Identification of Phytophthora nicotianae on the aerial portion of eucalypt seedlings

The pathogenicity, physiology and biochemical features of three isolates of Phytophthora. nicotianae var. nicotianae from Eucalyptus delegatensis and E. globulus seedlings were investigated. The acid phosphatase zymograms and total protein profiles confirmed the specific identity of the 3 isolates. Despite significant differences in mycelial growth and zoosporangium production, no differences were noted in pathogenicity. Artificial inoculations revealed a variability in resistance to P. nicotianae within Eucalyptus species.     

Capability of the European and North American race of Gremmeniella abietina to hydrolyse polygalacturonic acid in vitro

Polygalacturonase was found to be one of the first enzymes secreted by a pathogen during infection. The polygalacturonic-acid hydrolysing activity was compared between the North American and the European race of Gremmeniella abietina in vitro. Isolates were grown in pure pectin media from which the enzyme activity was analysed. Altogether, 29 isolates were tested in five experiments (experimental runs in a growing chamber). The data were analysed using variance-component models that included fixed-race effects and random-experiment, isolate, flask and measurement effects. The European race secreted more polygalacturonic-acid hydrolysing enzyme than the North American race and the mycelial dry weight produced was smaller for the European race. The differences between races were of the same order of magnitude as the variation between isolates within races; variance components relating to experimental errors were quite large. No correlation was found between the activity and mycelial dry-matter production within the races. Logarithmic transformation removed the apparent racial differences in the variability of the activity and mycelial dry weight. Results from the additionally tested A- and B-type of Finnish isolates indicated differences in dry-matter production.     

An in vitro method for screening Colletotrichum gloeosporioides as a biological control agent for western hemlock dwarf mistletoe

An in vitro system using detached western hemlock branches infected with dwarf mistletoe was developed to screen the virulence of five isolates of Colletotrichum gloeosporioides, a hyperparasite of dwarf mistletoe shoots and berries. Detached branches infected with dwarf mistletoe were placed in nutrient‐saturated rock‐wool blocks and mistletoe shoots were inoculated with a conidial suspension of C. gloeosporioides. One month after inoculation, lesions on mistletoe stems and berries were well developed. Infection levels for individual isolates varied from 40% to 60% of shoots and 60% to 80% of berries. Significant differences were found between the isolates and control (p = 0.02 and 0.001, respectively) while no differences were noted between the isolates for both the shoots and berries. Parallel inoculation of mistletoe shoots detached from hemlock branches on moist filter paper and in rock‐wool blocks failed because these shoots deteriorated rapidly, fragmenting into segments within a week. This in vitro test may provide an alternative method for rapid screening of potentially virulent C. gloeosporioides isolates.     

Cultural characters and growth of Cylindrocladium quinqueseptatum isolates1

Isolates of Cylindrocladium quinqueseptatum (CQ), which cause leaf blight of Eucalyptus in Kerala, differed significantly in growth and cultural characters on nine different agar media. Malt extract agar and yeast malt agar and yeast malt agar were the best media for discerning differences among the CQ isolates. Of the 11 carbon (C) and 13 nitrogen (N) sources evaluated in liquid media for their differential utilization by the five selected CQ isolates, five each of C and N sources differentiated them from each other. In general, organic N sources supported better growth and micro-sclerotia production over inorganic, and hexose and disaccharide over trisaccharide and polysaccharide.     

Cellulolytic activity and virulence of Ophiostoma ulmi and O. novo‐ulmi isolates

The cellulolytic activity (exoglucanase, endoglucanase and β‐glucosidase) of Ophiostoma ulmi (four isolates), O. novo‐ulmi (19 isolates) and ‘fast‐waxy’ (five isolates) was determined in growth media containing carboxymethylcellulose (CMC) and cellulose powder. Differences in enzyme activities were observed among isolates, irrespective of the species and substrate used. Inoculation experiments on Ulmus minor with randomly selected isolates of O. ulmi (two isolates), O. novo‐ulmi (five isolates) and ‘fast‐waxy’ (two isolates) were also performed. Disease was assessed as the percentage of leaves showing yellowing and browning. Ophiostoma novo‐ulmi and ‘fast‐waxy’ isolates exhibited a great variability in their capacities to cause the disease. In the presence of CMC, a significant correlation between the activity of exoglucanase and β‐glucosidase in vitro and virulence was found.     

Pathogenic variation in Cylindrocladium quinqueseptatum causing leaf blight of Eucalyptus1

A wide range of pathogenic variation was observed among the five isolates of Cylindrocladium quinqueseptatum leaf blight (CLB) which could be distinguished as different physiologic strains on 11 differential provenances of Eucalyptus. Susceptibility ranking of different provenances to five isolates also differed significantly indicating differential interaction between isolates and provenances. Analysis of variance showed that CLB severity of a provenance is mainly governed by the genetically different isolates and also that the provenances have closer genetical relationship. The results provide the first evidence for the existence of physiologic strains in C. quinqueseptatum.     

Tinctoporellus epimiltinus,a causal fungus of butt rot of Japanese cypress

One of the causal fungi of butt rot of Chamaecyparis obtusa in Kyushu Island, Japan, was identified as Tinctoporellus epimiltinus from cultural characters, and by mating and inoculation tests. The cultural characters of the isolates obtained from butt rot of C. obtusa and T. epimiltinus isolated from the basidiocarp tissue and from decayed wood were examined. The morphological characters and chemical reactions of the isolates from butt rot were typical for T. epimiltinus. Di‐mon mating tests were performed between five monospore isolates of T. epimiltinus and five heterokaryotic isolates from the butt rot. All of the monospore isolates examined were heterokaryotized by the isolates from butt rot. Tinctoporellus epimiltinus isolates were inoculated on roots of 24‐year‐old C. obtusa. Butt rot was observed on all inoculated trees 1–2 years after the inoculation, and the inoculated fungus was re‐isolated from the decayed parts of the inoculated trees.     

Multilocus phylogenetic analysis revealed a new cryptic lineage of Serpula himantioides in Japan

Serpula himantioides is a widely distributed saprotrophic fungus that causes root and butt rot in various tree species. In Japan, butt rot associated with S. himantioides is observed in Chamaecyparis pisifera and Abies sachalinensis. Previous studies have shown that S. himantioides includes five phylogenetically defined cryptic lineages, but the placement of Japanese isolates remains unclear. To clarify the phylogenetic relationship between Japanese S. himantioides and the five known lineages, we conducted a phylogenetic analysis using three newly collected Japanese isolates along with 74 S. himantioides isolates, based on the internal transcribed spacer (ITS) region, the 28 S large subunit (LSU) rDNA, the beta-tubulin (tub) gene, and the heat stress protein (hsp) gene. The concatenated phylogenetic tree showed that Japanese isolates composed a clade with a high bootstrap value distinct from the known lineages, indicating the Japanese isolates present a new cryptic lineage of S. himantioides.     

Resistance of new Belgian poplar clones to British isolates of the bacterial canker pathogen,Xanthomonas populi

Forty-nine new poplar clones, bred in Belgium and screened there for resistance to bacterial canker caused by Xanthomonas populi, were additionally screened in southern England, using British isolates of the bacterium. Thirty-five of the test clones ranked better than the clone ‘Boelare’, which is already registered in the UK as an approved clone, and is regarded as having acceptable resistance. The registered clone ‘Ghoy’ was more susceptible, and outranked only four test clones. Preliminary measurements of canker length and stem girdling index showed differences between five bacterial isolates.     

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.     

Molecular‐based identification and phylogeny of Armillaria species from Serbia and Montenegro

Armillaria causes problems of root rot, kill trees and decay wood in the forests of Serbia and Montenegro, but the species involved have not hitherto been identified. The aim of this study was to identify field isolates collected on 25 localities. Identification was based on restriction fragment length polymorphism (RFLP) analysis of intergenic spacer 1 (IGS1) region and comparisons of IGS1 sequence with those available on NCBI database. Phylogenetic analysis was performed on sequence information from selected isolates to determine possible interrelationships between isolates with different banding patterns and previously identified tester isolates of five European Armillaria species. Five Armillaria species were identified in 90 isolates obtained from forests in Serbia and Montenegro. Armillaria gallica was most frequently isolated, followed by A. cepistipes, A. mellea, A. ostoyae and A. tabescens; two isolates remained unidentified. Restriction digestion of IGS1 amplification products with AluI produced 10 RFLP patterns. Patterns G4 (400, 250, 180) for A. gallica and pattern X (400, 180, 140) for isolates 74 and 79 are reported for the first time in European isolates. Eight RFLP patterns were observed after restriction with TaqI. Two patterns each were observed for A. ostoyae and A. gallica, and one each for A. cepistipes, A. mellea, A. tabescens and isolates 74 and 79. Parsimony analyses based on the IGS1 region placed the isolates into four clades: one including A. mellea, the second containing A. gallica–A. cepistipes isolates, while isolates of A. ostoyae and A. borealis were in the third clade. Armillaria tabescens differed from all annulate species. Phylogenetic analysis supported the conclusion that European Armillaria species are closely related and separated from a common ancestor in the near past. According to this survey five European Armillaria species are present in the forests of Serbia and Montenegro, while A. borealis is not present in the studied ecosystems.     

Genetic characterization of Botrytis cinerea isolates collected from pine and eucalyptus nurseries in Bio‐Bio Region,Chile

A total of 55 Botrytis cinerea isolates collected from Pinus radiata and Eucalyptus globulus plants cultivated in nurseries located in the Bio‐Bio Region, Chile, as well as isolates collected from native plants such as Rubus spp and Aristotelia chilensis located near the nurseries were genetically characterized. All isolates carried the Bc‐hch2 allele, thus belonging to genetic Group II, which is now referred to as B. cinerea. Genotyping based on the presence of transposons Boty and Flipper showed differences between isolates related to the plant host. Thus, transposa isolates (containing both transposons) were detected in P. radiata and E. globulus, while vacuma isolates (containing neither transposon) were detected in all plants except E. globulus. Notably, boty isolates (containing just the Boty transposon) were detected at high frequencies in all plant hosts. Analyses to detect mutations involved in resistance to fungicides such as benzimidazoles (BZ), dicarboximides and QoIs also showed differences in the studied isolates. Isolates collected from E. globulus were shown to carry mutations for all tree fungicides, while those collected from P. radiata presented mutations involved in resistance to BZ only. Isolates collected from native plant hosts did not carry any of the mutations analysed.     

Virulence and double‐stranded RNA in Sphaeropsis sapinea

Forty wildtype isolates of Sphaeropsis sapinea were grouped into the morphotypes A and B based on previously defined differences in cultural and morphological criteria as well as restriction sites for Dde I and Bst UI endonucleases in nuclear ribosomal DNA amplicons. Thirteen of 20 type A isolates and nine of 20 type B isolates contained detectable dsRNA (55%) of different molecular weight and size. dsRNA was transmitted into conidia at a frequency of 71–100%. By selecting single conidia, dsRNA‐free subcultures were obtained from six of 22 isolates containing dsRNA. Pathogenicity tests on expanding buds of landscape trees of three species of Pinus showed highly significant statistical interactions between isolate virulence, Pinus species, and year. Pine species‐year had a profound impact on virulence. The pattern in the interactions was revealed by principal component analysis of the interaction sums of squares of the anova (Additive Main Effects and Multiplicative Interaction; AMMI). Pinus sylvestris was highly interactive in its susceptibility to S. sapinea with seasonal effects. P. nigra and P. resinosa were more stable. The interactivity analysis was used to apportion interaction to specific isolates to improve the accuracy of the estimates of virulence. Estimates of the relative virulence of isolates were predicted over five different Pinus species‐years. Isolates were ranked in virulence and interactivity using the AMMI model. This model permitted mean separation tests of the relative virulence among isolates over the combined Pinus species‐years. One isolate was identified as potentially having dsRNA‐mediated hypovirulence based on the significantly greater virulence of its isogenic, dsRNA‐free subculture, as expressed over the three Pinus species and 2 years. Type A isolates containing dsRNA ranged from stable to highly interactive and from low to high in virulence. Type B isolates containing dsRNA were similar in interactivity but virulence ranged from avirulent to moderate, seldom exceeding the mean for S. sapinea. dsRNA‐free isogenic subcultures tended not to express higher virulence than their dsRNA‐containing parent strains but often changed in interactivity. Therefore, in one year a dsRNA‐free subculture might be more virulent than its dsRNA‐containing parent. In another year the dsRNA‐free subculture might be less virulent.     

A versatile method for assessing pathogenicity of Hymenoscyphus fraxineus to ash foliage

We describe a method for inoculating rachises of Fraxinus excelsior (European or common ash) with Hymenoscyphus fraxineus, which is faster than previous methods and allows associated foliar symptoms to be assessed on replicate leaves. A total of ten ash seedlings were inoculated with five isolates of H. fraxineus and lesion development assessed over four weeks. A five‐point disease progress scale of symptom development was developed from no lesion (0), lesion on rachis (1), “pre‐top dead,” with curling of distal leaflets and bending of the rachis (2), top dead, with wilting and death of distal leaflets (3) to leaf abscission (4). The method revealed variation in aggressiveness of H. fraxinus isolates and may be suitable for assessing the resistance of F. excelsior and other Fraxinus species to dieback. The in vitro growth rate of H. fraxineus isolates was highly correlated with both disease progress and the length of rachis lesions on susceptible plants, indicating that it can be used as a preliminary step in selecting isolates with high aggressiveness for use in resistance screening.     

Comparison of procedures to evaluate the pathogenicity of Ceratocystisfimbriata sensu lato isolates from Eucalyptus in South Africa

Ceratocystis fimbriata sensu lato (s.l.) is an important pathogen of Eucalyptus. Pathogenicity of isolates has typically been evaluated by inoculating seedlings under greenhouse conditions. It is, however, not clear how accurately this reflects pathogenicity under field conditions. In this study, five techniques to potentially screen C. fimbriata isolates for their relative pathogenicity to Eucalyptus were compared. These included: in vitro growth comparisons on artificial media; inoculations on apples; inoculation on Eucalyptus seedlings in a greenhouse; inoculations on Eucalyptus bolts freshly cut from stems of young trees; and field inoculations on young trees. Eight isolates of C. fimbriata s.l. collected from various areas in South Africa were used. There was considerable variation in growth in culture and aggressiveness of the eight isolates. Field inoculations on young trees were best correlated with inoculations of bolts (r = 0.76). Lower correlation coefficients were obtained with seedlings (r = 0.59), apple inoculations (r = 0.56), and in vitro colony growth (r = 0.42). Inoculation of bolts provides a rapid and reliable method to screen isolates of C. fimbriata s.l. for pathogenicity to Eucalyptus.     

Sexual behaviour of Armillaria heimii and A. mellea isolates from Africa

Thirty isolates of Armillaria heimii from western, eastern and southern Africa were cultured for fruit body production in the laboratory. Most isolates fruited easily. Investigation of single-spore progenies revealed that all the isolates do not have the same sexual behaviour: some are heterothallic and unifactorial while others are homothallic. Two African isolates belonging to the species Armillaria mellea also appeared homothallic. Unifactorial heterothallism has not previously been described in Armillaria, species. Homothallic behaviour has been reported only in a rare European species Armillaria ectypa and in the Japanese subspecies Armillaria mellea ssp. nipponica.     

Pathogenicity of some isolates of Seiridium (Coryneum) cardinale,agent of cypress canker disease

Ramets of 200 clones of Cupressus sempervirens were inoculated with 6 isolates of Seiridium cardinale. No significant differences were found among isolates, while there were highly significant differences among clones when length and width of canker were assumed as discriminating traits.     

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.     

Geographical distribution of Erwinia salicis strains,the cause of watermark disease of willows

Eighty-six isolates of Erwinia salicis, the causal agent of watermark disease of willow, were obtained from culture collections and collected from various willow plantations in south-east England. These isolates were characterized by their reaction in a competitive enzyme-linked immunosorbent assay, by enzyme electrophoresis, and for lysis by a panel of five bacteriophage. Eighty-one isolates had similar characteristics and clustered at a level of similarity of at least 60%. Five isolates were found to have less than 20% similarity to the other E. salicis. Antiserum raised against E. salicis (NCPPB 2535) was found to be specific to E. salicis and did not cross-react with other bacteria tested. No geographical clustering of electrotypes was found: however, Dutch and English isolates could be distinguished by bacteriophage typing. The local distribution of electrotypes collected from diseased trees suggests that the bacterium is spread via the propagating material and that tree to tree spread is rare.     

Comparison of Gremmeniella abietina isolates from Pinus sylvestris and Pinus contorta in terms of conidial morphology and host colonization

Gremmeniella abietina isolates from Pinus contorta in northern Sweden produced, in vitro, shorter conidia with fewer septa compared with isolates from Pinus sylvestris in the southern part of the country. After mycelial inoculation of shoots with G. abietina isolates from both host species, the resulting necroses were longer in P. sylvestris than in P. contorta. Keeping seedlings in artificial mild winter climate or detaching shoots from the seedling before inoculation caused longer necroses. No host specificity in colonization was found. Isolates from P. sylvestris caused longer necroses than did isolates from P. contorta, and both types of isolates caused longer necroses in P. sylvestris than in P. contorta. The differences found between the two G. abietina populations probably reflect regional variation in the fungus.