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.     

Enzymatic interactions between Gremmeniella abietina var. abietina,European race,and two resistant hosts,Pinus banksiana and P. contorta

Ultrastructural observations showed that the European race of Gremmeniella abietina var. abietina can secrete several cell wall–degrading enzymes that help the invasion of Pinus banksiana and P. contorta shoot tip tissues. Alterations in the content or distribution of cellulose were obvious, indicating that colonization might be achieved primarily through the action of glucanases. Polygalacturonases seemed more abundant in the cortex and the phloem of the stem, suggesting they are among the first enzymes secreted by the pathogen during infection. Production of laccases and peroxidases was also revealed but always close to fungal cells or in areas where host wall degradation was obvious. In places where there was an accumulation of phenolic compounds in the infected zones of the shoot, particularly in or near the transition zone, immunolocalization of both enzymes was limited or absent. Laccases and polygalacturonases were regularly detected over the extracellular sheath of G. abietina, and this indicates that the sheath could play a significant role during host wall degradation. Finally, examination of G. abietina wall constituents, such as chitin and β‐1,3‐glucans, suggests that the composition of the pathogen wall changes during the infection process. This change may be associated with the secretion of fungal enzymes but could also be linked to host reactions altering the integrity of pathogen cell walls.     

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.     

Epiphytology of Scleroderris lagerbergii in a kettle frost pocket

Both the fungus Scleroderris lagerbergii Gremmen and summer frost were implicated in the death of seedlings of Pinus resinosa Ait. and Pinus banksiana Lamb. [=P. divaricata (Ait.) Dumont] in the Goulais Kettle Frost Pocket in central Ontario, Canada. This fungus was responsible for the preponderance of early seedling mortality, with the survival differential between inoculated and uninoculated seedlings decreasing slightly with increasing elevation in the depression. A mechanism whereby inoculum is recirculatcd to the seedlings by wind vortices was identified and described.     

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 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.     

Scleroderris canker in New York State: Attempts to justify and cope with regulatory action

Pinus resinosa, P. sylvestris, P nigra, Pseudotsuga menziesii, and Picea glauca seedlings in three sites and Pinus thunbergii in two sites in New York State were inoculated with conidia of Gremmeniella abietina twice in 1986. Percent infection therefrom was determined in spring of 1987, Some members of each species of Pinus became infected at one site within the current range of the disease and in other sites 84 and 300 km south of the current range. P. glauca and P. menztesii were not infected. Results of experiments to test efficacy of postharvest treatments to eradicate G. abietina from infected seedlings suggested that immersion in hot (55°C) water and applications of dilute sodium hypochlorite would eradicate the pathogen with little or no effect on retention or color of foliage of P. resinosa and P. sylvestris.     

Pathogenicity of A and B morphotypes of Sphaeropsis sapinea confirmed on American larch (tamarack) and European larch

Fungi identified as Sphaeropsis sapinea (or synonyms) have been reported from Larix. Lack of pathogenicity tests, confusion about the identity of isolates mentioned in previous literature, and existence of distinct populations (A and B morphotypes) of the pathogen indicated the need to evaluate the ability of S. sapinea to cause disease of larch. Elongating snoot tips of Larix decidua, Larix laricina, and known hosts of the pathogen, Pinus banksiana and Pinus resinosa, were inoculated with water-agar plugs colonized with an A or a B isolate, or sterile plugs (controls). Each of five replications included five seedlings for each treatment-species combination. After 6 weeks, no symptoms had developed on control seedlings, but two-way analyses of variance revealed significant effects of isolate morpnotype and host on both incidence and severity of disease (values of p ≤ 0.01). The A isolate killed almost all shoot tips, but the B isolate killed from no P. resinosa shoots to 56% of L. laricina shoots. The average length of shoot killed by the A isolate was also greater than that killed by the B isolate. Response to the B isolate again varied among species, with greater average lengths of shoot killed on the larches (compared with the pines). Both A and B morphotypes of 5. sapinea should be considered among the fungi encountered on L. decidua and L. laricina.     

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.     

Growth rate and shoot: Root allometry in Pseudotsuga menziesii (Mirb.) Franco and Pinus contorta Dougl. Seedlings raised under two photoperiodic regimes

Six provenances of both Pseudotsuga menziesii (Mirb.) Franco and Pinus contorta Dougl. from a range of locations in the interior of British Columbia between latitudes 49° and 58° were grown for 80 days in controlled environment chambers programmed to provide daylengths comparable with those at either latitude 48°, or latitude 58°, during the period from mid‐June to early September. Neither shoot : root ratio nor whole plant dry weight of P. menziesii were affected by photoperiod. However, P. contorta seedlings, which unlike P. menziesii set bud during the experiment, had a greater dry weight and a higher shoot:root ratio (p<0.05) under longer daylengths. The results appear relevant to the interpretation of effects of cross‐latitudinal provenance transfer on plantation productivity.     

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.     

Responses of conifer species of the Great Lakes region of North America to inoculation with the pitch canker pathogen Fusarium circinatum

Five conifer species grown in the Great Lakes region of North America were examined for their susceptibility to Fusarium circinatum, (syns. Fusarium subglutinans f. sp. pini and F. moniliforme var. subglutinans), the causal agent of pitch canker. Three‐year‐old (3‐0) seedlings of red (Pinus resinosa), jack (P. banksiana) eastern white (P. strobus), Scots (P. sylvestris) and Austrian (P. nigra) pine were planted in 4 l pots in a greenhouse at Auburn University in November 1998. In April and June 1999, seedlings were inoculated by removing a needle fascicle approximately 5 cm from the terminal bud and placing a drop containing F. circinatum conidia on the wound. Resin production, canker length and seedling mortality were recorded 12 weeks later. Jack, Scots and eastern white pine were the most susceptible with Austrian and red pine more resistant to the fungus. F. circinatum was re‐isolated from 37% to 96% of inoculated seedlings. The susceptibility of jack, Scots and eastern white pine indicates a potential risk to these important species of the region if F. circinatum were to be introduced into the area.     

Diplodia shoot blight and asymptomatic persistence of Diplodia pinea on or in stems of jack pine nursery seedlings

Diplodia pinea (syn. Sphaeropsis sapinea) is known as a major cause of damage to red pine (Pinus resinosa) seedlings in nurseries. The fungus can also be a latent pathogen of red pine seedlings, persisting in the absence of gross symptoms and later proliferating under conditions that induce host stress. In the fall of 2004, three nurseries in Wisconsin were surveyed to determine the potential for the occurrence of Diplodia shoot blight on jack pine (Pinus banksiana) seedlings and the persistence of D. pinea on or in asymptomatic seedlings of this species. Incidence of shoot blight was quantified in five 1 m long segments of an interior row in each of two survey areas in each nursery. The pathogen was identified on symptomatic seedlings collected in these areas on the basis of presence of characteristic pycnidia and conidia. Five groups of 20 asymptomatic seedlings were also collected in each of the two survey areas in each nursery. A segment of the lower stem/root collar from each of these asymptomatic seedlings was surface‐disinfested and culturally assayed using tannic acid agar. The mean incidence of shoot blight (as high as 9%) and mean frequency of cultural detection from asymptomatic seedlings (as high as 20%) were greatest in proximity to red pine windbreaks which are a source of inoculum. Only D. pinea was confirmed from subsets of symptomatic and asymptomatic seedlings which were tested using mt SSU rDNA polymerase chain reaction (PCR) primers that allow differentiation of D. pinea from D. scrobiculata and other fungi in the genus Botryosphaeria and related anamorphic fungi. Jack pine seedlings inoculated with D. pinea isolates obtained from asymptomatic nursery seedlings developed shoot blight symptoms in greenhouse trials. Thus, the ability of D. pinea to damage jack pine seedlings in nurseries has been documented and the potential for virulent strains of this latent pathogen to be distributed on asymptomatic jack pine seedlings from nurseries has been confirmed.     

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.     

Physiological performance of three pine species provides evidence for gap partitioning

Gradients of light and moisture availability peak at different positions within canopy gaps in northern latitudes providing the opportunity for niche partitioning in and around gaps based on differences in individual species’ life history attributes. This gap partitioning offers potential for increasing diversity in forests impacted by gap-creating disturbances. We examined resource availability and the physiological performance of three Pinus species with varying tolerances for shade and moisture stress across large (0.3 ha) canopy gaps to investigate relationships between gap position and species performance. Light availability was lowest in southern gap edges, while water availability was lowest in northern edges, and higher at gap interior positions than edges. Pinus banksiana seedlings had higher light-saturated CO₂ assimilation rates than P. resinosa or P. strobus seedlings at interior gap positions, and outperformed P. strobus at northern gap edges, but there were no differences between species at southern edges. Both transpiration and stomatal conductance were greatest for P. banksiana in gap centers, but showed few differences between species at edges. Foliar nitrogen concentrations were highest for P. banksiana, suggesting the dominance of this species in central gap locations may be due to a combination of high photosynthetic capacity and tight stomatal control to regulate moisture stress at drier gap positions. Our results suggest P. banksiana seedlings may be competitively superior in gap positions with high light and moisture availability, but P. resinosa and P. strobus become competitive under the drier conditions and moderate shade near gap edges. These findings support the concept of gap partitioning, and suggest silvicultural systems that incorporate patch cuttings could be used to promote diverse regeneration in northern pine forests.     

Water relations of pine seedlings in contrasting overstory environments

Overstory conditions influence understory microclimate and resource availability, leading to gradients in evaporative demand and moisture availability that influence seedling water relations. Partial canopies may either reduce seedling moisture stress by ameliorating environmental conditions, or increase moisture stress by reducing soil moisture availability. This study used stable isotope ratios of oxygen (δ¹⁸O) and carbon (δ¹³C) and mass-based foliar nitrogen concentrations to investigate changes in transpiration (E), stomatal conductance (g_s) and intrinsic water use efficiency (iWUE) of pine seedlings across an overstory gradient from open canopy gap environments to closed canopy forest. Foliar δ¹⁸O increased sharply from basal areas of 0–10 m² ha⁻¹ in Pinus banksiana, Pinus resinosa, and Pinus strobus seedlings, followed by a more gradual increase with further increases in basal area. Foliar δ¹³C followed a similar, but less pronounced pattern in P. banksiana and P. strobus seedlings, and had no apparent relationship with overstory basal area in P. resinosa seedlings. The slope of the δ¹⁸O:δ¹³C relationship was positive for every species. Foliar nitrogen concentrations were not correlated with overstory basal area. These results suggest seedling E declined as overstory basal area increased due to reductions in g_s, while iWUE increased slightly from open gaps to partial canopy environments. Open gap environments appear to provide sufficient moisture to sustain high leaf-level gas exchange rates in the species we studied, while relatively small increases in overstory basal area apparently promote rapid declines in g_s, leading to greatly reduced seedling water loss and small increases in iWUE.     

Effects of nickel and copper on growth and mycorrhiza of Scots pine seedlings inoculated with Gremmeniella abietina

Scots pine (Pinus sylvestris) seedlings were planted in soil originating from two localities with different background levels of nickel and copper. In addition, some of the seedlings were exposed to additional nickel (20.5 mg Ni/l of soil) or copper (63.5 mg Cu/l of soil), or a combination of both Ni and Cu, via soil without direct shoot exposure during their second growing period. The seedlings were either irrigated with spring water (pH 6) or got only natural rain during the whole field experiment. All seedlings were inoculated with conidia of a shoot‐pathogen Gremmeniella abietina during their third growing season, and harvested the following spring. Lengths of shoots of different year‐classes were used as growth estimates. In roots, the proportion of fungal (assumedly mycorrhizal) biomass was estimated by measuring ergosterol concentration. Guajacol peroxidase activity was measured. Short roots were classified into two groups according to their condition and the composition of the mycorrhizal community was expressed as a proportion of morphotypes in the roots. The seedlings exposed to additional Ni had higher shoot growth than the seedlings in the other treatments. The mean Ni concentration in the roots of seedlings exposed to additional Ni was 79 p.p.m. and in other seedlings 16 p.p.m. Additional Ni also decreased the frequency of clearly senescent short roots and the proportion of the mycorrhizal morphotype with the thinnest mantle. These results indicate that the Ni exposure levels used in this experiment had some positive effects on the seedlings. The relative fungal biomass was about 6% lower (p = 0.0981) in the fine roots of seedlings treated with additional Cu. The mean Cu concentration in the roots of seedlings exposed to additional Cu was 256 p.p.m. and in other seedlings 29 p.p.m. Peroxidase activity, which was used as a general stress indicator in this study, was not affected by any of the treatments. The shoot growth and the relative biomass of fungi in the fine roots were positively correlated in all seedlings, and this correlation was stronger in seedlings exposed to additional Ni that were not irrigated compared with seedlings not exposed to additional Ni that were irrigated. The frequency of asymptomatic infections of G. abietina was positively correlated with the proportion of senescent short roots in the irrigated seedlings but not in not‐irrigated seedlings. The general condition of seedlings may be an important factor for infection by G. abietina when moisture is high enough for the fungi to infect seedlings by conidia.     

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.     

Shoot dieback of lodgepole pine in Scotland caused by Ramichloridium pini

Research conducted on shoot dieback of lodgepole pine, Pinus contorta, during the early 1980's in west Scotland is summarised. The dieback symptoms and their stages of development are described. Ramichloridium pini de Hoog and Rahman was regularly isolated from bark and xylem tissue of dieased shoots. Artificial inoculations established that this fungus was the cause of the disease.     

Risk from Sirococcus conigenus to understory red pine seedlings in northern Wisconsin

Interest in development of multicohort stands of red pine (Pinus resinosa) in the Great Lakes region of North America prompted an investigation of the potential impact of the shoot blight pathogen Sirococcus conigenus (syn. S. strobilinus) on understory red pine seedlings. In May 2002 and 2003 healthy, 1‐year‐old red pine seedlings were planted in the understory of a maturing red pine plantation in northern Wisconsin in an area with a history of presence of this pathogen. Occurrence of shoot blight symptoms was recorded periodically during the summer, and in each year seedlings were harvested in fall and examined for signs of shoot blight pathogens. By fall 2002 and 2003, respectively, shoot blight incidence was 89% and 98% and most seedlings were dying. Pycnidia with conidia of S. conigenus were present on almost all of the symptomatic seedlings. The conifer shoot blight and canker pathogen Diplodia pinea (syn. Sphaeropsis sapinea) was also detected, though less frequently. Pycnidia of S. conigenus tended to be found more frequently on symptomatic current year's shoots than symptomatic previous year's shoots; the opposite was true for pycnidia of D. pinea. Risk from S. conigenus to understory red pine seedlings should be considered in any plans for development of multicohort red pine stands in areas where the pathogen is present.