility) towards H. abietinum in sympatry than in allopatry. We inspected whether this pattern was also present in H. abietinum by studying the inter‐sterility and gene flow amongst sympatric central European populations of H. abietinum and H. parviporum, and two isolated allopatric populations, Pyrenean H. abietinum and Scandinavian H. parviporum. Inter‐ and intra‐specific fixation index values were calculated from DNA sequence data of the GST‐1 locus and four microsatellite loci. Present allopatric and sympatric populations of H. abietinum were equally inter‐sterile towards H. parviporum, not suggestive of relaxed reinforcement. Several hypotheses explaining the observed pattern are presented. Genetic differentiation was observed between H. abietinum in the Pyrenees and in the Alps, suggesting geographical structure of H. abietinum in Europe.