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.     

Interactions between Melamspora larici‐epitea pathotypes and the mycoparasite Sphaerellopsis filum from willow rusts

Five pathotypes of the willow rust Melamspora larici‐epitea were inoculated with 12 isolates of Sphaerellopsis filum derived from Melampsora species/forms occurring on willows. On average, 20.5% uredinial pustules produced S. filum pycnidia and rust spore production was reduced by 38.4% on leaf discs inoculated with S. filum. Some rust isolates were more readily infected by S. filum than others while some S. filum isolates caused higher levels of infections than other S. filum isolates. In general, the suppressive effects of these S. filum isolates on rust spore production were similar on the majority of rust pathotypes tested. There appeared to be a positive link between the rust pustule area and the rate of infection by S. filum. Sphaerellopsis filum inoculum densities were positively correlated with the reduction in rust spore production but not with the number of rust pustules. Implications from the results were discussed in relation to the deployment of S. filum in biological control of willow rust in willow mixture plantations which harbour more diverse rust pathotypes compared with monocultures.     

Genetics of field resistance to Melampsora in Salix viminalis

The inheritance of resistance to Melampsora. spp. on Salix viminalis, the variation in resistance over space and time, and rust infection rate in relation to the geographic origin of the host was studied. The analysis showed that there was considerable additive genetic variation for rust resistance, that the results did not have high repeatability over space and time, and that the material from southernmost Sweden was most sensitive to the rusts occurring in south Sweden.     

Compatible and Incompatible Reactions of Melampsora Rust on Willow Leaves

Spore germination, growth and appresssoria formation for Melampsora willow rust were studied for compatible and incompatible hosts to elucidate penetration specificity and prehaustorial events in the infection process. Rust originating from Salix dasyclados was inoculated on a compatible (S. dasyclados) and an incompatible (Salix daphnoides) clone and, for comparison, on the non-host plants tomato, tobacco and poplar. The same experimental procedure was also carried out for rust originating from poplar. Rust development was followed for 5 days at 20 h intervals using a microscope with fluorescent equipment. The study showed that the Melampsora rust can develop on, and penetrate, the leaf regardless of whether the rust was inoculated on compatible or incompatible willow plants. However, the fungus was able to infect and reproduce itself only in compatible interactions. For willow rust in the prehaustorial stage, the study indicated that specific recognition mechanisms were unnecessary to start the infection process.     

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.     

Physiologic specialisation in the hyperparasitism of races of Melampsora larici-populina by isolates of Cladosporium tenuissimum

Three isolates of Cladosporium tenuissimum were prepared by culturing a field isolate of this organism separately on three races of Melampsora larici-populina. In a subsequent experiment, the production of uredinia by these three races on three cultivars of Populus X euramericana was usually lowest in the presence of the isolate of C. tenuissimum which had been cultured on the particular race. This apparent adaptation of the isolates of the hyperparasite to particular races of the rust, i. et. “physiologic specialisation” in the relationship, is confirmed by the significant race x C. tenuissimum isolate term in the ANOVA.     

Lesion development in stems of rough‐ and smooth‐barked Eucalyptus nitens following artificial inoculations with canker fungi

A study of lesion development in stems of Eucalyptus nitens following artificial inoculations with canker fungi was carried out on 16‐year‐old plantation trees. In a first trial cambium bark wounds on smooth‐ and rough‐barked trees were inoculated with the mycelium of nine species of canker fungi, including Endothia gyrosa. In a second trial spores or mycelium of E. gyrosa were applied directly onto undamaged or superficially wounded bark surfaces. Infection subsequent to artificial inoculation via wounding (whatever the wounding technique or type of inoculum) resulted in significantly larger external lesions (mean lesion area up to 35.6 cm² 20 months after inoculation) on smooth bark compared with those on rough bark (up to 19.0 cm²). Microscopic studies of infected rough and smooth bark suggest that, once smooth bark is compromised by wounding and artificial inoculation, the particular anatomical structure of smooth bark may offer less mechanical resistance to post‐penetration hyphal spread in comparison with rough bark. It is suggested that at a pre‐penetration stage under natural conditions spores of E. gyrosa more easily infect rough bark via cracks associated with this type of bark but not present in smooth bark.     

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’.     

Effectiveness of a detached‐leaf assay as a proxy for stem inoculations in backcrossed chestnut (Castanea) blight resistance breeding populations

A recently developed detached‐leaf blight resistance assay has generated interest because it could reduce the amount of time needed to evaluate backcrossed hybrid trees in the American chestnut blight resistance breeding programme. We evaluated the leaf inoculation technique on a sample of advanced progeny from the Indiana state chapter American Chestnut Foundation breeding programme, along with susceptible American chestnut (Castanea dentata), the recurrent parent, and resistant Chinese chestnut (Castanea mollissima), the donor parent for blight resistance. In experiments over 2 years using two pathogen isolates, we found no biologically meaningful relationship between leaf lesion size and the size (length and width) or severity (1–5 canker severity rating) of stem cankers on 5‐year‐old trees. Chinese chestnuts did develop significantly smaller leaf lesions than American or backcrossed chestnuts. We conclude that while the detached‐leaf assay may have utility in some chestnut breeding applications, it is not a suitable proxy for the established practice of stem inoculations.     

Effects of host plant hybridization on resistance to willow leaf rust caused by Melampsora sp.

Patterns of resistance to willow leaf rust caused by Melampsora sp. were measured on Salix eriocephala, Salix sericea, and their interspecific hybrids in natural field populations in 1991–94 and on potted plants in common garden experiments in 1994 and 1995. Testing was carried out to establish if the resistance of hybrids was intermediate between pure parental species (additive), equal to one of the parents (dominant), lower than both parents (susceptible), higher than both parents (resistant), or not different than either parent (null hypothesis). Support was generally found for hybrid susceptibility in the field and common garden experiments, with the null hypothesis of no difference having some support. The results from the common garden study suggest that the susceptibility of hybrids seen in the field is due to genetic differences. Hybridization between these species of willows results in increased susceptibility to this pathogen. The variation among years in the relative susceptibility of hybrid and parental willows may be due to environmental effects on the willows or to shifts in the abundance of Melampsora races.     

Effects of various densities of Ophiostoma ips inoculations on Pinus sylvestris in north‐western Spain

The aim was to determine the inoculation density above which Scots pine (Pinus sylvestris) is overcome by the blue‐stain fungus Ophiostoma ips that is associated with the bark beetle Ips sexdentatus. In north‐western Spain, stems of 16 Scots pines were inoculated at various densities (0, 400, 800 or 1600 inoculi/m²) along circumferential 100 or 150 cm wide inoculation belts. Each inoculum consisted of a 5 mm diameter cylinder of malt extract agar colonized by the fungus. Three months later, all trees were harvested and trunk resinosis and foliage colour were visually assessed. The percentage of healthy, desiccated, resin soaked, and blue‐stained sapwood, as well as growth productivity indices, were calculated from stem disks cut from within the inoculated zone of each tree. Sapwood‐specific hydraulic conductivity (Ks) of each tree was measured in the middle of the inoculated zone. All parameters of tree vigour changed dramatically to the worse when inoculation densities were above 400 inoculi/m², and foliage changed from green to yellow‐green or yellow when an inoculation density of 800 instead of 400 was used. The percentage loss of sapwood‐specific conductivity (PLC) increased from 30 to 90% and the percentage of healthy, conductive sapwood dropped from 85 to 35% at 800 inoculi/m². No effect of the width of the inoculation belt was observed, and there was no relationship between tree productivity indices and the level of resistance. A non‐linear negative relationship was found between PLC and the percentage of healthy sapwood. It is concluded that tree resistance was overcome and that trees were going to die when the inoculation density was ≥800 inoculi/m².     

Comparing Chilean and Swedish rust populations: possible impact of long‐distance transport of Melampsora rust

As willow (Salix) cultivation grows globally, worldwide monitoring of pathotypes for Melampsora rust, the most serious disease in willow, becomes increasingly important. We compared the pathotype composition of the Melampsora rust population in Chile with that in Sweden and assessed inocula exchange for the rust between the continents. For pathotyping rust isolates, a willow differential was used (a standardized set of willow test clones) to obtain clone‐virulence patterns. These patterns were used to identify virulence components involved in determining which willow clones a given individual pathogen isolate (genotype) can successfully attack. Each virulence component detected earlier in Sweden's rust population was present in Chile's rust population. Using isoenzyme analysis to classify willow species in Chile, a low genetic variation of Salix viminalis (an introduced willow species) was found, compared with the endemic species Salix humboldtiana. Comparison of rust populations for the two countries supports the present hypothesis that intercontinental inocula exchange can be a significant determinant of local pathotype structure, and consequently can be important for willow‐resistance breeding.     

Rapid differentiation of Ophiostoma ulmi and Ophiostoma novo‐ulmi isolates by matrix‐assisted‐laser‐desorption/ionization time‐of‐flight/time‐of‐flight mass spectrometry

The intact yeast phase spores of Ophiostoma ulmi and O. novo‐ulmi isolates were analysed by matrix‐assisted‐laser‐desorption/ionization time‐of‐flight/time‐of‐flight mass spectrometry. The results clearly showed that isolates of O. ulmi and O. novo‐ulmi have different mass peak profiles. The specific mass in m/z may vary slightly. One of the peaks from m/z 7623 to 7630 dominated (in high intensity) mass spectra for all isolates of O. novo‐ulmi tested in this study. This specific peak is believed to be CU (cerato‐ulmi) protein. However, no specific m/z peak can be found to distinguish between the isolates of ssp. novo‐ulmi and ssp. americana. On the contrary, all isolates of O. ulmi tested possessed one of a dominate peak at m/z 7337 to 7342, but none around m/z 7626 as shown in O. novo‐ulmi isolates.     

First report of Rhytisma filamentosum causing tar‐spot disease on Salix koriyanagi

The occurrence of previously unknown tar‐spot disease on Salix koriyanagi was observed during surveys for forest disease outbreaks in Korea. The fungus associated with tar‐spot disease was identified as Rhytisma filamentosum based on morphological characteristics of the fungus and molecular sequencing of the ITS and 28S rDNA regions of two samples. To our knowledge, this is the first report of R. filamentosum on S. koriyanagi from Korea, following its first discovery on Salix integra in Japan.     

Genetic diversity of apple‐ and crabapple‐infecting isolates of Venturia inaequalis in Pennsylvania,the United States,determined by microsatellite markers

Apple scab caused by Venturia inaequalis is the most destructive disease of apple worldwide. In this study, genetic diversity of 101 V. inaequalis isolates from cultivated apples and ornamental crabapples in Pennsylvania (PA, USA) was characterized using 14 microsatellite markers. A total of 157 alleles ranging from 5 (Vitg9/99) to 26 (Vica10/154) per locus were detected. Regardless of the host of origin, isolates were grouped into five clusters, which were largely supported by STRUCTURE and principal coordinate analysis. Cluster analyses based on genetic distances and population structure analysis suggest very small differentiation (PhiPT ranging from 0.016 to 0.103, depending on the population comparison) between apple and crabapple isolates of V. inaequalis. Pairwise comparisons among populations from different locations showed very low differentiation, and POPGENE analysis indicated frequent migration of alleles (Nm = 1.47). In pathogenicity tests using a detached leaf assay, isolates of V. inaequalis from crabapple caused characteristic scab symptoms on apple and were highly virulent. Results of the study indicate that scab lesions in crabapple trees in close vicinity to apple orchards could serve as reservoirs for spread of the pathogen. Movement of inoculum among locations and between hosts may be responsible for the limited population structure observed. Understanding the population structure of V. inaequalis isolates is significant for apple scab management as crabapples are often used as pollinizers and rootstock in apple orchards, and as ornamental trees.     

Growth and ultrastructural modifications to chestnut calli induced by culture filtrates of virulent and hypovirulent Cryphonectria parasitica strains

Callus cultures of two susceptible Castanea sativa cultivars, ‘Garrone rosso’ and ‘Clone 71’ were grown on culture medium supplemented to 50% with culture filtrates (CFs) from E4 virulent (E4-V) or E13 hypovirulent (E13-H) Cryphonectria parasitica strain, respectively. E13-H CFs caused a reduction in fresh weight and an increase in dry weight on calli of both genotypes. E4-V CFs did not induce any modification of these parameters compared with the control, but fresh weight of ‘Clone 71’ was reduced. Light and electron microscopy observation showed the presence of large osmiophilic aggregates in the vacuoles and, sometimes, the ruptured tonoplast in the E4-V treated calli. Calli grown on El 3-H CFs had a meristematic-like appearance, with small, scarcely vacuolated cells containing altered mitochondria and plasmalemma proliferations.     

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.     

Diversity of Cryphonectria parasitica in western Spain and identification of hypovirus‐infected isolates

Isolates of the chestnut blight fungus Cryphonectria parasitica were obtained from 44 localities in four provinces in Western Spain and characterized for vegetative compatibility (vc) types, mating types and the presence of Cryphonectria hypovirus 1 (CHV1). Among the 1232 isolates recovered from chestnut blight cankers, 11 vc types were identified: five known vc types included in EU1 to EU74 (EU1, EU11, EU12, EU28 and EU66) and six unknown vc types (CL4, CL5 CL6, CL8, CL9 and CL10). The number of vc types found per province varied between two and seven. The vc type EU11 was present in all provinces and accounted for 48.9% of all isolates. EU1 was detected in three provinces and accounted for 39.1% of the isolates. The vc types EU12, EU66, CL5 and CL6 were present in one or two provinces and comprised between 2.4% and 3% of the isolates. The other vc types were represented by only one or very few isolates. The mating type MAT‐1 was largely dominant in the provinces Leon and Avila, while both mating types MAT‐1 and MAT‐2 were found in Salamanca and Zamora. Fourteen hypovirus‐infected C. parasitica isolates were found, nine were in vc type EU1 and five in EU11, and they were detected only in the province León. All isolates analysed contained the French hypovirus subtype CHV1‐F1.     

Responses of Cupressus and Chamaecyparis callus tissues to inoculations with Seiridium cardinale

Inoculations of callus generated from micropropagated shoots of Cupressus sempervirens, Cupressus macrocarpa, Cupressus torulosa and Chamaecyparis lawsoniana showed that callus of species resistant or relatively resistant to Seiridium cardinale supported significantly less surface growth of the pathogen than callus of susceptible species. In dual cultures of Cupressus sempervirens or Cupressus torulosa callus with S. cardinale, inhibition of fungal growth towards the callus was correlated with known field resistance of these host species. Lignification, accumulation of polyphenolics and possible suberization of cell walls were detected in inoculated callus. This response was also correlated with known field resistance of the tested host species.