Genetic variation of the resin top fungus in Finland as determined by random amplified microsatellites (RAMS)

The analysis of random amplified microsatellite (RAMS) markers among aecia of the causal agent of the resin top disease (Peridermium pini) on Scots pine (Pinus sylvestris) suggested that the genetic variation between the populations of this rust fungus is low in Finland. The method used allowed the identification of heterozygotic aecia in two loci, where the degree of heterozygosity was, however, low. The RAMS patterns of the Finnish aeciospores, other aeciospores from Thetford, UK and Cronartium flaccidum from Italy were highly similar suggesting that the autoecious and heteroecious forms of these rust fungi would be genetically closely related.     

Studies on free amino acid contents in the aeciospores of Endocronartium pini and Cronartium flaccidum

Aeciospores of Endocronartium pini (Peridermium pini) and Cronartium flaccidum were analysed with regard to free amino acid content. As a rule the quantity in Endocronartium pini is greater than in Cronartium flaccidum. Twenty four amino acids were identified. Regarding thirteen amino acids, the quantity in Endocronartium was greater than in Cronartium; three amino acids were greater only in one year; two amino acids were about the same in the aeciospores of both species, and six occurred only in one analysis or as a trace. The beetle, Lagria hirta, has been observed to eat the acciospores of Endocronartium pini.     

Variation in virulence of Cryphonectria hypovirus 1 in Macedonia

We investigated variation in virulence of Cryphonectria hypovirus 1 (CHV‐1) to the chestnut blight fungus, Cryphonectria parasitica, in Macedonia by inoculating chestnut stems in the field. We inoculated trees with two isolates of C. parasitica, each infected with one of five isolates of CHV‐1, four of which were the same for both fungal isolates. Two virus isolates, [Sk28] and [Sk47], were significantly more virulent than the others when compared in the same fungal host isolates, as measured by reduced canker growth and increased callus formation. Mycelial growth rate in vitro was weakly correlated to canker growth or callus formation and is therefore not a reliable predictor for virulence. We found significant fungus × virus interactions for canker growth and callus formation, which seems due mainly to one virus isolate. Significant interactions were not expected because the two fungal host isolates are members of the same clone that is dominant in Macedonia and most of southeastern Europe. Phenotypic variation for response to viruses, therefore, is greater than variation revealed by the genetic markers used to define clones. More than half of the trees inoculated with virus‐free controls were dead within 2 years, and the 30% still alive after 5 years had cankers with extensive callus formation, indicating that natural virus transmission had occurred after inoculation. In contrast, only 2% of the trees inoculated with virus‐infected isolates were dead after 5 years. Hypoviruses naturally occurring in Macedonia reduce canker development and tree mortality similarly to those in other parts of southern Europe, and therefore, may have good potential for biological control of chestnut blight.     

Incomplete movement of Cryphonectria hypovirus 1 within a vegetative compatibility type of Cryphonectria parasitica in natural cankers on grafted American chestnut trees

American chestnut trees, grafted in 1980 from large survivors, were inoculated in 1982 and 1983 with four white (European) hypovirulent strains of Cryphonectria parasitica, infected with C. hypovirus 1 (CHV1); this hypovirus has been shown to be capable of moving rapidly within the mycelium of a vegetative compatibility (vc) type of C. parasitica in blight cankers. Using a 49‐cell lattice plot, 17.8×17.8 cm, the spatial patterns and frequencies of white and pigmented isolates and white and pigmented vc types were investigated within superficial cankers on the grafts located outside the hypovirulent‐strain‐inoculated zone. Four of six cankers assayed contained white isolates, and three of the four had random spatial patterns of white isolates, based on join‐count statistics. Vc tests, using pigmented isolates and pigmented single‐spore colonies of white isolates, indicated that the majority of white and pigmented isolates recovered from each of two cankers assayed were in one vc type. White and pigmented lattice‐plot cells of the same vc type were frequently in contact with each other, indicating incomplete movement of CHV1 within a vc type. Nine and 10 vc types were found in the two cankers; it is hypothesized that small, white vc type areas in each canker may be a source of CHV1 transmission to the major vc types. Based on join‐count statistics, the spatial pattern of the single, major vc type in one canker was non‐random (aggregated), whereas the other canker had a random major vc type pattern. White and pigmented in vitro variants (sectors) of C. parasitica, that resemble white and pigmented in vivo variants in spatial contact and vc compatibility, were intermediate hypovirulent and virulent on forest American chestnuts, and dsRNA positive and negative, respectively. Incomplete movement of CHV1 within a vc type could be a major cause of the prevalence of pigmented isolates in superficial cankers on chestnut trees.     

High genetic diversity of Fusarium circinatum associated with the first outbreak of pitch canker on Pinus patula in South Africa

The disease known as pitch canker results from infection of Pinus species by the fungus Fusarium circinatum. This fungus also causes a serious root disease of Pinus seedlings and cuttings in forestry nurseries. Pinus radiata and P. patula are especially susceptible to the pathogen, but there are no records of pitch canker on P. patula in established plantations. To date, only planting material of this tree species in nurseries or in plantations at the time of establishment have been infected by F. circinatum. Symptoms of pitch canker have recently emerged in an established P. patula plantation in South Africa and this study sought to determine whether the symptoms were caused by F. circinatum. Isolates from cankers were identified as F. circinatum using morphology and DNA-based diagnostic markers. Microsatellite markers were then used to determine the genetic diversity of a collection of 52 isolates. The entire population included 17 genotypes representing 30 alleles, with a greater number of genotypes collected from younger (three- to six-year-old) than older (12- to 19-year-old) trees. Both mating types of F. circinatum were present, but no evidence of sexual recombination was inferred from population genetic analyses. This is the first record globally of pitch canker on P. patula trees in managed plantations. It is of significant concern to South Africa, where P. patula is the most important Pinus species utilised for plantation forestry.     

Cultural and PCR‐based detection of Septoria musiva in inoculated hybrid poplar stems

The fungal pathogen Septoria musiva can be difficult to isolate from cankers that result from its colonization of poplar stems, and its persistence in these cankers has not been well studied. In order to compare cultural and polymerase chain reaction (PCR)‐based assays for detection of S. musiva in cankers, stems of susceptible hybrid poplar clone NC11505 were wounded and inoculated in August 2003. At 8, 16, 24 and 32 weeks after inoculation (October and December 2003, February and April 2004, respectively), 110 inoculated stems (plus controls) were harvested and a semiselective culture medium was used in attempts to detect the pathogen in bark and wood. Six chips of bark and six chips of underlying wood from one half of each canker were incubated on the semiselective medium for 2 weeks until pycnidia and conidia of S. musiva could be identified. The number of positive cankers and positive chips (out of six attempts per tissue per canker) was recorded. The remaining halves of cankers from subsets of 70 inoculated stems (plus controls) of those harvested in October 2003 and April 2004 were tested using a PCR‐based assay. Three chips of bark and three chips of underlying wood were ground, and DNA was extracted and then amplified using S. musiva‐specific primers designed from the internal transcribed spacer (ITS) region of nuclear rDNA repeats. The number of positive cankers and positive chips (out of three attempts per tissue per canker) was recorded. For both assays, the number of positive cankers and the number of positive chips per canker decreased with time. Using either assay, however, the pathogen was still detected from at least 49% of cankers at 32 weeks after inoculation.     

Susceptibility and lesion development in Scots pine saplings infected with Peridermium pini in northern Finland

Susceptibility of Pinus sylvestris provenances from northern Finland to Peridermium pini and lesion development caused by the rust were investigated by inoculating 768 saplings. Resin‐top disease symptoms on the stem including stem swelling, occurrence of spermatial fluid and rust aecia, were recorded over a period of 9 years. The disease incidence was low (3.6%), varying within the range of 0–9.4% among pine provenances and 3.4–3.9% among spore sources in 1994–2002. Aecia were produced for the first time 2–4 years after inoculation, appearing mainly in early June, 2–9 years after inoculation. The aecia ruptured in late July to early August, and the overall sporulation lasted for 1–8 years among the pine provenances. Spermatial fluid was observed regularly in the year prior to aecia development 2–7 years after inoculation. Annual fresh swelling was observed for 1–6 years after inoculation. No statistically significant differences occurred neither between the pine provenances, the spore sources nor the years with regard to all the major disease variables. The results suggest that Scots pine provenances from northern Finland are highly resistant to P. pini regardless of the pine provenance or spore source.     

Inoculation of young pine seedlings with Peridermium pini from north-east Scotland

Seedlings of Pinus sylvestris, P. nigra ssp. laricio, and P. mugo vars mughus, rostrata and pumilio were inoculated at their cotyledon stage with aeciospores of Peridermium pini from north-east Scotland. Infection was determined by the presence of both mycelia and haustoria in free-hand or paraffin sections. In the second growing season, infected seedlings showed symptoms such as stem-swelling, development of spermogonia and aecia, and death. In a second experiment, young P. sylvestris seedlings from seven seed sources collected in Great Britain were inoculated and the infection were examined after 6 weeks. In the samples of 50 seedlings from each source, 30–70% seedlings were infected.     

Susceptibility of Ribes spp. to pine stem rusts in Finland

Susceptibility of Ribes spp. to three pine stem rusts, Cronartium ribicola, Cronartium flaccidum and Peridermium pini, was investigated by inoculations both in laboratory and in greenhouse conditions, and by observing sporulation on Ribes spp. leaves under natural and artificial inoculum in 16 experiments. Twenty‐seven Ribes spp. cultivars were inoculated in 2000–2004 using 41 sources of C. ribicola aeciospores from a wide geographic range, and six Pinus spp.; 51 sources of C. flaccidum and 11 sources of P. pini from Pinus sylvestris. The results were very similar both after artificial inoculations and observations under natural inoculum in repeated experiments over the years. Cronartium ribicola uredinia and telia developed frequently or moderately on nine Ribes nigrum cultivars but were not present on two cultivars. Sporulation developed on five Ribes rubrum cultivars but was absent on two cultivars. Three Ribes uva‐crispa cultivars were only weakly susceptible to C. ribicola, while all R. alpinum cultivars were resistant to the rust. The other Ribes spp. tested, Ribes niveum, Ribes aureum, Ribes odoratum, Ribes sp. × nigrolaria, Ribes glandulosum and Ribes × culverwellii Jostaberry, were all susceptible to C. ribicola. Cultivars of R. nigrum were more susceptible than those of any other species. No significant variation in virulence of the aeciospores was observed between and among Ribes hosts over the years. Cronartium flaccidum and Peridermium pini did not form any uredinia or telia on Ribes spp. in any of the experiments.     

Endothia gyrosa associated with severe stem cankers on plantation grown Eucalyptus nitens in Tasmania,Australia

Severe stem cankers in Eucalyptus nitens, from a 14-year-old mixed provenance plantation, were associated with infection by Endothia gyrosa, present in its teleomorph state. Surveys of incidence among canker severity classes were carried out in a thinned and pruned stand and an adjacent unthinned and unpruned stand within the affected plantation. No differences in incidence among the canker severity classes were found between the thinned/pruned and unthinned/unpruned stands or between different crown dominance classes within the unthinned/unpruned stand. However, the incidence among canker severity classes was strongly associated with bark roughness with 97% of rough-barked trees developing either annual cankers or cankers causing cambial damage. Stem cankers were found on only 11% of trees with smooth bark. Bark roughness in E. nitens was shown to differ significantly between provenances. Deployment of provenances prone to rough bark in routine plantation establishment may pose a risk of damaging stem canker outbreaks.     

A method for estimating white pine blister rust canker age on limber pine in the central Rocky Mountains

Epidemiological studies of white pine blister rust on limber pine require a temporal component to explain variations in incidence of infection and mortality. Unfortunately, it is not known how long the pathogen has been present at various sites in the central Rocky Mountains of North America. Canker age, computed from canker length and average expansion rate, can be used to estimate infestation origin and infection frequency. To investigate relationships between canker lengths and canker ages for limber pine, we collected live white pine blister rust branch and stem cankers from three locations in Wyoming and two locations in Colorado. We quantified relationships between various measures of canker length and an estimate of canker age based on dendrochronological analysis. Total branch canker length was strongly, negatively correlated (r = ?0.79) with the first year of incomplete, annual ring formation (canker age). Mean longitudinal canker expansion rate was 8.4 cm year^?1 for branch and stem cankers where branches distal to the canker were either dead or alive. Annual longitudinal canker expansion, however, was significantly greater on a stem or branch where the portion distal to the canker was alive (11.5 cm year^?1) rather than dead (7.1 cm year^?1). For branches or stems, proximal expansion rate (i.e., toward or down stem) averaged 4.9 cm year^?1. The circumferential canker expansion rate (around branch or stem) was greater for stem cankers (8.3 cm year^?1) than for branch cankers (6.2 cm year^?1). Additional site and host tree covariates did not improve prediction of canker age. Two simple linear equations were developed to estimate a canker age from total length of a canker with the distal portion either alive or dead. An appropriate sample of canker ages can be used to determine how long a limber pine stand has been infested with white pine blister rust and how frequently infections have occurred.     

Development of microsatellite markers for Thekopsora areolata,the causal agent of cherry spruce rust

Cherry spruce rust is a fungal disease of Norway spruce cones caused by Thekopsora areolata and responsible for significant losses in seed production in Sweden and Finland. Here, we report the first set of nine microsatellites, which will allow an effective genetic fingerprinting of T. areolata. The markers were isolated using the FIASCO method and were characterized using DNA from 49 single aecia sampled from spruce cones in three different seed orchards in Sweden. Eight of the nine markers were shown to be polymorphic among the aecia. The markers were unlinked and are therefore suitable for future population genetic studies.     

Aeciospores of Cronartium flaccidum,C. ribicola and Endocronartium pini show no differences in morphology

The morphology of the aeciospores of the pine stem rust fungi Cronartium flaccidum, Cronartium ribicola and Endocronartium pini, collected from nine locations in Finland, Sweden and Italy, was compared. Surface structures of 900 aeciospores were visually classified using a scanning electron microscope, and dimensions of 1100 aeciospores were measured from micrographs. The data grouping was tested with discriminant analysis. The spores had well-defined smooth areas and annulate warts on their surface. E. pini, C. flaccidum and C. ribicola could not be distinguished by the surface structures of the spores. The grouping of spores by dimensions into sample locations was statistically significant by one discriminant function, while the grouping into species was not. The observed similarities corroborated the current views on the close relationship of C. flaccidum and E. pini.     

Formation and growth of stem cankers caused by Gremmeniella abietina on young Pinus contorta

From 1990 to 1995, the formation and growth of stem cankers caused by Gremmeniella abietina on Pinus contorta var. latifolia was studied in three stands in northern Sweden. The stands were planted in 1976–80. The total number of cankers on 756 trees that were individually followed increased from 233 to 477 during the 5‐year period. With 42.0% of the cankers, the pathogen entered through or from the base of diseased branches, and 33.6% through visually undamaged bark. Most of the cankers were within 100 cm of the ground. In one of the three areas, the cankers were evenly distributed within 180 cm of the ground. The frequency of cankers facing north exceeded those facing south. The average vertical length of cankers had increased, 55.6% of cankers had increased their percentage coverage of the stem girth; 13.8% had fully girdled the stem. At two of the sites, there was a negative correlation between canker coverage of the stem circumference and tree height increment.     

Relative susceptibility to blister rust caused by Cronartium flaccidum of several species of pine

At Florence, Italy, several species of pine were experimentally infected with blister rust. Inoculations were carried out on 3 and 15 months-old seedlings. After antificial and natural inoculation, Brutia, Aleppo, Austrian, Swiss mountain, Maritime and Italian stone pine showed pyenia and aecia. Spotted seedlings of Ponderosa pine showed only mycelium of C. flaccidum in needle and stem tissues. The exotic species seemed to have a very high degree of resistance to blister rust.     

Informative value of canker morphology on the presence or absence of virus infection in chestnut blight cankers

Chestnut blight destroyed the native chestnut forests in North America and also severely affected the European chestnut trees after its introduction in the 20th century. The ascomycete fungus Cryphonectria parasitica is responsible for this serious disease and causes lethal bark cankers on susceptible chestnut trees. In Europe, however, an infection of C. parasitica with Cryphonectria hypovirus 1 (CHV‐1) causes hypovirulence in C. parasitica and reduces the severity of the disease. Hypovirulence biologically controls chestnut blight in many regions to date. In this study, our goal was to determine morphological canker characteristics that are indicative of virus presence or absence in C. parasitica. We investigated 677 chestnut blight cankers from seven different geographic locations across Europe. For each canker, we assessed canker length, stem encircling, canker depth, presence of sporulation, canker activity and virus infection. We statistically analysed the informative value of these morphological characteristics for the presence or absence of CHV‐1. However, we did not find reliable indicators. Our logistic regression analysis revealed that virus infection of C. parasitica is not clearly related to canker morphology. This implies that fungal isolations from chestnut blight cankers and assessments in the laboratory are required to determine infection with CHV‐1 unequivocally.     

Botryosphaeriaceae species causing canker and dieback of English walnut (Juglans regia) in Italy

English walnut (Juglans regia) is an important nut crop worldwide and is currently considered emerging in Italy. Botryosphaeriaceae fungi cause symptoms including cankers, discoloration and dieback, and several species are reported across the world on walnut. In this study, symptomatic trees from an orchard in Southern Italy showing branch dieback, cankers, wood discoloration and gummosis were surveyed. Three different fungi from the Botryosphaeriaceae were consistently isolated from symptomatic tissues. Representative isolates were characterized using morphological and molecular approaches based on conidial morphology, optimum growth temperature and the comparison of DNA sequence data from the ITS, tef1‐α and tub2 loci. Three species were identified: Botryosphaeria dothidea, Neofusicoccum mediterraneum and Neofusicoccum parvum. Pathogenicity tests on detached fruits and potted plants showed that all three species were pathogenic. To our knowledge, this is the first report of these Botryosphaeriaceae species causing canker and dieback on English walnut in Italy.     

Bacterial canker of Maackia amurensis var. buergeri caused by a putative Pseudomonas syringae

A new disease of Maackia amurensis var. buergeri was recently found on the northern island of Hokkaido, Japan. Affected trees were heavily damaged and had cankers on both trunks and branches. After natural infection, a series of swellings on the bark surface developed longitudinally. These swellings burst and coalesced to become long cankers. It is proposed that the disease be designated ‘bacterial canker of Maackia’. The causal pathogen was isolated and characterized tentatively as Pseudomonas syringae on the basis of laboratory tests. Pathogenicity of the bacterium was confirmed by inoculation into the host.