Cedrus libani: the most susceptible Turkish conifer species to local Heterobasidion isolates in spring inoculations

Five‐year‐old seedlings of Pinus nigra, Pinus brutia, Abies nordmanniana ssp. bornmülleriana and Cedrus libani, 3‐year‐old seedlings of Pinus sylvestris, and 2‐year‐old seedlings of Juniperus excelsa were inoculated on the lower stem with Turkish Heterobasidion abietinum and Heterobasidion annosum s.s. isolates. In total, 300 seedlings were inoculated in April 2007 with five isolates of each Heterobasidion species and incubated in a glasshouse for 12 weeks. The daily maximum temperature increased gradually from 13°C to 31°C by the end of the incubation period. Infection incidence, mortality, lesion length in the inner bark and fungal growth in the xylem were examined. For H. abietinum, infection incidence ranged from 0% to 36%, and for H. annosum s.s. from 0% to 60%. Mortality was low; only 1% of the inoculated seedlings died. Mean lesion lengths varied between 7.6 and 11.9 mm for H. abietinum and 8.0 and 15.7 for H. annosum s.s. in all species apart from C. libani, in which the corresponding values were 42.2 and 42.6 mm respectively. Similarly, mean fungal growth in sapwood of C. libani was approximately 25 mm for both Heterobasidion species, in contrast to 0–3.2 mm in the other tree species. The H. abietinum isolates were not reisolated from P. nigra, P. sylvestris or J. excelsa. The H. annosum s.s. isolates did not infect Abies seedlings. Only P. brutia and C. libani were susceptible to both pathogens. Control seedlings showed no symptoms. The results indicated that C. libani is highly susceptible to Turkish isolates of Heterobasidion.     

Heterobasidion abietinum on Abies species in western Turkey

Forty‐five basidiocarp specimens of Heterobasidion were collected from native Abies species in three locations in western Turkey: A. nordmanniana ssp. bornmülleriana in Bolu province, A. nordmanniana ssp. equi‐trojani in Balikesir province and A. cilicica in Antalya province. Pure cultures were isolated from the basidiocarps and identified to the species level with the aid of mating tests. All the specimens proved to belong to the species Heterobasidion abietinum. This root rot fungus is common in the forests investigated and appears to be relatively virulent on Abies in Turkey. This is the first report of H. abietinum outside Europe.     

Heterobasidion on Abies nordmanniana in north‐eastern Turkey

Occurrence of Heterobasidion annosum s.l. was investigated in 15 coniferous stands in the Giresun and Pontic Mountains in north‐eastern Turkey. Basidiocarps of the fungus were found in 11 stands. Fifty‐two basidiocarp specimens of Heterobasidion were collected from stumps of Abies nordmanniana ssp. nordmanniana and two from stumps of Picea orientalis. Pure cultures were isolated from the basidiocarps and identified with the aid of mating tests. Forty‐five (90%) of the specimens collected from A. nordmanniana were identified as H. abietinum and five (10%) as H. annosum s.s. The former species was also found twice on P. orientalis. This is the first report of H. annosum s.s. for Turkey and the first report of H. abietinum on P. orientalis. Heterobasidion abietinum seems to be mostly a saprotroph on A. nordmanniana.     

Induction of tumours by various strains of Agrobacterium tumefaciens on Abies nordmanniana and Picea abies

Abies nordmanniana and Picea abies seedlings were inoculated with a wild‐type (C 58) or an attenuated strain (rooter or shooter mutants) of Agrobacterium tumefaciens. Large tumours were formed on A. nordmanniana in response to the wild‐type and rooter strains, 60–65 % of the seedlings being susceptible. Smaller tumours were formed on Picea abies in response to the wild‐type strain, at least 12% of the seedlings being susceptible. Tumours from both species induced by the wild‐type strain grew in vitro on medium without added phytohormones.     

Armillaria ostoyae in managed coniferous forests in Kastamonu in Turkey

Although several Armillaria species have been reported in Turkey, there is little information about their ecology in Turkish forests. In this study, we investigated five forest stands, approximately 5–74 ha in size, in Kastamonu province in the Black Sea Region of Turkey for the presence of Armillaria species in stumps and logs. The stands were mixed Abies nordmanniana ssp. bornmülleriana and Pinus sylvestris forests managed using a selective cuttings system; the proportion of fir in the total number of stems and stumps ranged from 36 to 98%. Based on sequence analysis of the internal transcribed spacer and intergenic spacer regions of the rDNA, all rhizomorphs sampled from the stumps and logs were of Armillaria ostoyae. The size of the genets was estimated with random amplified microsatellites analysis of the isolates and ranged from single stumps to approximately 450 m². One to seven genets were found in each stand. These results indicate that the genets had arisen from spores and vegetative spread was limited on most sites.     

Intraspecific variation in Heterobasidion annosum for growth in sapwood of Picea abies and Pinus sylvestris

Two greenhouse experiments were conducted to study intraspecific variation in growth of the root rot fungus Heterobasidion annosum in living host sapwood. In experiment 1, Scots pine (Pinus sylvestris) and Norway spruce (Picea abies) seedlings were inoculated with H. annosum isolates, 14 each of the S-and P-intersterility groups, collected from various parts of Sweden. In pine, the P-group isolates were more virulent than the S-group isolates both in terms of infection frequency, induced mortality rate (p < 0.05), and fungal growth in sapwood (p < 0.05). In spruce, the P-group isolates were also more virulent on average, but the difference was not statistically significant. Both S and P isolates had a higher infection frequency and a significantly longer sapwood growth on spruce than on pine. The P-group caused higher mortality on pine than on spruce. The length of the lesion in the inner bark was strongly correlated with fungal growth in spruce, but not in pine where the lesions were short or absent. In experiment 2, ten Norway spruce clones were inoculated with 18 S-isolates, originating from nine live-decayed trees and from nine spore-infected stumps in a single Norway spruce stand. The objective was to test whether any selection for growth rate in sapwood was detectable among individuals of H. annosum originating either from stumps or trees. The results gave no support for such selection since no difference in sapwood growth between the two groups of isolates was found.     

Studies on the biology of <Emphasis Type="Italic">Cryphalus piceae</Emphasis> (Ratz.) (Coleoptera,Scolytidae) in the Bartin and Karabük regions of Turkey

The purpose of this study is to investigate the biology of Cryphalus piceae (Ratz.) (Col., Scolytidae) living in Abies bornmülleriana (Mattf.) in fir forests in the Western Black Sea region in Turkey. The dates of swarming, tree attack, egg-laying, and different stages of development are given. The species had two generations per year.     

Variation in growth of Heterobasidion parviporum in a full-sib family of Picea abies

Abstract

Heterobasidion parviporum (Fr.) Niemelä & Korhonen and Heterobasidion annosum (Fr.) Bref. sensu lato are some of the major forest pathogens in the northern hemisphere causing root and butt rot to conifers. The relative susceptibility to H. parviporum was investigated in a full-sib family of Norway spruce [Picea abies (L.) Karst.] by inoculating a set of 252 cloned progenies from a controlled cross. Four ramets of each progeny were used and the 2-year-old rooted cuttings were incubated for 6 weeks under greenhouse conditions. The condition of the cuttings was assessed visually and all the plants were in excellent vigour with no mortality recorded during the experiment. To score the relative susceptibility, lesion length in the inner bark and fungal growth in the sapwood were measured. Among the progenies, significant differences were found for fungal growth in the sapwood (p<0.0005). There was no significant difference for lesion length; however, there was a significant positive correlation between fungal growth and lesion length. The broad-sense heritability was 0.11 for fungal growth. This shows that the genetic component for susceptibility to H. parviporum can be detected even within a full-sib family of Norway spruce and that there is a potential for mapping quantitative trait loci for this trait in Norway spruce.     

Differences in virulence of genets of Heterobasidion annosum and susceptibility of young plants of different conifer species and origins

Heterobasidion species are the most important pathogens causing root and stem rot on conifers in northern hemisphere forests. The host list of this complex is very wide and includes over 200 species of trees and shrubs. Among the members of this complex, Heterobasidion annosum s. s. has the largest host range. In this study, young plants of Pinus sylvestris, Picea orientalis, Abies nordmanniana, Cedrus libani and Pinus brutia (three different origins) were inoculated on the lower stem with known genets of Heterobasidion annosum s.s. collected from Pinus brutia stands in south-western Türkiye. Infection frequency, assessed as presence of the conidial stage in stem discs following incubation, in the inoculated seedlings was 100%. The Heterobasidion annosum s. s. isolates were re-isolated from all inoculated host species. Control seedlings showed no symptoms of disease. Mortality in inoculated plants was 11.5% of the 735 inoculated plants, which died over an 8-weeks incubation period. The isolates showed greater growth on Cedrus libani, Pinus sylvestris and Picea orientalis seedlings compared to other species tested. On the other hand, it was found that the least affected seedlings were Pinus brutia TB12 and Abies nordmanniana. This study proved that differences occur in aggressiveness of Heterobasidion annosum s. s. to host species. A striking point in the results is that, despite being the host species from which the isolates were obtained, Pinus brutia seedlings showed lower sensitivity to Heterobasidion annosum s. s. than the other conifer species tested. Inoculations of three different Pinus brutia provenances suggested there was no significant difference in mean lesion lengths and fungal growth values in Pinus brutia plants, except in Pinus brutia TB14, which was more susceptible to extension growth of the pathogen.     

Gene flow and inter‐sterility between allopatric and sympatric populations of Heterobasidion abietinum and H. parviporum in Europe

The tree pathogenic fungi Heterobasidion parviporum and H. abietinum are recently described sibling species that show marked host preference for Picea abies and Abies alba, respectively. H. parviporum shows a higher reproductive character displacement (i.e. higher inter‐sterility) towards H. abietinum in sympatry than in allopatry. We inspected whether this pattern was also present in H. abietinum by studying the inter‐sterility and gene flow amongst sympatric central European populations of H. abietinum and H. parviporum, and two isolated allopatric populations, Pyrenean H. abietinum and Scandinavian H. parviporum. Inter‐ and intra‐specific fixation index values were calculated from DNA sequence data of the GST‐1 locus and four microsatellite loci. Present allopatric and sympatric populations of H. abietinum were equally inter‐sterile towards H. parviporum, not suggestive of relaxed reinforcement. Several hypotheses explaining the observed pattern are presented. Genetic differentiation was observed between H. abietinum in the Pyrenees and in the Alps, suggesting geographical structure of H. abietinum in Europe.     

Extent of colonization by Raffaelea quercivora of artificially inoculated living and gamma‐ray‐sterilized seedlings of two Japanese and three American oak species

Mass mortality of Fagacean tree species caused by Raffaelea quercivora has occurred widely in Japan. Because conidia or other propagules of the pathogen have not been found in infected trees, pathogen spread is assumed to occur primarily by hyphae. To clarify the relationship between hyphal growth of the pathogen within trees and their vessel arrangements, we examined two native Japanese oaks, Quercus crispula and Quercus glauca, and three exotic American oaks, Quercus coccinea, Quercus palustris and Quercus rubra. Quercus glauca is a radial‐porous species, whereas the other four species have a ring‐porous wood structure. Hyphal growth within inoculated potted living seedlings and in cut, sterilized stem segments of these species was examined microscopically after fungal inoculation. Water conductance in the seedlings was examined using transverse stem sections. The proportion of non‐conductive sapwood in Q. crispula, Q. coccinea and Q. palustris differed between inoculation and control treatment, being much higher in inoculated seedlings. The proportions were positively correlated with the extent of the hyphal growth. In sterilized stem segments, the extent of fungal colonization varied among the foreign ring‐porous species Q. coccinea, Q. palustris and Q. rubra. It is hypothesized that the extent of colonization by R. quercivora reflects the extent of non‐conductive sapwood irrespective of tree species, but is little affected by vessel arrangements.     

Susceptibility of Abies pinsapo and its tree cohort species to Heterobasidion abietinum

Abies pinsapo, a rare fir found in the southernmost mountain region of Spain, is severely affected by Heterobasidion abietinum. It was hypothesized that spread of the disease might be favoured by woody species growing in the same habitat. In order to evaluate this hypothesis, artificial inoculation tests were performed on other potential hosts forming the tree cohort species with A. pinsapo. Inoculation test on seedlings demonstrated that H. abietinum was able to colonize stems of Pinus halepensis, P. pinaster and Quercus ilex subsp. rotundifolia, but failed on Q. faginea. Both Pinus species were colonized to a similar extent as the natural host, but Q. ilex subsp. rotundifolia showed a significantly lower susceptibility than conifers. These results suggest that Pinus spp. growing in mixed stands with A. pinsapo might favour spread of H. abietinum. As a consequence, natural replacement of pure A. pinsapo stands by mixed forest Abies‐Quercus should be facilitated.     

Pinus taeda L. response to differential inoculum density of Leptographium terebrantis colonized toothpicks

Bark beetle‐vectored ophiostomatoid fungi, Leptographium terebrantis, is inoculated on the roots and lower stems of stressed Pinus species during the feeding activity of bark beetle. To determine the exact host response following inoculation, it is critical to challenge the host with a realistic amount of fungal inoculum. Thus, we designed a series of stepwise experiments using L. terebrantis colonized toothpicks which focused on the inoculum transfer from the toothpicks to excised Pinus taeda stem segments and living saplings, respectively, at different inoculum densities. The toothpicks served as a substrate for fungal growth and sporulation and the inoculation showed their utility in eliciting host's response to the pathogen. The inoculated fungus caused blue‐stain and sapwood occlusions in P. taeda stems and saplings, respectively. The volume of occluded, visually damaged sapwood increased by 1.96 cm³ per radial inoculation point on average. Fungal colonized toothpicks can be used as a suitable alternative to agar discs for studying bark beetles vectored fungi and their host interactions.     

Variations in virulence and hyphal growth of four Raffaelea quercivora isolates within Quercus crispula

Mass mortality of fagaceous trees caused by Japanese oak wilt has occurred widely in Japan. Although virulence of the causal fungus, Raffaelea quercivora, appeared to differ among isolates, its relation to the fungal growth within trees was unknown. To clarify the differences in fungal virulence against susceptible Quercus crispula, we examined fungal growth of four R. quercivora isolates within trees and the resulting virulence. In our study, the isolates were multiple‐inoculated in seedlings and single‐inoculated in twigs of mature trees. In the multiple‐inoculation test, mortality rates were examined by the observation of external symptoms. In the single‐inoculation test, water conductance and hyphal growth within the trees were examined by applying aqueous dyes and fluorescence microscopy, respectively. Mortality rates, the proportion of the cross‐sectional area comprising non‐conductive sapwood and horizontal hyphal growth differed significantly among the isolates. Univariate logistic regression analyses showed that both the proportion of non‐conductive sapwood and hyphal growth were significantly positively related to mortality rates. For three isolates, hyphal growth was significantly positively correlated with the proportion of non‐conductive sapwood. These results suggested that the virulence against Q. crispula varies among R. quercivora isolates and that the extent of fungal colonization of the tree determines fungal virulence.     

Testing species of genus Abies for Christmas tree production in Norway

Ten exotic Abies species were tested in two field trials, where the aim was to find suitable species and provenances for Christmas tree production in coastal and fjord areas in Norway. The material included 14 provenances of Abies nordmanniana, 3 provenances of Abies bornmuelleriana, 3 provenances of Abies koreana, 2 provenances of Abies amabilis, and 1 provenance each of Abies equi-trojani, Abies alba, Abies procera, Abies homolepis, Abies nephrolepis, and Abies forrestii. Field trials were established at Gulen in Sogn og Fjordane County and at Verdal in Nord-Trøndelag County. Christmas tree classification was done seven and eight growing seasons after establishment. The Christmas tree yield was higher in Gulen (64%) than in Verdal (45%), which is situated further north. Also, the survival and the height growth were higher in Gulen than in Verdal. A more humid climate and a longer growth season in Gulen may explain some of the differences. Of the 10 species, A. nordmanniana, A. homolepis, and A. bornmuelleriana produced the highest Christmas tree yield at Gulen, while A. homolepis and A. koreana had the highest yield at Verdal. Due to early bud burst, A. equi-trojani and A. bornmuelleriana are only suited for Christmas tree growing in the best climatic areas.     

Pathogenicity of fungi associated with ash dieback symptoms of one‐year‐old Fraxinus excelsior in Montenegro

In addition to Hymenoscyphus fraxineus, two fungi identified as Diaporthe eres aff. and Fusarium sambucinum aff. were also isolated from necrotic bark lesions on declining one‐year‐old Fraxinus excelsior in a forest stand in Montenegro. To examine their involvement in ash decline, a pathogenicity test was performed using under bark inoculations on one‐year‐old Fraxinus excelsior. Hymenoscyphus fraxineus was included as comparison. All three fungal species proved highly pathogenic towards one‐year‐old seedlings although lesion sizes differed significantly between the different species. Hymenoscyphus fraxineus was most aggressive, followed by F. sambucinum aff., while D. eres aff. caused the smallest lesions. This study demonstrates for the first time the ability of isolates in the D. eres and F. sambucinum species complexes to cause decline on one‐year‐old common ash seedlings.     

Weed control measures in Christmas tree plantations of <Emphasis Type="Italic">Abies nordmanniana</Emphasis> and <Emphasis Type="Italic">Abies lasiocarpa</Emphasis> on agricultural land

Abies nordmanniana and Abies lasiocarpa, established for Christmas tree production in southwestern Norway (58°44′N, 5°38′E), were treated with different weed control methods, including chemicals, use of black plastic mulch, grass or clover as ground cover, living mulch and mechanical hoeing. Ground cover with black plastic mulching resulted in the best growth and quality in A. lasiocarpa; the least favourable treatment was when grasses were allowed to grow close to the trees. Unless measures were taken to remove this competing vegetation, the ground cover grew over the trees, smothering 94% of them. A. nordmanniana plants grew to the same height on plots with no weed control as on plots with thorough weed eradication. Stem diameter decreased on plots with ground vegetation compared to seedlings grown in the weed-free environment. The use of Trifolium repens as ground cover decreased height growth by 30% compared to thorough weed control. Both grasses and clover sown as living mulch, damaged the trees more than did natural weed vegetation.     

Pathogenicity of Heterobasidion annosum (Fr.) Bref. sensu stricto on coniferous tree species in Turkey

Two‐year‐old seedlings of Pinus brutia, P. brutia var. eldarica, Pinus pinea and 3‐year‐old seedlings of Pinus radiata, Pinus sylvestris, Pinus nigra and Cedrus libani were inoculated on the lower stem with isolates of Heterobasidion annosum s.s. collected from the Black Sea and Mediterranean regions of Turkey. In total, 315 seedlings were inoculated in April 2014 and incubated in a growth chamber for 7 weeks at 18–20°C. All isolates were pathogenic on the seven different hosts and had the ability to grow in living sapwood. The isolates had a greater growth on C. libani, P. sylvestris and P. radiata seedlings compared to plants of the other species tested. The least affected species were P. brutia and P. nigra. The isolates originating from the Black Sea region caused longer lesions on the hosts. Overall mortality during 7 weeks of incubation was 4%.     

Variation in resistance of loblolly pine (Pinus taeda L.) families against Leptographium and Grosmannia root fungi

Pine decline poses a serious threat to forest sustainability in the south‐eastern United States. Complex interactions between biotic and abiotic factors are involved in the decline and include root‐feeding bark beetles and their associated fungal genera, Leptographium and Grosmannia. A study was conducted to determine the relative tolerance of loblolly pine (Pinus taeda L.) families when challenged with either Leptographium or Grosmannia species. In the first study, bare root seedlings from 23 loblolly pine families were screened with L. procerum, L. terebrantis, G. huntii and G. alacris using an artificial inoculation method. In a second study, containerized seedlings from 27 loblolly pine families were screened with G. huntii and L. terebrantis. Measured seedling responses to the inoculations included lesion length, lesion width and occlusion of vascular tissues, measured 8 weeks after inoculations. The most common host response was dark brown lesions and resinous occluded stem tissue. Seedling families had a wide range of host responses to the different Leptographium and Grosmannia species and showed that it could be possible to select the families that may be tolerant to Leptographium and Grosmannia fungal species based on these results.     

Ophiostomatoid fungi associated with bark beetles onAbies veitchii in wave-regenerated forests

Relationships between tree mortality and bark beetle infestation onAbies veitchii at the wave-regenerated forest in Mt. Asahi, Okuchichibu area were investigated. Most of the firs with green needles and newly developed current year’s shoots in the dieback zone were heavily infested by bark beetles before the death of the trees. After heavy infestation of beetles, about half of the infested firs died within the year, and the other half died in the next year. When the species composition of bark beetles and associated ophiostomatoid fungi were investigated in Mt. Asahi and also at a typical wave-regenerated forest in Mt. Shimagare, Yatsugatake area,Cryphalus montanus andC. piceae were dominant beetle species for Mt. Asahi and Mt. Shimagare, respectively.Ophiostoma subalpinum andO. europhioides were dominant fungal species at both wave-generated forests. BecauseO. subalpinum was detected more frequently from deeper areas of sapwood thanO. europhioides, it was suggested that the fungal species may accelerate the death of stressed firs in wave-regenerated forests. Contribution No. 169, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba.