Conidial germination and formation of necrosis in pine seedlings by Gremmeniella abietina at low temperatures

The germination of conidia of G. abietina on water agar reached the same germination percentage at 0°C as at 5 °C to 12.5 °C, but required more time. The fungus caused necrosis even at - 4 °C in pine seedlings.     

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.     

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.     

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.     

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.     

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.     

Effect of temperature on the ability of Gremmeniella abietina to survive and to colonize host tissue

Gremmeniella abietina was able to survive in vitro for four weeks at average temperatures less than 30° C. At higher temperatures, survival time was inversely proportional to temperature. The fungus survived for 23–28 months inside asymptomatic trees. Freezing, or freezing and thawing, of inoculated trees, may have promoted symptom development.     

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.     

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.     

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.     

Interactions between callus cultures of Pinus sylvestris and pine fungi with different trophic properties

Fungal virulence may be studied using tissues cultures of host plants in dual cultures in vitro, enabling analyses of interactions with undifferentiated cells of their host plants. Three genotypes of Pinus sylvestris callus, initiated by somatic embryogenesis, were used for establishing dual cultures with fungi pathogenic, endophytic or saprotrophic on pine needles or shoots. Fungal growth towards the plant callus tissue differed, depending on the life strategy of the fungus. The pathogen Gremmeniella abietina proved the slowest colonizer of callus whereas the saprotrophic Phacidium lacerum was the fastest. Gremmeniella abietina partially overgrew the callus, causing extensive necrosis and death within 10 days after inoculation. Anthostomella formosa, an endophyte of pines, did not cause evident symptoms of callus degradation: after 10 days of dual culture, the callus cells remained greenish and at least 50% of cells were alive. In dual cultures Ph. lacerum, callus remained alive until the end of the experiment, maintaining a white‐creamy colour with a loose cell structure. Electrophoresis of protein extracts from the callus showed the presence of additional bands of 25–35 kDa only in host tissues challenged with the pathogen G. abietina, possibly indicating the production of pathogenesis‐related proteins. This work has shown that pine callus does not respond equally to challenge with different fungal isolates. In general, one‐third of the isolates of each fungus examined showed greater virulence compared to other isolates.     

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.     

Temperature sensitivity of qualitative race-cultivar interactions in Melampsora medusae Thüm. and Populus species

Six mono-uredospore isolates (races) of Melampsora medusae Thiim. produced qualitatively distinct reactions, in vitro, when incubated at 15 °C on leaf disks of certain cultivars of Populus deltoides Marsh. The infection types in some race-cultivar combinations were very temperature sensitive with less distinct reactions when incubated at 20 or 25°C. The significance of differential race-cultivar-temperature interaction in the epidemiology of M. medusae is discussed.     

Mountain pine beetle-associated blue-stain fungi are differentially adapted to boreal temperatures

Mountain pine beetles (MPB) are the most serious pest of lodgepole pine in Canada and are likely to invade boreal jack pine forests. MPB vector three blue-stain fungi, Grosmannia clavigera, Ophiostoma montium and Leptographium longiclavatum, which contribute to beetle success. Fungal survival at extreme boreal temperatures will contribute to their success in jack pine. Growth, sporulation and survival of the three fungi at −20 to 37°C were tested in vitro. Overwintering survival of G. clavigera and O. montium was assessed in vivo. All species grew at 5–30°C, with optimal growth at 20–25°C. Grosmannia clavigera and L. longiclavatum survived at −20°C, but O. montium died. Growth of G. clavigera and L. longiclavatum was inhibited at 30°C, but O. montium grew well. Grosmannia clavigera and O. montium overwintered in living pines. These results suggest that G. clavigera and L. longiclavatum were adapted to cold boreal winters but not hot summers, with the converse true for O. montium. Temperature tolerance varied among G. clavigera isolates. British Columbian and Californian isolates grew faster at 25°C than Albertan isolates. Isolates from Alberta and Idaho/Montana grew optimally at 20°C, while British Columbian and Californian isolates grew optimally at 25°C.     

Variability in the Italian population of Gnomonia leptostyla,homothallism and resistance of Juglans species to anthracnose

Isolations of Gnomonia leptostyla were carried out in 13 plantations of Juglans regia distributed throughout Italy, including Sardinia and Sicily. Mean diameter of colonies grown in vitro at 22°C and sporulation of 191 isolates, grouped by site of collection, were compared. Four groups of isolates, from environmentally diverse sites, were more thoroughly investigated for the effect of temperature (from 10 to 30°C, 5°C increment) on growth and sporulation. Colony growth of isolates within groups correlated with temperature and environmental parameters at the site of collection, such as temperature, rainfall, number of rain days per month and per year, and altitude. Altitude and mean temperature in April correlated with colony growth. In general, the isolates that grew significantly more slowly were from sites with colder early springs and higher altitudes. More significant data were obtained comparing isolates from different sites obtained from the same walnut cultivar or population. The host source was more significantly correlated with colony growth within the same site of collection. Acervular conidiomata were abundantly produced by all isolates at 22°C in darkness after 21 days, while protoperithecia were produced within 2 months by most isolates, under the same conditions. Similarly, the four groups of isolates tested at different temperatures produced conidiomata at 20 and 25°C, but not at 10, 15 and 30°C. After 2 months, protoperithecia were present in most isolates at 20°C, very few at 25 and 15°C, and no production was recorded at 10 and 30°C. Fertile perithecia with asci and ascospores were produced, after 3 months at 10°C in darkness, by six homothallic isolates out of 38 tested. In general, perithecia were larger than protoperithecia both in ascocarp diameter and in neck length. Ascocarp diameter, width of asci and length of ascopores of in vitro-produced perithecia were larger than those of perithecia produced in nature. The latter showed a neck length longer than in vitro-produced perithecia. Virulence of isolates when inoculated on J. regia was correlated with colony growth rate, but rated independently for homothallic or heterothallic isolates. A preliminary screening for sources of resistance to anthracnose in the genus Juglans showed J. sieboldiana and J. cinerea to be highly resistant and both J. nigra and J. hindsii to be highly susceptible to the disease. Juglans regia showed an intermediate response of susceptibility to anthracnose.     

Detection and partial characterization of extracellular proteases of the pathogenic fungi Endocronartium pint,Gremmeniella abietina and Heterobasidion annosum

Protease activities from 10 homokaryotic isolates of Heterobasidion annosum., two isolates of Grem-meniella abietina and six isolates of Endocronartium pini were studied. All the fungi showed in vitro protease activity with denatured casein. The greatest caseinolytic activity was at acidic pH, but Heterobasidion annosum and Gremmeniella abietina showed caseinolytic activity at basic pH also. Protease preparations from Heterobasidion annosum were able to degrade proteins from total phloem extracts of Pinus sylvestris, Picea abies, Betula pendula and Juniperus communis. At basic pH the artificial substrates hippuryl arginine (HA) and hippuryl phenylactic acid (HPLA), were hydrolysed most rapidly by Heterobasidion annosum at pH 8.1, indicating high exopeptidase activity at this pH. According to inhibitor studies with EDTA (ethylenediaminetetraacetic acid), E64 (L-trans-epoxy-succinyl-leycylamide-(4-guanidino)-butane-agmatine) and pin2 (potato trypsin/chymotrypsin inhibi-tor) both cysteine and serine proteases were present in proteases secreted by these pathogens, although only very low protease activity at basic pH was detected with Endocronartium pini.     

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.     

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.     

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.