Infection of Norway spruce container seedlings by Gremmeniella abietina

First‐ and second‐year containerized Norway spruce seedlings were inoculated with conidia of type A (large tree type) and type B (small tree type) of Gremmeniella abietina var. abietina at different times during the summer. The appearance of symptoms after artificial inoculation and natural infection on spruce seedlings were recorded the following spring and compared with the disease symptoms on Scots pine seedlings. The proportion of diseased seedlings after inoculation reached as high as 80%. The susceptible period during the summer began later on the first‐year seedlings than on the second‐year seedlings, and was similar for the pine seedlings. Susceptibility of first‐year seedlings was highest in August and on second‐year seedlings in July. The accumulated temperature sum, relative humidity and height growth for first‐ and second‐year seedlings was assessed. Natural infection in 2002 caused more disease on pine than on spruce seedlings. Experimental thinning of seedlings had no effect on disease incidence. In a preliminary comparison between the ability of A and B types to cause disease in Norway spruce seedlings, type B caused more damage than type A after inoculation. However, type A caused a high disease frequency in other experiments in this study. Symptoms on Norway spruce seedlings often first occurred in the mid‐section of the shoot, and were similar to those observed on pine seedlings: needles turned brown, starting at the needle base, in the spring following inoculation. On first‐year spruce, diseased needles were shed rapidly, in contrast to a slower rate of shedding on first‐year pine seedlings. Pycnidia developed about 2 years after inoculation (on pine 1 year after inoculation). On Norway spruce seedlings the lower part of the shoot, including the lateral shoots, often remained alive. The experiments show that G. abietina can cause disease on containerized Norway spruce seedlings under nursery conditions in Finland. The coincidence of spore dispersal, seedling susceptibility and predisposing factors are important in disease development.     

Effects of associated fungi Sclerophoma pythiophila and Cenangium ferruginosum on Gremmeniella abietina dieback in Spain

The interactions between Gremmeniella abietina and either Sclerophoma pythiophila or Cenangium ferruginosum, fungi frequently isolated from diseased twigs along with G. abietina, were studied under laboratory (dual cultures) and greenhouse conditions (double‐inoculations). Virulence of each species was also evaluated in greenhouse experiments by means of single‐inoculations. In vitro interactions were assessed on Petri dishes containing malt agar with pine needle extract, and greenhouse experiments were performed on 1‐year‐old Pinus halepensis seedlings. In vitro growth of G. abietina was inhibited by both fungi when grown in dual culture. In single‐inoculations, G. abietina caused the greatest necrosis length on P. halepensis seedlings, followed by S. pythiophila, whereas C. ferruginosum did not cause significant necrosis. In double‐inoculations, C. ferruginosum was able to reduce the length of necrosis caused by G. abietina on the P. halepensis seedlings. In contrast, necrosis length was greater in seedlings inoculated with both S. pythiophila and G. abietina than in those inoculated with G. abietina alone. Therefore, S. pythiophila seems to play a role in disease expression caused by G. abietina on P. halepensis in Spain.     

Virulence of two Finnish Gremmeniella abietina types (A and B)

Thirteen Finnish Gremmeniella abietina isolates of types A and B were compared for differences in their virulence. Three kinds of inoculations were made: one with conidia on young Scots pine (Pinus sylvestris) seedlings and two with mycelia on the stems and shoots of young Scots pine trees. Inoculations with conidia were carried out in August 1992 and inoculations with mycelia were carried out six times between 11 August and 20 October, 1992. The experiments were evaluated in the late spring and early summer of 1993. The results showed that there was a difference in virulence between the two types. In the conidial inoculations type A infected 34.7% and type B infected 11.0% of the inoculated seedlings. For mycelial inoculations with type A the mean canker (stems) and necrosis (shoots) lengths were 19.3 mm and 8.6 mm longer, respectively, than with type B inoculations. In shoot inoculations there also was a clear difference between the two types in the number of such inoculations where no symptoms were observed. For type B shoot inoculations there was no fungal growth in 21.5% whereas for type A inoculations the figure was only 3.7%.     

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.     

In vitro growth of Gremmeniella abietina isolates (European race) at different temperatures

The mycelial growth of 24 isolates of types A and B of Gremmeniella abietina (Lagerb.) Morelet, collected in northern Finland and the Kola peninsula, Russia, was studied on malt agar plus pine needle extract at 18°C and 5°C. Great variation occurred within both types, and the results suggest that several isolates per stand are needed to represent one local isolate, if responses of G. abietina mycelia are studied on artificial media.     

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.     

Effects of previous forest types and site conditions on species composition and abundance of naturally regenerated trees in young <Emphasis Type="Italic">Cryptomeria japonica</Emphasis> plantations in northern Japan

Naturally regenerated trees in young Cryptomeria japonica plantations were investigated in 141 quadrats of 10 × 50 m within a watershed of 1,000 ha, and factors affecting their composition and abundance were examined. The species composition of naturally regenerated trees was classified into four types. Dominant species were Swida controversa, Magnolia hypoleuca and Pterocarya rhoifolia in Type A1, Betula maximowicziana, M. hypoleuca, Quercus crispula and Castanea crenata in Type A2, Q. serrata and C. crenata in Type B1, and Pinus densiflora in Type B2. The results of path analysis showed significant influences of previous forest type and geology among the factors that correlated with the species composition of naturally regenerated trees; previous forest type in particular showed a higher absolute path coefficient value. Species composition types of naturally regenerated trees corresponded to the previous forest types: Types (A1 and A2) corresponded to the natural forests composed of Thujopsis dolabrata var. hondai, Fagus crenata, Aesculus turbinata, etc., Type B1 corresponded to the secondary Q. crispula and Q. serrata forest, and Type B2 corresponded to the secondary P. densiflora forest, respectively. The abundance of naturally regenerated trees was strongly affected by geology; i.e., plantations on soft-sedimentary dacitic tuff contained great amounts of colonizing P. densiflora trees.     

Hybridization between two biotypes of Gremmeniella abietina var. abietina in artificial pairings

Summary The ability of the European biotypes A and B of Gremmeniella abietina var. abietina to hybridize in artificial pairings was tested. Using three types of molecular markers progenies with parental markers mixed indicating that the two biotypes can hybridize were observed. However, the resulting progeny had a low rate of successful germination, grew poorly on artificial medium and one was most probably a partial diploid or heterokaryon. Thus, both biotypes are genetically distinct populations and should be described as separate species as soon as sufficient discriminating information is available.     

Genetic variation in Polish strains of Gremmeniella abietina

Thirty-nine strains of Gremmeniella abietina were isolated between 1980 and 2005 from diseased or symptomless Pinus sylvestris and Pinus nigra, or sporadically Pinus jeffreyi and Pinus armandii from four regions of Poland with varying climatic conditions. Thirty-five of the strains were genetically similar to the G. abietina A type found in Scandinavia, whereas four were similar to the B type. On the basis of random amplified microsatellite markers, very high intrapopulation and interpopulation genetic variation was detected among the strains. The average value of the Jaccard genetic similarity coefficients among strains was 0.56 for A type and 0.47 for B type, and the monomorphic loci partition was small, at 14% of all analysed loci for the A type. The degree of genetic variation of the fungal strains within each of the four regions was similar, showing no significant differences. The Jaccard coefficient for the strains isolated from P. sylvestris and P. nigra differed significantly, at 0.57 and 0.54 respectively. The genetic distance among A type strains from the investigated regions, expressed using Nei’s coefficient, was not correlated with geographic distance; however, it was highly dependent on elevation (correlation coefficient r = 0.92).     

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.     

New morphometric ranges for Bursaphelenchus willibaldi Schönfeld et al. 2006 (Nematoda: Parasitaphelenchidae) from Iran

Bursaphelenchus willibaldi, associated with trees in two separate locations in forests of northern Iran, are characterized and illustrated based on morphological, morphometric and molecular data. The Iranian population of B. willibaldi has a body length of 392–595 μm, stylet length of 12–14 μm, c‐index of 9.0–14.5 and PUS length of 51–82 μm. Males have spicules 15–18 μm long along the arch line. The Iranian population showed morphometric variations compared with the originally described samples. For example, it has a shorter body in females and males, greater c‐index, slightly greater range of V, slightly smaller range of PUS and tail length. Molecular phylogenetic analysis of the recovered populations revealed both sequenced isolates forming a clade with one European isolate of the species using Bayesian inference (BI) analysis with full Bayesian posterior probability (BPP).     

Molecular and morphological characterization of Ektaphelenchoides maafiae n. sp. (Nematoda: Ektaphelenchinae) from northern forests of Iran

Ektaphelenchoides maafiae n. sp. was isolated during a survey of nematodes associated with bark samples of an oak tree (Quercus castaneifolia) in Gorgan, Golestan Province, northern Iran. The new species has a body length of 480–546 μm (in females) and 431–480 μm (in males). The cuticle is weakly annulated, with three lateral lines. Lip region offset. The stylet is 13–15 μm long without basal swellings. The excretory pore is located at the level of the metacorpus base to slightly posterior, and hemizonid is at 15–17 μm posterior to the excretory pore. The post‐uterine sac is short, 6–8 μm long. Spicules having rounded condylus, rostrum short, conical with bluntly pointed tip, a cucullus (apophysis) presented on the dorsal distal end. Male tail bearing four (2 + 2) caudal papillae, conical, with sharply pointed terminus. The new species is close to four known species of the genus, including E. hunti, E. ruehmi, E. caspiensis and E. poinari, but differs from them by body size, shape of tail terminus, stylet length, shape and size of spicules, length of post‐vulval uterine sac and number of caudal papillae. Phylogenetic analysis based on small subunit (SSU) and partial large subunit (LSU) sequences of rDNA confirmed its status as a new species.     

Differences between provenances of pinus contort a var. latifolia in resistance to attack by gremmeniella abietina

A provenance trial of lodgepole pine (Pinus contorta var. latifolia) planted in 1969 at Matrand, Eidskog, Norway was apparently attacked by Gremmeniella abietina in 1984/85. In late autumn the damage was recorded separately on the lower and upper halves of the trees. The material ranged in latitude from 40.25°N to 56.28°N, and in altitude from 2 850 to 450 m a.s.l., highest in interior western United States and lowest in western Canada. The most heavily attacked sources were those from southern latitudes and from high elevations. The attack frequency changed gradually from south to north. In order to avoid attack by G. abietina, only well‐adapted provenances should be used.     

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.     

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.     

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.     

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.     

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.     

Differential reaction in Pinus spp. to inoculation by various isolates of Ascocalyx (Gremmeniella) abietina

Stem inoculations of 3 species of pole-size pines with 4 diverse isolates of Ascocalyx (Gremmeniella) abietina revealed Pinus pinea to be more susceptible than either P. pinaster or P. nigra in terms of canker length. Isolate type and geographical aspects considered more briefly, also have some effect.