Phloem parenchyma cells are involved in local and distant defense responses to fungal inoculation or bark-beetle attack in Norway spruce (Pinaceae)

The anatomical response of Norway spruce bark polyphenolic parenchyma cells (PP cells) to inoculation with the phytopathogenic fungus Ceratocystis polonica and attack by its bark-beetle vector Ips typographus was examined. Fungal inoculation on the periderm surface had no effect, while inoculation just below the periderm or halfway into the phloem (mid-phloem) generated detectable responses within 3 wk. The responses included increase in PP cell size and in periodic acid-Schiff's staining of PP cell phenolics, wound periderm initiation from PP cells, and cambial zone traumatic resin duct formation. Fungi were not seen in samples 3 wk after subperiderm or mid-phloem inoculation, but were found in some samples 6 and 9 wk after mid-phloem inoculation. In contrast, inoculations into the cambium resulted in partial (3 wk) or complete (6 and 9 wk) fungal colonization and death of tissue in the infected area. This indicates that PP cells have defenses capable of inhibiting fungal growth. Samples taken near bark-beetle galleries had similar anatomical responses as inoculated samples, validating the inoculation approach to studying defense responses in spruce. These results show that PP cells represent not only a constitutive defense system, but are also involved in local and remote inducible defenses against fungal and beetle attack.     

Application of methyl jasmonate on Picea abies (Pinaceae) stems induces defense-related responses in phloem and xylem

Application of 100 mmol/L methyl jasmonate (MJ) to the intact bark of 30-yr-old Norway spruce induced anatomical reactions related to defense. Within 30 d, a single MJ treatment induced swelling of existing polyphenolic parenchyma cells (PP cells) and an increase in their phenolic contents and formation of additional PP cells and of traumatic resin ducts (TDs) at the cambial zone. These changes occurred up to 7 cm away from the application zone. Treatment enhanced resin flow and increased resistance to the blue-stain fungus, Ceratocystis polonica. Methyl jasmonate application to the oldest internode of 2-yr-old saplings also induced TD formation, and, more surprisingly, TDs were formed in the untreated internode. Traumatic ducts were not formed in branches, ruling out an effect of volatile MJ on the upper internode. Methyl jasmonate application never gave rise to a hypersensitive response, cell death, tissue necrosis, or wound periderm, indicating the amount of MJ transported across the periderm was very low relative to the application concentration. This is the first report of a single compound giving rise to major cellular features related to acquired resistance and previously shown to be induced by wounding, fungal infection, and bark beetles in Norway spruce.     

Induced responses in stilbenes and terpenes in fertilized Norway spruce after inoculation with blue-stain fungus, Ceratocystis polonica

In conifers, attacks by bark beetles and associated pathogenic fungi cause an induced wound response, which is characterized by accumulation of antifungal compounds and morphological changes that aid wound healing. In this article the stilbene and terpene concentrations of Norway spruce phloem were monitored as symptoms of induced wound responses in relation to changed nutrient conditions caused by fertilization. Plots of mature Norway spruce were fertilized with N, P or NPK. One year after fertilization the trees were artificially infected with Ceratocystis polonica, a pathogenic fungus associated with the bark beetle Ips typographus. The response of stilbenes to fungal inoculation was mainly qualitative. The concentration of stilbene glycosides in the phloem decreased, and in the immediate vicinity of the site of fungal inoculation, stilbene glycosides were less frequent than in mechanically wounded or unwounded phloem. Corresponding stilbene aglycones were most frequent inside the reaction lesion. The concentration of total stilbene aglycones near the inoculation site was significantly lower in N-fertilized trees than in unfertilized trees. Fungal inoculation caused a strong quantitative response in terpenes. The total terpene concentration of the phloem increased significantly, to almost 100 times greater near the inoculation site compared to the constitutive values. N fertilization significantly reduced the total terpene and total stilbene aglycone concentrations near the inoculation sites. Thus, N fertilization may reduce the ability of Norway spruce to defend itself against fungal pathogens.     

Immunofluorescence localization of levopimaradiene/abietadiene synthase in methyl jasmonate treated stems of Sitka spruce (Picea sitchensis) shows activation of diterpenoid biosynthesis in cortical and developing traumatic resin ducts

Conifers produce terpenoid-rich oleoresin in specialized resin ducts as a main line of defence against pests and pathogens. In spruce species (Picea spp.), axial resin ducts are either present constitutively in the cortex tissue (cortical resin ducts, CRDs) or are formed de novo as traumatic resin ducts (TRDs) in the cambial zone upon attack by insects, fungi or treatment with methyl jasmonate (MeJA). Using immunofluorescence localization we tested if previously formed CRDs respond to MeJA treatment with increased capacity for diterpenoid biosynthesis. We also tested the dynamics of diterpene synthase localization in the cambial zone. Immunofluorescence localization was performed using an antibody against a diterpene synthase, levopimaradiene/abietadiene synthase (LAS), in stem cross-sections of untreated and 0.1% MeJA-treated 4-year old Sitka spruce (P. sitchensis) trees. No fluorescence signal was observed in untreated stem cross-sections; however, signal was present 2 days after treatment with MeJA exclusively in the epithelial cells of CRDs. Fluorescence steadily increased in the CRD epithelial cells 4 and 8 days after treatment. At 8 days, additional fluorescence was observed in developing epithelial cells of traumatic resin ducts TRDs in the cambial zone. These results confirm that resin duct epithelial cells are the main site of diterpene biosynthesis in Sitka spruce, diterpenoid biosynthesis is induced in CRD epithelial cells early upon treatment with MeJA, and immature developing TRD epithelial cells produce diterpene synthase enzyme. Overall, the results of this work improve our understanding of spatial and temporal patterns of induced diterpene resin acid biosynthesis in conifers.     

华山松大小蠹及其伴生蓝变真菌对华山松木质部危害的解剖学特征

为揭示华山松大小蠹和伴生蓝变真菌引起秦岭华山松枯萎的机制,选择秦岭北坡沣峪林场境35年树龄的健康华山松Pinus armandi为研究对象,对接种华山松大小蠹Dendroctonus armandi及与其伴生的蓝变真菌Ceratocystis polonica引起的寄主树木木质部形态变化进行了解剖观察。结果表明：接种致病性蓝变真菌C. polonica 1周后的4株华山松 的木质部组织内,蓝变区域显著增加,4～6周后蓝变区域不再增加; 而在接种无菌琼脂的2株对照华山松的木质部组织内,没有检测到蓝变区域。研究结果提示蓝变真菌C. polonica是致死秦岭华山松的重要病原菌,该伴生菌随华山松大小蠹入侵健康寄主华山松木质部组织,在木质部定居并分解木质部,堵塞树脂道,致使寄主华山松树脂代谢和水分代谢紊乱。该研究结果表明,虽然华山松大小蠹长期以来被认为是致死华山松的毁灭性小蠹虫,但是其共生蓝变真菌C. polonica对成熟华山松的致害作用不应该被忽视。     

SITES OF CALLUS PRODUCTION IN PRIMARY EXPLANTS OF SPRUCE TISSUE

When explants for tissue cultures taken from mature spruce trees, consisting only of xylem-cambium-phloem without pith or cortex, are placed on nutrient agar, callus forms first from the cambium, then from phloem parenchyma, and finally from the lining of xylem resin ducts. The proliferating cells of the phloem are the tannin cells which in situ give rise to sclereids in the bark.     

Methyl jasmonate induces traumatic resin ducts,terpenoid resin biosynthesis,and terpenoid accumulation in developing xylem of Norway spruce stems

Norway spruce (Picea abies L. Karst) produces an oleoresin characterized by a diverse array of terpenoids, monoterpenoids, sesquiterpenoids, and diterpene resin acids that can protect conifers against potential herbivores and pathogens. Oleoresin accumulates constitutively in resin ducts in the cortex and phloem (bark) of Norway spruce stems. De novo formation of traumatic resin ducts (TDs) is observed in the developing secondary xylem (wood) after insect attack, fungal elicitation, and mechanical wounding. Here, we characterize the methyl jasmonate-induced formation of TDs in Norway spruce by microscopy, chemical analyses of resin composition, and assays of terpenoid biosynthetic enzymes. The response involves tissue-specific differentiation of TDs, terpenoid accumulation, and induction of enzyme activities of both prenyltransferases and terpene synthases in the developing xylem, a tissue that constitutively lacks axial resin ducts in spruce. The induction of a complex defense response in Norway spruce by methyl jasmonate application provides new avenues to evaluate the role of resin defenses for protection of conifers against destructive pests such as white pine weevils (Pissodes strobi), bark beetles (Coleoptera, Scolytidae), and insect-associated tree pathogens.     

Distribution, Development and Structure of Resin Ducts in Guayule (Parthenium argentatum Gray)

The distribution, development and structure of resin ducts inguayule (Parthenium argentatum Gray), the second best sourceof natural rubber, have been studied. Resin ducts are widelydistributed in stem, root, leaf, petiole and peduncle. The ductsin the primary tissues are initiated schizogenously and theirfurther development is schizolysigenous. The ducts in the cortexof the root do not have a well-defined epithelium. Ducts developedfrom the vascular cambium are initiated and develop schizogenously.Both resin and rubber are produced in the epithelial cells ofresin ducts. While resin is secreted into the duct lumen, rubberis stored within these cells. Epithelial cells store more thanneighbouring parenchyma cells. Guayule, rubber, resin, ducts, epithelial cells     

Specialized phloem parenchyma cells in Norway spruce (Pinaceae) bark are an important site of defense reactions

The bark anatomy of Norway spruce clones that were resistant or susceptible to Ceratocystis polonica, a bark-beetle-vectored fungal pathogen, was compared. The major difference concerned the axial parenchyma cells, called polyphenolic parenchyma (PP cells) because of their vacuolar deposits. The phenolic nature of the deposits was indicated by autofluorescence under blue light, and immunocytochemical studies demonstrating PP cells are enriched in phenylalanine ammonia lyase (EC 4.3.1.5), a key enzyme in phenolic synthesis. Susceptible-clone PP cells occurred as single rows filled with dense deposits. The resistant clone had 40% more PP cells, which occurred in rows two cells thick plus as individual cells scattered among the sieve cells and had lighter deposits. Trees inoculated with fungus were analyzed but a distinct fungal response could not be separated from the general wound response. In the resistant clone, phenolic bodies were reduced in size and density or disappeared completely 12 d after wounding, and PP cell size increased. The susceptible-clone phenolics and cell size changed only slightly. These data show that PP cells are active in synthesis, storage, and modification of phenolics in response to wounding, providing an important site of constitutive and inducible defenses.     

White pine weevil performances in relation to budburst phenology and traumatic resin duct formation in Norway spruce

1 As the phenological window hypothesis was reported to be significant in influencing the fitness of many herbivores feeding on tree foliage, could it also explain the performance of an insect such as the white pine weevil Pissodes strobi mainly attacking the bark phloem of conifers? 2 Under field conditions, adult weevils were caged on Norway spruce trees presenting a natural variation in their shoot growth phenology. 3 We evaluated white pine weevil biological performances, including oviposition, the number of emerged insects, survival, adult mean weight and tree defense responses as reflected by the production of induced resin canals. 4 None of the white pine weevil biological parameters was significantly affected by Norway spruce phenology. 5 The number of eggs per hole, the number of oviposition holes per leader, the number of emerged adults and their mean weight were not affected by host phenology. 6 The intensity of the traumatic response observed was variable and not correlated with budburst phenology. 7 Trees with higher traumatic responses, forming two or more layers of traumatic ducts, had lower adult emergence and estimated survival. 8 The distance between the first layer of traumatic resin ducts and the start of the annual ring was not correlated with the number of emerged weevils. 9 Norway spruce, which is an exotic tree in North America and a relatively recent host for the white pine weevil, might not possess the defense mechanisms necessary to fight off the white pine weevil.     

Differential anatomical response of Norway spruce stem tissues to sterile and fungus infected inoculations

The anatomical defense responses in stems of Norway spruce (Picea abies) clones of different resistance to pathogenic fungi were characterized over time and distance from small mechanical wounds or wounds inoculated with the root rot fungus Heterobasidion annosum. Common responses for both treatments included division of ray parenchyma and other cells in the cambial zone, accumulation of phenolic inclusions in ray parenchyma cells, activation of phloem parenchyma (PP) cells, and formation of traumatic resin ducts (TDs) in the xylem. TD formation occurred synchronously from a tangential layer of cells, or symplasmic domain, within the zone of xylem mother cells. TD induction is triggered by a signal, which propagates a developmental wave in the axial direction at about 2.5 cm per day. TDs are formed at least 30 cm above single inoculations within 16–36 days after inoculation. The size and number of TDs is attenuated further away from the inoculation site, indicating a dose-dependent activity leading to TD development. Compared to sterile wounding, fungal inoculation gave rise to more and larger TDs in all clones, and multiple rows of TDs in weak clones. Fungal inoculation also induced the formation of more new PP cells, increasing the number of PP cells in the phloem in the year of inoculation up to 100%. TD and PP cell formation was greater in susceptible compared to resistant clones and after fungal versus sterile inoculation. Potential mechanisms responsible for this variable response are discussed.     

Induced Resistance to Pathogenic Fungi in Norway Spruce

Norway spruce (Picea abies) trees (approximately 16 m high) of a single clone were used to study the effects of fungal infection and wounding on induction of resistance to the bark beetle-associated bluestain fungus Ceratocystis polonica. A dose-response experiment was designed involving three different dosages of fungal (fungus and wound) and sterile agar (wound) pretreatment inoculations (10, 50, or 100 inoculations/m² on the stem between 0.8 and 2.0 m high). Three weeks after pretreatment, trees were challenged with a massive C. polonica inoculation (400 inoculations/m²). Control trees that received no pretreatment were heavily colonized and killed by the challenge inoculation. The high and medium fungal pretreatments reduced subsequent fungal colonization success by 76% to 97% relative to the control, and fungal pretreatments protected the trees much more efficiently than sterile agar pretreatments. The protection was demonstrated to be local and not systemic in a subsequent experiment, where trees were pretreated with the medium fungal dosage on the lower bole and challenge inoculated further up the stem. Protection was also demonstrated to be pathogen nonspecific, as trees that had been pretreated with a medium dosage of the root rot fungus Heterobasidion annosum showed enhanced resistance to challenge inoculation with C. polonica.     

The influence of Ceratocystis polonica inoculation and methyl jasmonate application on terpene chemistry of Norway spruce,Picea abies

Constitutive and inducible terpene production is involved in conifer resistance against bark beetles and their associated fungi. In this study 72 Norway spruce (Picea abies) were randomly assigned to methyl jasmonate (MJ) application, inoculation with the bluestain fungus Ceratocystis polonica, or no-treatment control. We investigated terpene levels in the stem bark of the trees before treatment, 30 days and one year after treatment using GC–MS and two-dimensional GC (2D-GC) with a chiral column, and monitored landing and attack rates of the spruce bark beetle, Ips typographus, on the trees by sticky traps and visual inspection. Thirty days after fungal inoculation the absolute amount and relative proportion of (+)-3-carene, sabinene, and terpinolene increased and (+)-α-pinene decreased. Spraying the stems with MJ tended to generally increase the concentration of most major terpenes with minor alteration to their relative proportions, but significant increases were only observed for (?)-β-pinene and (?)-limonene. Fungal inoculation significantly increased the enantiomeric ratio of (?)-α-pinene and (?)-limonene 1 month after treatment, whereas MJ only increased that of (?)-limonene. One year after treatment, both MJ and fungal inoculation increased the concentration of most terpenes relative to undisturbed control trees, with significant changes in (?)-β-pinene, (?)-β-phellandrene and some other compounds. Terpene levels did not change in untreated stem sections after treatment, and chemical induction by MJ and C. polonica thus seemed to be restricted to the treated stem section. The enantiomeric ratio of (?)-α-pinene was significantly higher and the relative proportions of (?)-limonene were significantly lower in trees that were attractive to bark beetles compared to unattractive trees. One month after fungal inoculation, the total amount of diterpenes was significantly higher in putative resistant trees with shorter lesion lengths than in putative susceptible trees with longer lesions. Thus, terpene composition in the stem bark may be related to resistance of Norway spruce against I. typographus and C. polonica.     

The effect of environmentally induced changes in the bark of young conifers on feeding behaviour and reproductive development of adult Hylobius abietis (Coleoptera: Curculionidae)

Young plants of Sitka spruce, Scots and Corsican pine were subject to high and low light, and high and low nitrogen treatments in a polyhouse experiment. The effect of treatments on resin duct size and nitrogen concentration in stem bark was determined together with feeding by Hylobius abietis Linnaeus on the stems of 'intact' plants and on 'detached' stems cut from the plant. Resin duct size was largest on Corsican pine and smallest on Sitka spruce and inherent variation in duct size between the three conifer species appears to determine the pattern of weevil feeding between species. Resin ducts and the flow of resin from them protect the stems of young conifers from weevil feeding not by affecting the total amount of bark eaten but by limiting the depth of feeding and so protecting the inner phloem and cambium. Shallow feeding may increase the likelihood of effective wound repair. Duct size was positively related to plant growth and in particular increased with bark thickness. Overall, ducts were largest in the high light treatment although species differed in their response to the treatment. It is suggested that the effects of plant size, growing conditions and transplantation on susceptibility to attack by H. abietis, reported in various studies, may be due to underlying variation in resin duct size or flow rate. The effect on weevils of superficial feeding on stems is to increase the time for reproductive maturation by reducing consumption of the inner bark which has a higher nitrogen content.     

果胶酶的细胞化学定位：马尾松树脂道发生的细胞化学证据

运用薄切片和电镜细胞化学方法观察了马尾松(Pinus massoniana Lamb.)茎皮层树脂道的发育及发育过程中果胶酶的变化。树脂道的发育过程一般可分为4个阶段,即原始细胞阶段、胞间隙形成阶段、腔道扩大阶段和树脂道成熟阶段。在原始细胞阶段,果胶酶的反应产物首先出现在原始细胞膨胀的细胞壁角隅处,然后沿细胞壁中层分布。胞间隙形成后,果胶酶的反应产物分布在细胞壁和胞间隙的交界面。随着胞间隙的扩大,反应产物的密度逐渐降低。当树脂道成熟后,上皮细胞壁上则没有果胶酶的反应产物出现。细胞化学证据表明: 在树脂道的发育过程中,果胶酶降解树脂道原始细胞细胞壁中层,支持树脂道以裂生方式形成。果胶酶的细胞化学定位技术还可用于其他植物中与果胶水解有关的发育过程。     

Can Norway spruce trees be ‘vaccinated’ against attack by Ips typographus?

1 A field experiment was carried out to test the hypothesis that treatment of Norway spruce trees with the Ips typographus-transmitted blue-stain fungus Ceratocystis polonica enhances tree resistance to later mass attack by this bark beetle. 2 Twenty-five mature trees were pretreated by inoculating a non-lethal dose of the fungus into the bark, while 18 trees served as untreated controls. Three and a half weeks after treatment a bark beetle attack was initiated by attaching dispensers with I. typographus pheromone to the tree trunks. 3 A significantly larger proportion (67%) of the control trees than of the pretreated trees (36%) were killed by the beetle attack. The result is discussed in relation to recent results regarding defence mechanisms in Norway spruce trees.