Differential effects of nutrient availability on the secondary metabolism of Austrian pine (Pinus nigra) phloem and resistance to Diplodia pinea

Nutrient availability can modulate the incidence and severity of plant diseases. We examined the effects of fertility on (i) host resistance and (ii) secondary metabolite production in the Pinus nigra Arnold –Diplodia pinea (Desm.) Kickx pathosystem. Following preconditioning of trees to one of three fertility levels for one growing season, phloem tissue of one branch was harvested for chemical analyses while another branch on the same whorl was inoculated with the fungus to assess resistance. Nutrient availability had concave and convex quadratic effects on concentrations of total monoterpenes and total phenolics respectively, but had no effect on fungal lesion lengths. Lesion lengths were negatively correlated with lignin, suggesting that this compound is an important factor in resistance. Counteracting effects of nutrient availability on phenolic and terpenoid pathways may explain why fertility had no effect on expression of disease resistance.     

Detection of Diplodia pinea in asymptomatic pine shoots and its relation to the Normalized Insolation index

Diplodia pinea (syn. Sphaeropsis sapinea), a common pathogenic fungus, causes considerable damage in Italy, particularly to pine stands in which trees are subjected to environmental stress. The occurrence of D. pinea in symptomless Pinus nigra shoots was investigated and related to the amount of radiation received by the trees growing on a site in a year, expressed as the Normalized Insolation index (NIi). Twenty‐seven pines were selected from nine locations in Trentino (northern Italy). For each pine the incidence of the fungus in apparently healthy shoots was determined by both culturing on an agar medium and application of real‐time PCR. The incidence of D. pinea determined by culturing samples taken from asymptomatic trees was 59% (16 of 27 trees), compared with 85% found using real‐time PCR (23 of 27 trees). Detection of the pathogen in healthy pine tissue was positively correlated (p < 0.05) with the NIi values, using both detection methods.     

Toxicity of monoterpenes from Pinus radiata cortical oleoresin to Diplodia pinea spores1

The effect on spore germmation and germ tube growth of Diplodia pinea produced by (+)- α-pinene, (?)-β-pinene, (+)-Δ³-carene, (?)-limonene, β-phellandrene, and terpinolene was determined over a range of vapour concentrations. Spore germination was most inhibited by A³-carene; germ tube growth was inhibited by all these monoterpenes except (?)-limonenc and ß-phellandrene. In contrast to the low toxicity of (?)-limonene, (+)-limonene (not known to be present in Pinus radiata cortical oleoresin) was extremely toxic to both spore germination and germ tube growth. The results arc discussed in rotation to an earlier observation that trees free of dieback in the field had high levels of certain monoterpenes.     

First report of Sydowia polyspora causing disease on Pinus pinea shoots

The fungus Sydowia polyspora is frequently isolated from conifers worldwide and is considered a pathogen on several hosts. Stone pine (Pinus pinea) is one of the most important forestry species throughout the Mediterranean basin due to the value of the edible pine nut. Stone pines showing tip dieback, needles with tan‐ to yellow‐coloured lesions and shoot death, observed in stands in Portugal, were sampled for analysis. Fungal colonies covered with cream‐coloured spore masses, were consistently obtained. Morphological and phylogenetic analyses of the ITS rDNA region enabled identification of these isolates as S. polyspora. Inoculation tests showed that the fungus caused lesions on excised P. pinea shoots. The symptoms observed might have a negative effect on pine nut production, and thus, evaluation of the impact of this disease is of relevance to future research. This paper is the first to report S. polyspora causing disease on P. pinea.     

Effect of pruning intensity and season on Diplodia pinea infection of Pinus radiata stem through pruning wounds

In a five-year-old Pinus radiata stand, trees were pruned to remove 50%, 40% or 25% of the crown and pruning stubs inoculated with Diplodia pinea. Infection occurred in 65–68% of the intensely pruned trees some of which also developed crown wilting and died, but in lightly pruned trees (25% crown removal) only 8% of the trees were infected and no mortality occurred. Pruning and inoculation every two weeks for a whole year cycle showed an infection peak in summer, but extremely low infection level during the rest of the year. A vertical gradient of resistance of infection was apparent along the stem with the basal part being extremely resistant, becoming more susceptible towards the top. Within limits infection also increased with increasing stub diameter.     

Sources of Diplodia pinea endophytic infections in Pinus patula and P. radiata seedlings in South Africa

Diplodia pinea, an opportunistic and latent pathogen, can significantly affect Pinus productivity worldwide. Despite being studied in South Africa for almost 100 years, the source of D. pinea inoculum responsible for seedling infection is unknown. The aim of this study was to determine the role of seed in vertical transmission of D. pinea and to investigate sources of inoculum leading to horizontal transmission to pine seedlings. Surface‐disinfected seeds were inoculated with spore and mycelium suspensions of D. pinea to determine its effect on germination. In addition, isolation of the fungus was performed from surface‐disinfected seeds, asymptomatic seedlings collected from nurseries, plantations where pines naturally regenerate and recently established fields, to assess transmission and incidence of endophytic D. pinea infections. Inoculation of seeds with D. pinea spore suspensions affected speed and rate of germination. The fungus was isolated from surface‐disinfected seeds in only a few instances (2–3%) and was not found in healthy seedlings collected from greenhouses and nurseries, suggesting that vertical transmission of the fungus does not occur or is rare. In contrast, D. pinea was isolated from 40% of seedlings obtained from the understory of mature P. patula trees showing that horizontal transmission from mature to young trees sustains the D. pinea inoculum in South African pine plantations.     

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.     

Systemic effects of Heterobasidion annosum s.s. infection on severity of Diplodia pinea tip blight and terpenoid metabolism in Italian stone pine (Pinus pinea)

Three-year-old seedlings of Pinus pinea L. were inoculated near the stem base with one of two Heterobasidion annosum (Fr.) Bref. sensu stricto (s.s.) strains belonging to two populations: the North American P-group (NAm-P) and the European P-group (Eur-P). The NAm-P strain caused smaller H. annosum stem lesions than the Eur-P strain. Three weeks after the stem inoculations with H. annosum, apical shoots were inoculated with Diplodia pinea (Desmaz.) J. Kick. Basal stem infection with H. annosum resulted in D. pinea causing longer necrotic lesions in the shoots, indicating systemic induced susceptibility (SIS) to this shoot blight pathogen. Furthermore, stem induction with the NAm-P strain resulted in higher susceptibility to D. pinea than stem induction with the Eur-P strain. Total terpene accumulation was suppressed by about 50% in the shoots under attack by D. pinea when seedlings were induced with H. annosum. Total terpene concentration in shoots inoculated with D. pinea was negatively correlated with lesion size, both overall and by stem treatment. Stem base inoculation with H. annosum induced whole-plant changes in ter-pe-noid profiles, but these were not associated with the SIS phenotype. We discuss our findings on modulation of systemic response of P. pinea to fungal attack in the context of tripartite ecological interactions.     

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.     

Resistance levels of Spanish conifers against Fusarium circinatum and Diplodia pinea

Pitch canker, caused by Fusarium circinatum, and Diplodia shoot blight, caused by Diplodia pinea, are both damaging to pines (Pinus spp.) grown in plantations throughout the world, including Spain. To assess the potential for interspecific differences in susceptibility to contribute to the management of pitch canker and Diplodia shoot blight in the Atlantic region of Spain, the present study was undertaken to characterize the susceptibility of six pine species (P. sylvestris, P. nigra, P. pinaster, P. radiata, P. halepensis and P. pinea) and Douglas‐fir (Pseudotsuga menziesii) to F. circinatum and D. pinea. Based on inoculations of 2‐year‐old trees, Ps. menziesii, P. pinea and P. nigra were the most resistant to F. circinatum, with lesion lengths ranging from 3.7 to 21.5 mm, 2.2 to 12.6 mm and 2.8 to 30.9 mm, respectively. At the other extreme, Pinus radiata was the most susceptible, sustaining lesions that ranged from 8.5 to 74.8 mm in length. Pinus sylvestris, P. pinaster and P. halepensis showed an intermediate response to F. circinatum. Broadly similar results were observed in inoculations with D. pinea, with Ps. menziesii being relatively resistant and P. radiata being highly susceptible. Consistent with these results, field surveys revealed no pitch canker in stands of Ps. menziesii and low severity of Diplodia shoot blight, whereas P. radiata was severely affected by both diseases. Our findings suggest that selection of appropriate species can greatly reduce the risk of damage from two important canker diseases affecting pine plantations in the Atlantic region of Spain. Furthermore, intraspecific variation in susceptibility implies that selection may allow for the enhancement of resistance in otherwise susceptible species.     

Organ-dependent induction of systemic resistance and systemic susceptibility in Pinus nigra inoculated with Sphaeropsis sapinea and Diplodia scrobiculata

Systemic induced resistance (SIR) is a well-known host defense mechanism against pathogen attack in herbaceous plants, but SIR has only recently been documented in conifers. We tested if inoculation of Austrian pine (Pinus nigra Arnold) with Sphaeropsis sapinea (Fr.:Fr.) Dyko and Sutton or Diplodia scrobiculata de Wet, Slippers and Wingfield results in SIR or systemic induced susceptibility (SIS) to subsequent colonization by S. sapinea. Induction at the stem base resulted in significant (P < 0.01) SIR in the upper stem, and induction in the upper stem resulted in significant (P < 0.05) SIR at the stem base, indicating that SIR is bidirectional in Austrian pine. However, inoculation at the stem base resulted in significant (P < 0.01) SIS in shoot tips, demonstrating that, in the same host species, the expression of resistance can be organ-dependent, resulting in either SIR or SIS depending on the site of challenge infection. Systemic induced resistance in the stem was associated with induced lignification, supporting a potential role for this defense mechanism in disease resistance. Systemic induced susceptibility has been documented before, but this is the first demonstration of organ-dependent expression of both SIR and SIS in a tree or any other plant.     

Histology of Pinus maximinoi cones infected by Cronartium conigenum1

Apparently healthy and C. conigenum infected female strobili of Pinus maximinoi, collected in Guatemala, were histopathologically studied by light microscopy. Scales from infected cones were fused and fertile scales had aborted seeds. Infected cone scales lacked fibre cells in the cortex, which are abundant in healthy cone scales. Intercellular spaces of cortex contained abundant hyphae compared to phloem and xylem. The cell walls of infected tissues in the ovules were darkly stained, while those of comparable healthy tissue were colourless under Pianeze's IIIB stain. Infected ovules of different developmental stages with nucellus, or nucellus plus spongy tissue, were observed. The cells of each tissue type contained many haustoria and did not develop into female gametophyte and embryo. Ovule colonization by hyphae appeared after pollination and subsequently caused their early abortion.     

Longevity of inoculum production by Diplodia pinea on red pine cones

In summer of 2004, pycnidia of Diplodia pinea were observed on cones of Pinus resinosa that had matured and opened during previous years, but had been retained in canopies of trees at a mature red pine plantation in southern Wisconsin. Surveys during the winter and early summer of three consecutive years (2005–2007) to determine incidence and abundance of D. pinea conidia on cones of different ages in this stand. Cones from each age class consistently bore pycnidia with conidia of D. pinea. Although cones collected in June of the year after their maturation tended to yield more D. pinea conidia than older cones, large numbers of conidia were obtained from cones even 3 years after maturation. Perennial availability of inoculum due to persistence of D. pinea on cones of several ages in the overstory or in adjacent stands should be considered when regenerating red pine in areas where this pathogen is known to be present.     

意大利石松在我国的引种栽培

意大利石松是一种优良的园林绿化及防风固沙树种,在世界各地被广泛种植。通过从国外引进优质种子,在民勤治沙综合实验站温室大棚内进行意大利石松引种栽培试验,结果表明:种子处理方式对意大利石松容器育苗的发芽表现、发芽势等影响差异性不显著,但发芽指数表现为浸种催芽高于堆放催芽,通过催芽处理可使得意大利石松种子缩短发芽时间;土壤基质对容器苗冠幅和生长量的影响具有显著的差异性,表现为细沙土中沙粗沙腐殖质混合物耕作土。     

Leptoglossus occidentalis and Diplodia pinea: a new insect‐fungus association in Mediterranean forests

Leptoglossus occidentalis, an insect native to North America, was inadvertently introduced into Italy about 1999. The insect damages the cones of conifer trees, especially Pinus pinea (Italian Stone pine). Pinus pinea is also affected by Diplodia pinea, a fungus native to Italy, which is becoming an increasing threat because pine trees are becoming more susceptible to it as a consequence of global warming. Because the insect and the fungus both have the pine cones as a common habitat, a possible interaction between them has been postulated. The aim of this study was to ascertain whether L. occidentalis and D. pinea interact on P. pinea cones. The interaction was studied using real‐time PCR on a group of naturally infected insects collected from a forest, and a group raised in the laboratory and artificially inoculated with D. pinea conidia. Molecular analysis showed that D. pinea DNA occurred on both naturally infected and inoculated insects, but with significant differences between the two groups. The rapid and sensitive molecular technique made it possible to detect D. pinea DNA on the bodies of the insects, and to show that the native D. pinea occurred on the exotic insect.     

Container characteristics influence Pinus pinea seedling development in the nursery and field

The growth and development of Pinus pinea seedlings grown in different containers was followed through one growing season in the nursery and 3 years following outplanting in the field. The variables studied in the nursery were height, diameter, biomass of shoots and roots, nutrient uptake and root density. The measured field variables, height and diameter increment and survival, were correlated with the nursery variables. Container volume had the greatest influence on plant morphology. Containers with larger rooting volume had seedlings with larger height and diameter, greater nutrient content, and better field performance. Growing density was correlated with seedling morphology and nutrient concentration in the nursery. Among the variables that influenced container volume, the diameter of the container was the most important, while the depth of the container had a minor influence on seedling morphology.The best indicator of seedling development in the nursery was the ratio of container depth to container diameter, and the optimum ratio was 4. All containers produced seedlings with some root spiralling, including those containers with ribs. There was no relationship between either the number of spiralling roots or the angle of spiralling and container characteristics. Furthermore, root spiralling did not influence seedling performance following outplanting. Root density (root biomass/cm³) was inversely correlated with container volume but there was no correlation with either depth or growing density. The largest plants were produced with container volumes of 300–400 cm³, depth/diameter ratios of 4, and growing densities of 200–300 seedlings/m². These growing conditions will result in larger Pinus pinea seedlings coming out of the nursery, which will increase growth following outplanting.     

奥地利黑松生长特性的研究

本文对延安树木园引种的奥地利黑松的生长特性进行了研究。结果表明,奥地利黑松叶芽开放期比乡土树种油松早8 d;展叶期比油松晚11～16 d;新梢开始生长期,奥地利黑松与油松相差近1个月时间,封顶期9月6～10日。奥地利黑松幼苗根系发达,直根长、侧根粗壮、层次明显,鲜质量大,苗木地径生长量显著大于油松,与油松形成鲜明对照,是荒山造林的好树种。31 a生奥地利黑松高生长速生期出现在第15～20 a,生长量占树高的32.1%,比较而言,奥地利黑松树高生长较慢,胸径大于油松,材积年生长量是油松的2倍。