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 four conifer species to Gremmeniella abietina

G. abietina causes severe dieback in pole-stage stands of Scots pine in Britain. The susceptibility of ll provenances of Scots pine and of three other conifer species was tested by inoculations. Significant differences in susceptibility were found amongst Scots pine provenances, with those originating from a native Scottish pinewood at Loch Maree showing the highest levels of disease. Corsican pine was shown to be the most susceptible species tested although sporulation was greatest on Scots pine. Norway spruce was also shown to be susceptible when inoculated late in the growing season. Infection in lodgepole pine was negligible. High levels of β-phellandrene were tentatively linked with resistance among trees in a single stand of Scots pine.     

An outbreak of Gremmeniella abietina in central Finland

G. abietina has damaged P. sylvestris forests in central Finland. The denser the stand, the greater the number of recoverable trees. Artificially regenrated stands were more infected than naturally regenerated ones. Forest fertilization decresed the proportion of recoverable 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.     

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.     

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).     

The ability of Gremmeniella abietina to hydrolyze polygalacturonic acid

Gremmeniella abietina grew well in media with pectin as the sole carbon source. The fungus secreted at least one enzyme with polygalacturonase activity, and this was induced by addition of pectin to the culture medium.     

Physiological and morphological variation of Gremmeniella abietina from Spain

Gremmeniella abietina isolated from Pinus halepensis in Spain was characterized based on disease symptoms and conidia morphology. The disease symptoms, which became more evident in the spring, included drying up of needles and branches with some distortion of terminal twigs, resulting in dieback and sometimes to death of trees of all sizes. Variation in conidia size and growth rate was investigated between 15 isolates from Spain, and the conidia morphology was compared with four isolates from Finland, four from Switzerland and three from the US. Great variation occurred between Spanish isolates both in growth rates and in conidia size. The growth rates of the isolates were greatest on malt agar amended with pine needle extract and at 15°C. The length and the width of the conidia of the isolates from Spain ranged between 10.7–44.8 and 1.5–4.4 μm, and most of them had three septa. The results suggest that the isolates from Spain do not belong to the Alpine biotype, and the disease symptoms caused by these isolates resembled those of the European biotype.     

Precipitation and temperature as factors in Gremmeniella abietina epidemics

The combination of temperature and precipitation during the growth season could be correlated with the occurrence of Gremmeniella abietina epidemics in Denmark. The Gremmeniella index value (GIV) is the result of the monthly rain in mm divided by average monthly temperature in centigrade (mm/°C) and it was created to facilitate easy prediction of epidemics in Denmark. The two most severe outbreaks of G. abietina in pine and other conifers in Denmark took place in 1963–64 and 1984–85, following years where low average temperatures and high precipitation in May combined with the same weather patterns in either August or September. These meteorological conditions were not fulfilled from 1874 to 1900, in which period Pinus nigra was pronounced unsuitable for Danish forestry because of recurring problems with fungal disease. However, the earlier epidemics seemed to coincide with cold and rainy weather (high GIV) in August. In addition, severity of epidemics may also depend on the presence of dense stands between 30 and 40 years of age, as high GIV in August has occurred without disease outbreak in periods with low frequency of susceptible stands. Gremmeniella abietina attacks in Denmark are initiated by the climatic conditions expressed by the Gremmeniella index value, although it does not explain the biological process behind this phenomenon. Nevertheless, the method could be useful for other regions, where epidemics of G. abietina may be related to weather.     

Conidial dispersal of Gremmeniella abietina: climatic and microclimatic factors

The conidia dispersal started in Suonenjoki, in central Finland, in 1997–99 by the end of May or beginning of June, and continued occasionally at least to the middle of September. The temperature sum, day degrees (d.d., threshold temperature = 5°C) was between 100 and 165 d.d. at the beginning of dispersal. In years 1997–99, 80, 94 and 82% of the dispersal had occurred by the end of July – beginning of August when the temperature sum reached 800 d.d. All the spore data are coming from the spore traps. Cumulative number of conidia increased linearily with logarithm of temperature sum. A binary logistic regression model with temperature sum and rainfall as explanatory variables predicted accurately the date of the first spores in the spring: the prediction error was at most 3 days. The model classified 69% of all the days in the analysis correctly to the spore‐free days and correspondingly 74% to the days of at least one spore caught. A regression model for the number of spores per day explains 21, 5 and 51% of the within‐season variation in 1997–99 (24, 37 and 62% on a logarithmic scale). The explanatory weather variables in the model were d.d., rain and year. The very low explanatory coefficient of determination in 1998 results from one exceptionally high number of conidia. The between‐differences in the total number of spores were large and could not be explained by the measured weather variables. In the regression model, these differences were taken into account by adding a constant for each year in the model. Rain increased conidia dispersal significantly but conidia were found also in consecutive rainless days.     

Resistance of Pinus contorta to the European race of Gremmeniella abietina

Pinus contorta seedlings, together with Pinus resinosa and Pinus banksiana seedlings, were planted adjacent to 25‐year‐old red pine trees infected by the European (EU) race of Gremmeniella abietina. Resistance to this race was assessed over 5 years. All P. resinosa seedlings were dead after that period while 65% of P. contorta and 86% of P. banksiana seedlings appeared resistant to the disease. The tip blight that occurred on P. contorta was slightly longer than that observed on P. banksiana. In microscopy, one, two, or even more suberized boundaries were seen to be initiated near the surface of the shoot at the base of healthy needles where they extended downward in the direction of the vascular cambium. Suberized boundaries occasionally crossed the xylem and joined together in the pith region to form continuous barriers around necrotic tissues. However, in most cases, these suberized barriers were not continuous across the shoot and compartmentalization was then completed by other barriers mainly constituted of parenchyma cells and xylem tracheids that accumulated phenolic compounds. Meristematic‐like cells were observed adjacent to the necrophylactic periderm. Tissue regeneration, restoration of cambial activities and formation of traumatic resin canals also seemed to be associated with the defence system of P. contorta against the EU race of G. abietina.     

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.     

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.     

Susceptibility of Six Scots Pine Clones to the Pathogenic Fungus Gremmeniella abietina

Genotypic variation in susceptibility to Gremmeniella abietina, an economically important fungal pathogen of conifers, was studied by artificially inoculating 23 - yr - old grafted plants of six Scots pine (Pinus sylvestris L.) clones in a seed orchard. Two fungal isolates were used. The mycelium was inserted into the current year shoots in late winter and the length of the necrotic lesion caused by the fungus was measured the next spring. The growth and male flower production of the experimental shoots were also measured. The development of symptoms did not vary among the clones, but the location of the grafts within the orchard statistically significantly affected the length of necrosis. The clones differed significantly in regard to height and the production of male and female flowers. Differences in flowering intensity among the clones were not connected with the development of G. abietina. At the level of the single shoot, the production of male flowers correlated positively with the length of necrosis. The length of necrosis correlated negatively with the length of current and previous year shoots. The ability of the two fungal isolates to cause necrosis differed significantly.     

Wachstumshemmung von Stammfäulepilzen und von Gremmeniella abietina durch Bacillus subtilis

Inhibition of butt rot causing Basidiomycetes and of Gremmeniella abietina by Bacillus subtilis . A zone of inhibition was observed when the fungi were inoculated into the test plates with malt agar either together with B. subtilis (fast growing Basidiomycetes) or two weeks before B. subtilis (slow growing fungi). With some of the fast growing Basidiomycetes a zone of inhibition was observed only during the early growth phase. Sparassis crispa, either not growing or with restricted growth when transferred to the plates together with B. subtilis, completely suppressed the Bacillus culture when inoculated into the plates two weeks before B. subtilis.     

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.     

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.     

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.     

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.     

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.