Pathogenicity of Spanish isolates of Heterobasidion annosum s. s. in Pinus pinaster seedlings

The aim of this study was to test the pathogenicity of two Spanish isolates of Heterobasidion annosum sensu stricto in 2‐year‐old Pinus pinaster seedlings. Two types of inocula (woodchips and sawdust) were used to infect the seedlings by two different routes (stem inoculation and soil infestation). The mortality rates of the stem‐inoculated seedlings differed significantly from controls, but those of the seedlings infected via soil infestation did not differ. For both types of inoculation, the lesions were longer, and wilting symptoms were more severe in the seedlings inoculated with H. annosum than in control seedlings. For stem inoculation, biomass allocation did not differ significantly between the infected and control seedlings. However, the percentage of fine roots was lower in seedlings infected via soil infestation than in the control seedlings. To our knowledge, this is the first pathogenicity test with H. annosum isolates and P. pinaster.     

Covariances between morphological characteristics in bareroot Pinus sylvestris nursery stock

In this study an attempt was made to gather information on the relationships of morphological attributes of bareroot Pinus sylvestris seedlings in order to determine the possible use of these attributes for grading purposes. The covariance structure of different morphological variables was studied in commercially produced crops. From each selected batch of Scots pine seedlings, 300 seedlings were sampled, three successive living seedlings from each of the 100 systematically selected sampling points ("clusters"). Variance in the seedlings crop was described using a linear model: total covariance was divided into variance between batches, between clusters and between individual seedlings (error).

Most of the correlations between different plant parts were large and positive. Only needle length and root length seemed to vary independently of other “length”; variables. Within one batch, at the end of the growing season, height‐diameter was more clearly size‐independent than shoot‐root ratio. Needle biomass increased exponentially with seedling height but the smallest seedlings had relatively less needle biomass than the biggest seedlings. It is not necessary to include dry mass in the requirements for seedling quality since it correlates well and can be described with other variables that can be measured more easily. Of the principal components for morphological characteristics, the “stem”; component explained 69% of the height variation and 77% of the diameter variation, the “needle”; component explaining 79% of the variation in current needle length, and the “root”; component 58% of the variation in root length. Together the three components explain 76% of the total variance in the original crops.     

Correlation of seedling size and branch number with disease resistance of Pinus thunbergii seedlings to Bursaphelenchus xylophilus

Japanese black pine (Pinus thunbergii) seedlings resistant to pine wood nematode (PWN; Bursaphelenchus xylophilus) are routinely selected in Japanese field inoculation trials. Correlations between morphological factors (such as height, stem diameter at ground level and number of branches on seedlings) and disease resistance were examined to improve the production efficiency of 1‐year‐old black pine seedlings for inoculation. Family relatedness and environmental conditions strongly affected seedling resistance; accordingly, logistic regression analysis was used to separate effects of these two variables. Height and stem diameter at ground level significantly correlated with disease resistance in seedlings inoculated with PWN. Because (a) interactions between stem diameter at ground level and environmental condition were significant and (b) height did not interact with any other factor, it was concluded that height of 1‐year‐old Japanese black pine seedlings independently correlated with PWN resistance. Thus, field inoculation tests should use tall seedlings to achieve enhanced survival rates.     

Artificial inoculation and susceptibility of Pinus armandii to Dothistroma septosporum

Dothistroma needle blight (DNB) is a serious needle disease of conifers that primarily affects pine species (Pinus spp.). Dothistroma septosporum is one of the DNB pathogens that has a diverse range of host species excluding Pinus armandii. In 15 inoculated P. armandii seedlings, D. septosporum acervuli were observed in 43 infected needles of ten seedlings with a mean disease severity of 1.11% at 25 weeks after inoculations, demonstrating the potential of D. septosporum to cause symptoms on the needles of P. armandii via artificial inoculation. The disease severity of P. armandii was similar to the positive control, Pinus nigra (median 0.75 for P. armandii to 0.70 for P. nigra), thus, P. armandii acts under artificial conditions as a susceptible host species.     

Seedling shoot,needle and bud development in three provenances of Pinus sylvestris and Pinus contorta cultivated in northern Sweden

The patterns of current‐year shoot, needle and terminal bud elongation in seedlings of three Scots pine (Pinus sylvestris L.) and three lodgepole pine (Pinus contorta Dougl. var. latifolia Engelm.) provenances were compared during the third and fourth growing seasons after planting. Lodgepole pine produced longer shoots and buds than did Scots pine, mainly because lodgepole pine formed more stem units and elongated at a faster rate. Stem unit length and the duration of shoot and bud elongation differed relatively little between species and provenances. Lammas or polycyclic growth occurred in some lodgepole pine provenances, but not in any Scots pine provenance, and was associated with enhanced shoot elongation. Needle elongation commenced earlier, proceeded at a faster rate, and was greater in lodgepole pine than in Scots pine, but ceased about the same time in all species and provenances. The heat sum required to attain 50% of final length was lower for shoots and needles in lodgepole pine than in Scots pine, and for shoots in northern provenances than in southern ones. Mitotic activity in the apical meristem of the terminal bud, which occurred less than one week after the seedlings were free from snow, started and ceased about the same time in each species, but was higher in lodgepole pine than in Scots pine early in the shoot elongation period.     

Pathogenicity of canadian isolates of the Bursaphelenchus xylophilus (pinewood nematode) to provenances of Pinus sylvestris and Pinus contorta as grown in Finland: A greenhouse study

Seedlings of Scots pine (Pinus sylvestris L.) and lodgepole pine (P. contorta Dougl.) provenances, as grown in Finland, were inoculated with “m”; and “r”; “forms”; of the pinewood nematode (Bursaphelenchus xylophilus) from Canada, an m form isolate from France and an r form isolate from Missouri, USA. Scots pine was highly susceptible to an Alberta r form and a British Columbia m form isolate and moderately susceptible to two Quebec m form isolates. Lodgepole pine was higly susceptible to the two r form (Alberta and Missouri) isolates and moderately susceptible to the British Columbia m form and the two Quebec m form isolates. Mortality of both pines after inoculation with the French isolate was inconsistent. Mortality of both pines occurred more rapidly following inoculation with r form than with m form nematodes. Large numbers of nematodes were generally found in the tissues of both pines. Our results with seedlings need to be corroborated by inoculating larger field‐grown trees.     

Effects of forest floor planting and stock type on growth and root emergence of Pinus contorta seedlings in a cold northern cutblock

A two-year field trial was conducted to determine the growth response, and root emergence pattern of interior lodgepole pine (Pinus contorta Dougl. var. latifolia Engelm.) seedlings in response to container type and forest floor removal. Seedlings were grown in Styroblocks^TM, Copperblocks^TM, or AirBlocks^TM, and were planted directly into the undisturbed forest floor or into manually prepared planting spots where the forest floor had been scraped away to expose the mineral soil. Seedlings planted into scalped planting spots exhibited marginally but significantly (7%) greater above-ground growth rates (seedling stem volume); whereas seedlings planted into the forest floor produced significantly more (11%) new roots. There were no differences in above- or below-ground biomass. Seedlings grown in Copperblock^TM containers produced a higher proportion of roots near the top of the plug when tested at lifting, however this pattern was not observed in the field. Given that scalping is more costly than forest floor planting, and that the increased shoot growth was relatively small, we recommend that forest floor planting be considered as an alternative to manual spot scalping for sites, such as the site tested here: those with cold, but well-drained soils and where competition from other plants is not a serious problem.     

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.     

Pre‐ and post‐inoculation water stress affects Sphaeropsis sapinea canker length in Pinus halepensis seedlings

In order to assess the influence of water stress on the development of Sphaeropsis sapinea cankers in Pinus halepensis, the stems of 4‐ to 5‐year‐old potted seedlings were artificially inoculated with the fungus before and after being kept at controlled water regimes from April 1997 to March 1998. In the pre‐water‐stress inoculation experiment, the canker length, measured 5 months after inoculation (September 1997), was greater in seedlings predisposed to extreme water deficit (midday needle water potential between ?4.5 and ?5.5 MPa). In the post‐water‐stress inoculation experiment, the fungus was inoculated in April 1998, after irrigation had enabled the seedlings to resume normal needle water potential. In this case also, at 5 months after inoculation, longer cankers were visible in seedlings that had been subjected to extreme water deficit. These findings suggest that the occurrence of marked water stress, although apparently tolerated by Aleppo pine, can enhance the development of S. sapinea cankers in this species, regardless of whether the stress occurs before or after infection by the fungus.     

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.     

Enhancing resistance in Pinus radiata seedlings to terminal crook (Colletotrichum acutatum) using methyl jasmonate and ultraviolet‐C radiation

Colletotrichum acutatum is a fungal pathogen that causes terminal crook disease in radiata pine (Pinus radiata) seedlings in New Zealand forest nurseries. Symptoms of infection include malformation or death of the growing tip and a stiffening and thickening of the stem. Although the disease can be managed effectively using fungicides, the New Zealand forest industry is interested in alternative control options such as induced resistance. The aim of this study was to evaluate spray application of chitosan (1.4 g/l) or 2.25 mm methyl jasmonate (MeJA) and irradiation with UV‐C (2.16 kJ/m²) for their potential to induce resistance to terminal crook. The treatments were applied to 4‐month‐old seedlings at 1 week before pathogen inoculation. By the end of the experimental period (42 days after inoculation), there was 80% disease incidence in the controls, with 48% of seedlings exhibiting severe terminal crook symptoms. The most effective treatment (p < 0.05) was MeJA with 16% disease incidence and none with severe symptoms. UV‐C also significantly (p < 0.05) reduced infection with 52% incidence and 20% of seedlings exhibiting severe symptoms. Chitosan did not reduce disease incidence (72%) compared with the control (80%) but did significantly reduce (p < 0.05) disease severity with 28% exhibiting severe symptoms. MeJA was the only treatment that significantly (p < 0.05) reduced the detrimental effects of infection on seedling apical growth and stem diameter. To our knowledge, this is the first report demonstrating the potential for MeJA and UV‐C to control terminal crook in radiata pine.     

A new Leptographium species from the roots of declining Pinus sylvestris in Switzerland

Scots pine (Pinus sylvestris) trees have been declining in the Rhône valley, Switzerland, for almost three decades. In an assessment of the role of fungi in this syndrome, the dominant fungus isolated from stained roots was a Leptographium species, morphologically similar to the asexual state of Grosmannia serpens. We examined isolates of this fungus based on DNA sequences of four protein‐coding genes including actin, β‐tubulin, calmodulin and translation elongation factor‐1 alpha. The results showed that they were of a distinct, undescribed taxon related to species in the Grosmannia serpens and G. wageneri complexes. The fungus, described here as Leptographium rhodanense sp. nov., resembles other species in the two species complexes morphologically, and most probably ecologically, as is suggested by the fact that it was isolated from stained pine roots.     

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.     

Effects of ectomycorrhizal fungi on damping‐off and induction of pathogenesis‐related proteins in Pinus tabulaeformis seedlings inoculated with Amanita vaginata

The formation of mycorrhiza on Pinus tabulaeformis (Pt) seedlings by ectomycorrhizal fungi (ECMF), the protective effect of the ECMF on damping‐off caused by Rhizoctonia solani and the expression of pathogenesis‐related proteins (PR‐proteins) induced by Amanita vaginata and/or R. solani in Pt seedlings were investigated. The results indicated that: (i) among the 10 ECMF tested, five species (Gomphidius viscidus, Suillus laricinus, Suillus tomentosus, A. vaginata and Basidiomycetes no. 058) formed mycorrhiza with Pt seedlings. There was a positive correlation between mycorrhizal colonization levels and protective effects (r = 0.9207). Among the five species, A. vaginata had the highest colonization level and the best protective effect against damping‐off; however, G. viscidus had no protective effect even though it had a high mycorrhization level. (ii) Chitinases, β‐1,3‐glucanases and PR‐proteins in the seedlings were not induced by A. vaginata colonization, but were induced when A. vaginata‐mycorrhizal seedlings were attacked by R. solani. It is possible that chitinases, β‐1,3‐glucanases and 108, 97, 78, 44, 37 and 14‐kDa PR‐proteins induced both by A. vaginata and R. solani protect Pt seedlings from R. solani infection.     

Autumn frost hardiness of Pinus sylvestris offspring from seed orchard grafts of different ages

Autumn frost hardiness of offspring from clones (originating from 66.0–68.8° N) in six southern (62° N) and one northern (66.5° N) Pinus sylvestris seed orchards in Finland, was evaluated. One‐year‐old seedlings derived from open pollinated grafts of young, intermediate, and fairly mature age, and natural stand seedlings, were artificially freeze tested. The northerly located orchard produced the least injured offspring. There were no significant systematic differences between age classes. Neither were there any indications of decreased freezing injury with later age classes. The family variance was significant in four orchards. The estimated injury levels of orchard offspring were, in general, closer to those of natural stands in the neighbourhood of the orchard than natural stands at the place of clonal origin. It is suggested that not only differences in mother clone hardiness but also clonal differences in pollen contamination rate caused the observed family variation.     

Trichoderma atroviride promotes growth and enhances systemic resistance to Diplodia pinea in radiata pine (Pinus radiata) seedlings

Root drench application of Trichoderma atroviride isolates R32, R33, R40 and R84 promoted the growth of potted radiata pine seedlings. After 6 weeks, seedlings treated with R33 and R84 had thicker stems and greater stem and root biomass (p < 0.05) than untreated controls. Treatment with R32 increased seedling root biomass whilst R40 increased stem diameter. None of the isolates affected seedling height. One isolate, R33, induced systemic resistance to stem inoculation with Diplodia pinea and reduced dieback incidence by 20% compared with untreated controls. To our knowledge, this is the first report of systemic induced resistance by Trichoderma in a pine species. Furthermore, seedlings that were treated with R33 (root drench) plus foliar application of methyl jasmonate (MeJA) expressed elevated peroxidase activity in their stems 2 weeks later, compared with seedlings treated only with MeJA. Because R33 itself did not affect peroxidase activity, this may be indicative of treatment synergy or defence potentiation by R33. Curiously, R33 + MeJA induced terpenoids but suppressed phenylalanine ammonia‐lyase activity suggesting possible trade‐offs between phenolic and terpenoid defence pathways in the treated seedlings.     

Histopathology of primary needles and mortality associated with white pine blister rust in resistant and susceptible Pinus strobus

White pine blister rust caused by Cronartium ribicola is a damaging non‐native disease of five‐needled pines in North America. Efforts to control the disease and mitigate damage to date have been only somewhat effective. Recent efforts to improve the health of eastern white pine and reestablish the tree as a dominant species in the North Central United States have focused on identification and propagation of disease‐free eastern white pine (Pinus strobus) growing in areas with a high incidence of blister rust. Many of these selections have been shown to resist infection following artificial inoculation with C. ribicola. In this study, 13 eastern white pine families derived from controlled pollination of selections previously determined to possess putative resistance as well as susceptible selections were inoculated with C. ribicola. Mortality data from inoculation studies show superior survivability in three families with over 60% of seedlings able to survive the 52 week post‐inoculation monitoring period compared to 0–10% survival of the most susceptible families. Primary needles were collected for histological analysis from all inoculated families 4 weeks after inoculation and from selected families 6.5 weeks and 38 weeks after inoculation. Histological observations of infection sites show distinct resistance reactions in the families more likely to survive infection based on mortality data. Analysis of the reactions in susceptible families revealed extensive hyphal colonization of the vascular bundle and adjacent mesophyll cells that appear uninhibited by tree responses. In resistant families, collapsed cells adjacent to infection sites, heavy deposition of phenolic compounds and abnormal cell growth were documented more frequently and appear to play an integral role in the ability of these eastern white pine families to impede growth of C. ribicola in primary needle tissue.     

Inoculation of several Bursaphelenchus xylophilus group nematodes into adult trees of Pinus thunbergii and their survival in the trees

To clarify the pathogenicity of Bursaphelenchus nematodes to adult pine trees, inoculation experiments using six species of B. xylophilus group nematodes and ca. 10‐year‐old trees of Pinus thunbergii were conducted. Trees inoculated with an avirulent isolate (C14‐5) of B. xylophilus did not die during the survey, but showed a decline in oleoresin exudation compared with the controls. Fifteen months after the inoculation, a small number of B. xylophilus survived in a tree inoculated with B. xylophilus C14‐5. Trees inoculated with B. mucronatus, B. doui, B. luxuriosae, B. conicaudatus and Bursaphelenchus sp. NK224 (undescribed) showed no decline in oleoresin exudation and no external symptoms of wilt. However, 9 months after the inoculation, a small number of B. luxuriosae survived in a tree inoculated with the nematodes, although four other nematode species were not isolated from trees inoculated with them. These results were approximately consistent with our previous results (Kanzaki, N.; Aikawa, T.; Maehara, N.; Ichihara, Y., 2010, J. For. Res.; in press), in which an avirulent isolate (OKD‐1) of B. xylophilus and B. luxuriosae caused water flow inhibition without external symptoms in 3‐year‐old seedlings. Therefore, to examine the pathogenicity of the nematodes to pines, it is useful to use 3‐year‐old seedlings in inoculation experiments when adult trees cannot be used.     

Blue‐stain infections in roots,stems and branches of declining Pinus sylvestris trees in a dry inner alpine valley in Switzerland

Increased mortality rates in Scots pine (Pinus sylvestris) forests have recently been observed in the inner alpine Swiss Rhone valley. Drought, in combination with stand competition, mistletoe infections as well as nematode and insect infestations, appears to be the main factor for the decline. In focus of this study was the occurrence and role of fungal pathogens in the decline dynamics. Branches, stems and roots of 208 trees in five different crown transparency classes were collected and inspected for blue stain and fungal infections. Neither Armillaria species nor Heterobasidon annosum s. str. were detected, but blue stain was commonly observed. Visible blue stain increased with increasing crown transparency. Among the recently dead trees, 80% showed visible blue stain in the branches, 90% in the roots and 100% in the stems. In the crown transparency classes 2 and 3 (25–60% crown transparency), five of the 103 trees showed visible blue stain in the roots, one of 130 trees in the stem but none in the branches. Blue‐stain fungi were isolated from all parts of the trees and from all crown transparency classes. Overall incidence of blue stain was highest in the roots and lowest in the branches. In class 2, roots of 60% of the trees were visibly blue‐stained or developed blue stain in culture, but stems of only 24% and branches of 14% of the trees. In the roots Leptographium species, mostly L. serpens, dominated. From stems and branches, mainly Ophiostoma species were isolated. The positive relationship between the incidence of blue stain and crown transparency, in combination with the high infection levels of roots of fairly vigorous Scots pines, indicates the pathogenic potential of the blue‐stain fungi. Hence, these fungi together with their insect vectors may well act as an important contributing factor involved in pine decline.     

Microbial communities in roots of Pinus sylvestris seedlings with damping‐off symptoms in two forest nurseries as determined by ITS1/2 rDNA sequencing

A methodological molecular procedure, which included extraction and cloning of the ITS1/2 rDNA of root‐associated organisms with subsequent transformation and sequencing of representative clones, was effective for detection, discrimination and determination of the frequency of the main damping‐off pathogens in roots of Pinus sylvestris seedlings growing in different forest‐tree nursery soils and exhibiting different rates of disease progress. Roots exhibiting slower damping‐off progression were colonized by Fusarium oxysporum, Neonectria radicicola (Ascomycota) and Pythium spp. (Oomycota), which comprised 50% of the microbial community. Roots exhibiting faster damping‐off progression were dominated by Thanatephorus cucumeris (Basidiomycota), which comprised 80% of the microbial community. The microbial community was more diverse in roots with slower damping‐off progression (14 species) than in roots with faster disease progression (seven species).