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.     

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.     

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.     

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.     

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.     

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.     

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.     

Rust scorings in a plantation of Salix viminalis clones during ten consecutive years

Among pests and diseases occurring in coppiced willow plantations established to produce biofuel, Melampsora rust is the most widespread and severe. In the present study, the variation in rust attacks in a set of Salix viminalis clones was investigated. The aim was to look for trends possibly arising from changes in rust genotype composition, to get some idea about the risks of rust overcoming resistance bred into new willow varieties. Rust levels on 92 S. viminalis clones belonging to 18 different families were scored once each year during 1987–96 in a plantation in southern Sweden. Rust scorings in 1993 deviated most from the average scoring pattern of the clones. Furthermore there was a successive change, such that the longer the time interval between scorings the lower was the correlation between rust scores. Willow families were identified that deviated from the others over time. Various explanations for inconsistency in rust scores over time are discussed.     

Phylogeographic variation among isolates of the Sirococcus conigenus P group

In this study the phylogeographic variation among isolates of the Sirococcus conigenus P group and the phylogenetic relationships of S. conigenus with Sirococcus clavigignenti‐juglandacearum and other species previously placed in the genus Sirococcus were investigated. A collection of 33 isolates originating from Picea, Pinus and Larix in Europe, North America and Bhutan were characterized by sequence analyses of the internal transcribed spacer (ITS) region (including ITS1, 5.8S ribosomal DNA, ITS2) of the nuclear rDNA and a portion of the β‐tubulin gene. In phylogenetic analyses most isolates from pine, spruce and larch formed a distinct clade, representing the P group of S. conigenus, which was separated from the T group of this pathogen. Four isolates from Picea in Europe and Canada formed a third clade within S. conigenus and these isolates are referred to as the S group. The P group consisted of five distinct ITS haplotypes, which partly differed in their optimum growth temperature and their growth rates at 25°C on malt extract agar. Nested clade analysis resolved the five haplotypes into three distinct clades and revealed significant genetic/geographic associations for some of the haplotypes. Parsimony analysis of the small subunit (18S) ribosomal DNA sequences confirmed the phylogenetic affinities between S. conigenus and S. clavigignenti‐juglandacearum. In contrast, Godronia cassandrae and Hormococcus conorum, which formerly had been placed in the genus Sirococcus, were found to be only distantly related to S. conigenus and S. clavigignenti‐juglandacearum.     

Characterization of Pyrofomes demidoffii from Ethiopian Afromontane forests

Pyrofomes demidoffii is a basidiomycetous fungus that causes a white heart rot in living junipers (Juniperus spp.). Morphological characters and genetic variation in species of Pyrofomes were investigated for specimens from Ethiopia, Tanzania, Macedonia, USA and Costa Rica. In addition, the behaviour of isolates from Ethiopia was studied in vitro. The Ethiopian specimens had relatively larger basidiospores than collections from other countries and showed a high sequence similarity in the ITS region of rDNA sequences with most of strains evaluated in this study (95–100%), but only 89–92% with P. demidoffii from the USA. Phylogenetic analysis of ITS‐5.8S rDNA sequences resulted in two well‐supported (99–100% bootstrap value), separate clades of P. demidoffii. Deeper branches were less well supported (bootstrap < 90%), but the two geographically and phylogenetically separated clades within P. demidoffii may still represent sister groups. For Ethiopian strains, the temperature optimum was between 20 and 25 °C and the pH optimum was around 5.0. A phylogenetic study with additional genetic markers and with a more representative collection of samples is needed.     

Host‐related variation among isolates of the Sirococcus shoot blight pathogen from conifers

Variation among isolates of the Sirococcus shoot blight pathogen (attributed to Sirococcus conigenus, Sirococcus sp., or one of many synonyms) was studied. Inter‐simple‐sequence‐repeat‐anchored polymerase chain reaction fingerprints, nuclear rDNA internal transcribed spacers and 5.8S sequences, and 18S rDNA sequences were analysed. In addition, culture morphology as well as sizes and shapes of conidia produced on seed cone scales in culture were compared. Two main groups were distinguished based on both genetic markers and cultural characteristics. The P group consists of isolates mostly from pine and spruce from Europe and both eastern and western North America; the T group consists of isolates mostly from hemlock from western North America. Results provide a benchmark for comparison of other isolates of these fungi and indicate the need for additional sampling and comparative research of these pathogens.     

Chrysomyxa ledi,a new rust fungus sporulating in cone scales of Picea abies in Finland

Abstract

Seasonal fruiting and sporulation of cone rusts were investigated in Norway spruce cones during the growing season of 2007, a year after an excellent cone crop and a severe outbreak of rust. Current-year and 1-year-old cones were collected bimonthly to monthly from case-stands in southern and northern Finland and checked for rust fruitbodies. A previously unreported species of rust formed spermogonia and aecia, and sporulated on the cone scales of Norway spruce in two study areas. Morphologically, the rust was distinct from other spruce cone rusts and, based on ultrastructure of aecia and aeciospores, most closely resembled Chrysomyxa ledi. Molecular analysis of the aeciospores confirmed that the rust belonged to the C. ledi/C. rhododendri complex. The rust fruited frequently but only colonized a few scales per cone and presumably had a minor effect on cone development and seed crop.     

A new species of Melampsora rust on Salix elbursensis from Iran

A rust fungus was found causing stem cankers on 1‐ to 5‐year‐old stems of Salix elbursensis in the north west of Iran. The rust also forms uredinia on leaves and flowers of the host willow. Light and scanning electron microscopy revealed that the new rust is morphologically distinct from several Melampsora species occurring on the willows taxonomically close to S. elbursensis, but indistinguishable from Melampsora larici‐epitea. Examination of the internal transcribed spacer (ITS) region of the ribosomal DNA suggested that the rust fungus is phylogenetically close to Melampsora allii‐populina and Melampsora pruinosae on Populus spp. Based on both the morphological characteristics and the ITS sequence data, the rust is described as a new species –Melampsora iranica sp. nov.     

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.     

Identification of the genus Armillaria by specific amplification of an rDNA-ITS fragment and evaluation of genetic variation within A. ostoyae by rDNA-RFLP and RAPD analysis

Genetic variation among Armillaria ostoyae isolates was studied by rDNA-restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) analysis. A total of 20 A. ostoyae isolates, mainly obtained from Picea spp. of different geographical origins, were examined. Southern hybridization of whole-cell DNAs digested with AvaII and probed with biotin-labelled cloned rDNA from Saccharomyces carlsbergensis allowed the differentiation of five RFLP groups. UPGMA cluster analysis of RAPD profiles (138 scorable bands) generated by 10 decamer primers (OPA 01-OPA 10) grouped the isolates in subclusters at similarity levels between 40% and 96%, indicating high intraspecific genetic variability. Some isolates of different geographical origins subgrouped together, suggesting that similar mutational events have occurred independently and that genetic exchange and recombination occurs among the DNAs in natural populations. The potential role of historical and current spread of spruce plants on the genetic variation of A. ostoyae isolates in Europe is discussed. Using the primer pair ARM-1 and ARM-2, an Armillaria-specific ITS-DNA fragment of about 660 bp was obtained. No intraspecific RFLP of this amplicon could be revealed, indicating low genetic variability of this region. The established informative RFLP and RAPD markers and also the Armillaria-specific ITS-DNA fragment may be powerful tools for further epidemiological, phylogenetic and host-pathogen interaction studies with A. ostoyae.     

Infection of Populus alba var. hickeliana by Melampsora medusae Thüm.

The first record of infection of Populus alba var. hickeliana by the American rust, Melampsora medusae Thum. is reported. The morphology of uredospores of rust on P. alba is compared with that of other Melampsora spp. infecting white poplars. The implications of these observations for the widespread establishment in the future of white × black hybrid poplars are discussed.     

Clonal response in Salix to Melampsora rusts in short rotation coppice plantations

The appearance of Melampsora spp. rust on a range of Salix spp. clones used for short rotation coppice was recorded. The progress of the disease and its subsequent effect on leaf fall was also recorded. Rust pustules from all the clones described were examined using the Stereoscan Elec-tron Microscope. This revealed no differences in uredospore architecture which could be consis-tently related to host.     

Spread of Cryphonectria hypovirus1 into 45 vegetative compatibility types of Cryphonectria parasitica on grafted American chestnut trees

A mixture of hypovirulent strains of Cryphonectria parasitica, including four white (European) strains infected with Cryphonectria hypovirus1 (CHV1), was used in 1982 and 1983 to inoculate natural blight cankers located within a zone ranging from the ground to 183 cm on grafted American chestnut trees. These four white strains belonged to three vegetative compatibility (vc) types. Using pigmented, single‐spore colonies from white isolates, 48 vc types were identified among 110 white isolates recovered in 1996, 1998, and 1999 from cankers located outside the inoculated zone. Twenty‐five of the 48 white vc types consisted of two or more isolates. The 25 major white vc types were vegetatively incompatible with all four of the original white hypovirulent strains, providing evidence for spread of CHV1 but not for spread of the original inoculated strains. Forty‐five vc types represent the minimum number of `new' vc types into which CHV1 had spread. The ratio of white vc types to white isolates tested (S/N) and Shannon diversity index were 0.436 and 3.64, respectively. The spatial pattern of white vc types on the grafts was found to be non‐random (p=0.019). White single‐spore colonies of white isolates were placed into four cultural morphology (CM) groups. The two largest groups contained 37 (CM group 3) and 33 (CM group 1) isolates. Single‐spore colonies from the original, white inoculated strain, Ep 49, were classified into CM groups 3 and 1, and colonies of Ep 51 W were classified into CM group 1.     

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’.     

Assessment of intrapathovar variability of Xanthomonas axonopodis pv. commiphorae through phenotypic and molecular markers

Gummosis of guggal (Commiphora wightii) caused by Xanthomonas axonopodis pv. commiphorae (Xac) is one of the major reasons for drastic reduction in guggal population under natural plant stand. The pathogen spreads mainly through human activities (tapping). We isolated 43 Xac strains from 14 locations representing four different regions spread over three districts of Gujarat state, India. A polyphasic approach was followed to characterize these strains and to measure phenotypic and genetic variations among them. All strains were identical in colony morphology and were virulent on guggal. Some of the strains showed differential reactions towards certain biochemical tests viz., acid production from carbon sources. Whole cell protein profiles were apparently uniform for all strains with similarity coefficients ranging between 0.75 and 1.00. Clustering based on sodium dodecyl sulphate–polyacrylamide gel electrophoresis banding patterns showed heterogeneity among the isolates, but strains originating from same zone had similar protein profiles. Inter simple sequence repeats (ISSR)‐based and repetitive elements (rep)‐based PCR analyses revealed genetic heterogeneity among the strains. Both these methods were equally effective in deciphering variability among the strains and indicated similar types of variability with highly significant correlation (r = 0.91) on the similarity matrices. The variation matrix analysis on combined ISSR‐ and rep‐PCR data suggested 66.1% variability among Xac strains. The present study established that Xac strains from different geographical locations had profound genetic heterogeneity.