RAPD variation in Gremmeniella abietina attacking Pinus sylvestris and Pinus contorta in northern Sweden

Genomic DNA from 81 isolates of Gremmeniella abietina collected from eleven plantations each of Pinus sylvestris and Pinus contorta in northern Sweden was studied using RAPD markers. The DNA variation between and within populations and the race and type distribution of G. abietina populations, causing symptoms similar to those of the North American race, were studied. The degree of genetic similarity was greater among G. abietina isolates from the same geographical areas than among isolates from different geographical areas, regardless of whether they were isolated from P. sylvestris or P. contorta. RAPD variation was greatest in the central parts of northern Sweden, suggesting that sexual reproduction has been somewhat more important there than further north or south. Only the RAPD fragments characteristic of the EU race of G. abietina were found in the material tested. The RAPD pattern described as characteristic of the northern type within the EU race was identified in 62% of the isolates. Divergence from the expected profile was due to differences in occurrence of fragments OPA12-1400 and 12-1500. This indicates that this part of the RAPD profile cannot be treated as diagnostic for the northern type. A conclusion of practical importance is that there is a considerable risk of G. abietina spreading from infected P. contorta plantations to adjacent areas with indigenous P. sylvestris regeneration, and vice versa, owing to the indicated lack of host-specificity of the pathogen. It is possible, however, that host-specific strains exist, but do not differ in their RAPD profiles.     

From the Editor

Abstract

In 2001 large areas of Pinus sylvestris L. stands in Sweden were attacked by Gremmeniella abietina (Lagerb.) Morelet. The resultant damage was assessed in five genetic field trials and one clonal seed orchard, with the aims of quantifying the genetic variation in tree responses to G. abietina, studying the genetic relationship between G. abietina susceptibility and growth traits, and examining the implications of the results for P. sylvestris breeding. A comparison of G. abietina damage between plus-tree progenies and seed stand check-lots in each of four tree classes (dominant, co-dominant, subordinated and suppressed) found no significant differences except in the suppressed tree class, where plus-tree progenies were damaged less severely than trees from the seed stands. Narrow- and broad-sense heritability for G. abietina damage traits were in the range 0.22–0.42. Genetic correlations between G. abietina damage and growth traits were weak and not significantly different from zero. The results imply that selection for growth capacity in P. sylvestris breeding programmes using plus-tree selection or field progeny tests does not increase susceptibility to G. abietina. In addition, there is scope for selecting genotypes with increased resistance if this is required in the future.     

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.     

Growth reactions of a Gremmeniella abietina isolate and Scots pine embryogenic tissue cultures differ in a host–parasite in vitro system

Summary The aim of this study was to screen Scots pine embryogenic tissue culture material to Gremmeniella abietina. According to the results Scots pine (Pinus sylvestris) embryogenic tissue lines (27 lines) could be ranked based on significant differences in G. abietina effects estimated by changes in glucosamine concentrations, growth, and dry matter contents of inoculated tissue pieces. The ranking order of the extreme tissue lines stayed basically similar, regardless of the variable measured. These findings imply a possibility to use the ranked embryogenic tissue lines in more detailed investigations of interactions between G. abietina and its host. This is the first report of using embryogenic tissue cultures in studying host–parasite interaction between Scots pine and G. abietina.     

Development of fruiting bodies of large tree type of Gremmeniella abietina var. abietina and timing of infection on Scots pine inn northern Finland

The formation and maturing of the large tree type Gremmeniella abietina var. abietina fruiting bodies and their sporulation were investigated for 3 years on Scots pine (Pinus sylvestris) in northern Finland. This was done by monthly assessment of shoots in the field and in the laboratory. Infection caused by G. abietina var. abietina was dated on Scots pine by monthly covering with pollination bags and exposing branches during the growing season. Pycnidia appeared between August and September, 1 year after infection, and they started to release conidia between late June and early July, 2 years after infection. Fresh pycnidia and microconidia were formed during the following August and September in the infected shoots. The causal large tree type of G. abietina var. abietina did not produce apothecia on branches within 3 years of infection. Monthly covering and exposing branches showed that infection took place mainly between June and July.     

Susceptibility of six pine species to Gremmeniella abietina isolates from Spain

Inoculation experiments were performed in order to evaluate the virulence of Gremmeniella abietina isolates from Spain on the main pine species planted in the Iberian Peninsula, as well as the influence of seedling age on this virulence. Two different experiments were carried out with four isolates of G. abietina from Spain. The greenhouse experiments consisted of seedling inoculations. One‐ and 2‐year‐old seedlings of the following five pine species were used: Pinus halepensis, Pinus pinea, Pinus pinaster, Pinus sylvestris and Pinus uncinata; also, 1‐year‐old seedlings of P. nigra were inoculated. The relative necrosis length (RNL) caused by the pathogen after 130 days was used as a response variable. The laboratory experiments were performed on 2‐ to 6‐year‐old internodes of the above pine species excluding P. uncinata. The necrosis length after 6 weeks of incubation was measured. The results have shown that all G. abietina isolates were pathogens on seedlings of these six pine species and seedlings of P. halepensis were consistently the most susceptible ones, although it is important to take into account that all the isolates used in the present work were isolated from P. halepensis, the only pine species in Spain where G. abietina has been recovered up to now. The susceptibility of the other pine species depended on the age of the seedlings.     

Incidence of Gremmeniella abietina in planted seedlings of Picea abies and Pinus sylvestris in northern Finland

The ascomycete Gremmeniella abietina causes a disease (scleroderris canker) on conifers. The pathogen kills terminal shoots and branches of Pinus sylvestris and Picea abies, sporulates on host shoots and causes leader changes (i.e. dieback). Damage caused by G. abietina was investigated in young P. abies and P. sylvestris plantations in northern Finland. Side branches from seedlings of both species and the main stems of P. sylvestris were collected from three sites in two locations (Poksa 1 and 2, Kivalo) in northern Finland. The number of cankers and leader changes was counted in branches of each age (i.e. year) in a total of ca. 6300 shoots of P. abies and 1200 shoots of P. sylvestris. Cankers were common on both P. abies and P. sylvestris in Poksa 1 (2002–2008) and on P. abies in Poksa 2 (1998–2001). In Kivalo, cankers occurred only sporadically on P. sylvestris. Leader changes were most frequent in 1999–2009 in Poksa 1, in 2001–2009 in Poksa 2 and in 2003 in Kivalo. Cankers and other symptoms of infection were more obscure on P. abies than on P. sylvestris. According to both conidial morphology and molecular analysis, the strain of G. abietina infecting both P. abies and P. sylvestris was small‐tree type (STT). This is the first report of G. abietina, STT or B type, injuring P. abies in plantations.     

Survival and vitality of Gremmeniella abietina on Pinus sylvestris slash in northern Sweden

Survival and vitality of Gremmeniella abietina on Pinus sylvestris slash was studied in northern Sweden during 2003 and 2004. Once a month between September 2003 and April 2004, two to three trees were cut down and debranched. Shoots with pycnidia were sampled at the felling date and then at every consecutive month. The percentage of germinated conidia from each shoot was calculated after 24, 48 and 72 h incubation. The vitality of G. abietina pycnidia in the slash remained high the whole period. Intact pycnidia were found on slash several months after the time of conidial sporulation, which indicates that new pycnidia may be produced on dead pine branches. Sampling of shoots from slash on 13‐ to 18‐month‐old clear‐cuts showed conidial germination capacity as high as in pycnidia collected in fresh slash. Due to survival of G. abietina in slash it is recommended to postpone planting of P. sylvestris seedlings in northern boreal areas to the third vegetation period after sanitary clear‐cuts.     

Infection experiments with Gremmeniella abietina on seedlings of Norway spruce and Scots pine

Conidia of Gremmeniella abietina infected and caused disease symptoms in annual shoots of both Scots pine (Pinus sylvestris) and Norway spruce (Picea abies) seedlings. In Norway spruce shoots the infection remained largely latent, with only a few seedlings showing symptoms. Mycelial growth inside the shoots was faster in Scots pine than in Norway spruce and was favoured by low temperature in both hosts. The shoots of Norway spruce seedlings had higher endophyte populations than those of Scots pine, and the populations were decreased by low temperatures. Reductions in the normal epiphytic or endophytic flora by acid mist treatments seemed to favour the development of G. abietina.     

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 of the life cycle of Gremmeniella abietina on Scots pine in southern Sweden

Aspects of the life cycle of Gremmeniella abietina (Lagerb.) Morelet were studied from 1988 to 1990 in stands of Pinus sylvestris L., 16–32 years old, in southern Sweden, initiated in 1988 with a widespread outbreak of the disease. Pycnidia started to release conidia in late spring and apothecia began to release ascospores in summer. Latent infections could still be detected one year after their establishment by cultivation of healthy looking shoots on agar petri dishes. G. abietina appeared to have a mainly biennal life cycle, as most spores were released two years after infection of the shoot.     

Infection of Scots pine seedlings by Gremmeniella abietina during summer under different inoculum potential

The connection between natural conidia dispersal of Gremmeniella abietina (A‐type) and the disease incidence and severity in first and second year conifer seedlings of Pinus sylvestris was studied in central Finland. The seedlings where exposed to natural infection for a 3‐week period throughout the growth season, followed by a 3‐week incubation period in a growth chamber to promote symptom expression. In second year seedlings the infection periods in May–June had a similar effect as regards disease outbreak and roughly half of the successful infections occurred during these periods. However, the first year seedlings were more severely diseased after the infection period in late July/early August, although the inoculum density was lower during this time compared with the period in late June/early July. This result is consistent with earlier studies. The effect of age and growth phase of the seedlings, temperature sum and the number of conidia on the disease occurrence is discussed and compared with the results of earlier studies in which artificial inoculation was employed.     

Susceptibility of different provenances of Pinus sylvestris,Pinus Contorta and Picea abies to Gremmeniella abietina

In a randomized block factorial experiment, 1200 seedlings from four provenances, each of Pinus sylvestris, Pinus contorta and Picea abies were inoculated with conidia (2 × 10⁴ and 1 × 10⁶ conidia/seedling) of Gremmeniella abietina (Brunchorstia pinea), isolated from P. contorta plantations in northern Sweden. A further 600 seedlings were left as controls. The occurrence of symptoms and the extension of dead tissues on the annual shoots were recorded 13 months after inoculation. Only the higher spore dose resulted in significant infection. P. sylvestris and P. contorta seedlings were equally susceptible (53% infected), and significantly more infected than P. abies seedlings (39%). The annual shoots of P. sylvestris and P. abies were affected to 43% and 37% of their length, which was significantly more than 15% of the length of P. Contorta shoots. The frequency of affected seedlings differed between the most southern and northern provenances of P. sylvestris and P. abies, and between the northwestern and the south-eastern provenance of P. contorta Twenty-six months after inoculation, a higher proportion of P. contorta seedlings than P. sylvestris and P. abies seedlings had recovered, and a lower proportion of P. contorta than P. sylvestris and P. abies seedlings had died. This paper discusses why P. sylvestris was found to be more susceptible to G. abietina than P. contorta in this experiment, while the reverse is found in plantations in northern Sweden.     

Effect of fungicides,endophytes and fungal filtrates on in vitro growth of Spanish isolates of Gremmeniella abietina

The effect of eight fungicides and 15 endophytes isolated from twigs of healthy Pinus halepensis trees on the growth rate of four Spanish isolates of the pathogen Gremmeniella abietina was evaluated in vitro. In the fungicide experiments, four doses of each fungicide tested were added to the growth medium. In the endophyte experiments, dual cultures endophyte‐pathogen were paired in Petri dishes. Furthermore, growth of three G. abietina isolates was evaluated on malt agar with pine needle extract amended with filtrates from cultures of endophyte E14, which produced a brownish compound apparently inhibiting G. abietina growth. The results obtained suggested that chlorothalonil and daconil were the most suitable fungicides at low doses to reduce growth of G. abietina isolates from Spain. Four of the endophytes tested in vitro showed strong antagonistic activity against G. abietina and deserve further testing in vivo. The endophyte E14 produced in vitro a brownish compound which almost completely inhibited mycelial growth of G. abietina isolates from Spain.     

Rapid living crown reduction caused by Gremmeniella abietina affects foliar nutrient concentrations of Scots pine

The foliar chemistry of diseased and healthy trees was studied one growing season after severe reduction in living crown caused by Gremmeniella abietina in four young Scots pine (Pinus sylvestris) stands. Sample trees were chosen pairwise on the basis of the living crown length: a diseased tree with about 50% live crown reduction and a healthy tree in each pair. Fifteen elements were determined in the youngest healthy needles on the lateral top shoots of each sample tree. Diseased trees had higher foliar boron, manganese and sodium concentrations and lower magnesium, iron, zinc, copper, potassium, nitrogen and sulphur concentrations compared to the healthy trees. Foliar calcium, aluminium, phosphorus, carbon and hydrogen concentrations did not differ between the diseased and healthy trees, except for P and A1 in two of the stands when the stands were analysed separately. Significant correlations between the needle element concentrations and crown ratio (length of the living crown/tree height) were found especially for B (increasing B with decreasing crown ratio) and for Mg, Fe and Zn (decreasing concentrations with decreasing crown ratio). The effect of G. abietina-induced living crown reduction on tree nutrient status and the role of these mineral nutrients in the susceptibility are discussed.     

Microspatial genetic diversity of Dothistroma septosporum

The distribution of genetic diversity in a local population of Dothistroma septosporum was determined on a microspatial geographical scale. The study was conducted in a seed orchard of Pinus nigra divided into four plots, each of 0.84 ha in area. Ninety‐two isolates were obtained from needles with red band needle blight symptoms. Molecular variance, Mantel test and autocorrelation spatial analyses were performed with the use of random amplified microsatellites markers to test the null hypothesis of a random distribution of genotypes. The groups of isolates from individual plots demonstrated small differences as regards intrapopulation variation. The mean contribution of polymorphic loci for isolate groups was almost 77%, genetic diversity 0.270, while the Shannon coefficient was 0.389. The Mantel test demonstrated a significant, positive correlation between Huff's genetic distance and geographical distance. Significant spatial genetic structure was detected with autocorrelation coefficients being significant in the first two distance classes up to about 8–12 m.     

Gremmeniella abietina,Phacidium infestans,and other causes of damage in alpine,young pine plantations in Norway

Field studies of damage and mortality were carried out through 16 years in six young plantations, mostly of Pinus contorta, but also P. sylvestris and P. sibirica. Differences between pine species, P. contorta provenances, sites, and soil treatments are reported and discussed, as are influences of environment, climatic conditions, snow cover, and nutrient deficiency. Gremmeniella abietina was by far the most important cause of mortality; there were great differences between the sites as to development and seriousness. Other important causes of mortality and damage were Phacidium infestans, elk (Alces alces L.), voles, and snow pressure.     

Marker trait association for autumn cold acclimation and growth rhythm in pinus sylvestris

The main objective of this study was to identify quantitative trait loci (QTLs) involved in the genetic control of growth rhythm and autumn cold acclimation in Pinus sylvestris L. Eighteen full‐sib families of P. sylvestris with parental origins between latitudes 62 and 67° N in Sweden were assessed for autumn cold acclimation (frost injury measured from artificial freeze testing) and growth rhythm during the first two growing seasons. One family north × south with 108 individuals was used for the construction of one linkage map for the male and one for the female parent using 286 markers from random amplification of polymorphic DNA. Marker trait associations (QTLs) for shoot elongation, growth cessation and cold acclimation were found on both maps. Some QTLs were verified in two additional north × south families. Proportions of the additive genetic variance explained by the markers (R ²/_G) indicated that genetic factors of large effect were involved in all investigated traits. Single or multiple markers accounted for between 16 and 47% of the additive variance in annual shoot elongation, between 42 and 79% of the additive variance in growth termination, and between 7 and 31% of the additive variation in autumn cold acclimation. The study confirms that there are major QTL regions on different linkage groups controlling a large part of the variation for growth rhythm and autumn cold acclimation in P. sylvestris.     

The effect of acid rain treatments on the susceptibility of Pinus sylvestris to Gremmeniella abietina

Scots pine (Pinus sylvestris) seedlings were planted in a nursery bed 20 cm thick. The substrate had been removed from an illuvial horizon in the soil underlying a stand of Scots pine in Finland, which had suffered from severe infection by Gremmeniella abietina. The seedlings were irrigated for 3 years, applying three pH levels (2.5, 3.5, and 4.5) using sulphuric acid, nitric acid, and a 3:1 mixture of sulphuric and nitric acid. The pH of the pure water used in the control treatment was 6.1–6.4. The seedlings were inoculated with the conidiospores of G. abietina to find out their susceptibility to infection with the Scleroderris canker disease. Because to the adequacy of its buffering capacity, the soil was observed to become more acidic only in the treatments at pH 2.5. The concentrations of extractable Ca and Mg cations decreased while the concentration of Al and Fe increased with soil acidification during the 3-year experiment. Acidification did not increase the susceptibility of Scots pine to infection by the Scleroderris canker. Seedling growth was at its maximum in the acidification treatments where most nitrogen was added.     

Tree mortality caused by Gremmeniella abietina in a subalpine afforestation in the central Alps and its relationship with duration of snow cover

Tree survival and causes of mortality were studied in an experimental afforestation in the upper subalpine forest zone in the Swiss Alps. A total of 59.8% of Pinus cembra and 45.6% of Pinus mugo were killed by Gremmeniella abietina during the first 20 years after planting, compared to 1.5% of Larix deddua trees. The mortality rates caused by G. abietina were highly correlated with the duration of snow cover in spring. Tree losses were lowest at sites where the snow melted early and highest at sites where the snow ablation was delayed in spring. Tree mortality varied greatly between years. In the year after the coldest summer of the observation period mortality due to G. abietina infections was highest, suggesting high susceptibility of trees in poor condition. Phaddium infestans, which was the second most important factor for mortality in P. cembra, killed trees irrespective of their condition. Other biotic and abiotic causes of tree mortality had negligible influence compared with the impact of G abietina and P. infestans. Excluding anthropogenic impacts, the diverse spatial pattern of forested and treeless sites close to the subalpine timberline may predominantly result from the action of parasitic fungi, depending on the ablation pattern of the snow cover in spring.