Physical and chemical responses of Sitka spruce (Picea sitchensis) clones to colonization by Heterobasidion annosum as potential markers for relative host susceptibility

Forty-one 2-year-old clones of Picea sitchensis (Bong.) Carr. from three full-sib families (14 clones from each of two families and 13 clones from a third family) were either wounded and inoculated with an isolate of Heterobasidion annosum (Fr.) Bref. or wounded without inoculation. Lesion lengths on the inner bark from the point of inoculation varied among clones 35 days after treatment. There was no relationship between lesion length and relatedness within families. Two clones (21342 and 25202) with the shortest lesions, tentatively designated as less susceptible to H. annosum, and two clones (21176 and 27166) with the longest lesions, designated more susceptible, were selected for comparison of host anatomical and chemical responses to infection. The position and structure of the ligno-suberized boundary zone (LSZ) in the bark of the clones suggested that the less susceptible clones formed thicker layers of suberized cells in the LSZ following wounding plus inoculation. No LSZ was observed in two ramets of the more susceptible Clone 27166 following wounding and inoculation with H. annosum. Compared with more susceptible genotypes, clones of P. sitchensis with low susceptibility to H. annosum had high relative proportions of (+)-alpha-pinene, (-)-beta-pinene and one unidentified terpene constituent (Unknown-15) in cortical resin sampled 25 cm from the lesions. In contrast, more susceptible clones had higher relative proportions of (-)-limonene, Unknown-16, Unknown-18 and Unknown-19. In the secondary resin produced in bark tissues surrounding the lesions, proportions of several monoterpene constituents varied; these changes included a decrease in the relative amount of beta-phellandrene and corresponding small increases in some minor constituents. The concentrations of the monoterpenes, except a few minor constituents, increased in the infected tissues. Wounding plus inoculation with H. annosum resulted in varied monoterpene responses, with distinct differences between less susceptible and more susceptible clones. In less susceptible clones, Unknown-19 increased following wounding plus inoculation, whereas in more susceptible clones, concentrations of delta-3-carene and Unknown-13 and Unknown-16 increased. Differences in both constitutive and induced resin monoterpene profiles may provide useful markers for resistance to H. annosum in selection and breeding programs.     

Inducible anatomical defense responses in Norway spruce stems and their possible role in induced resistance

Norway spruce (Picea abies (L.) Karst.) trees were preinoculated with a sublethal dose of the blue-stain fungus Ceratocystis polonica Siem. (C. Moreau) 1 to 52 weeks before they were mass inoculated with the same fungus. Trees pretreated 1 week before mass inoculation had similar, severe symptoms of fungal infection as the control trees. Pretreatment 3, 6 or 9 weeks before mass inoculation resulted in effective protection of the trees, reducing pathogenic symptoms by 63-90% relative to the control trees, whereas pretreatment 52 weeks before mass inoculation gave intermediate protection (44-71% reduction in symptoms). Thus, pretreatment induced resistance to the blue-stain fungus in Norway spruce by a process that requires more than 1 week to become activated and protects trees for at least one year after pretreatment. Pretreatment induced formation of traumatic resin ducts (TDs) in the sapwood and swelling and proliferation of polyphenolic parenchyma cells (PP cells) in the phloem. Trees pretreated 3-9 weeks before mass inoculation had more TDs and showed greater swelling of existing PP cells than control trees or trees pretreated 1 week before mass inoculation. We conclude that induced disease resistance in Norway spruce is probably associated with PP cell activation and TD induction, because resistance was enhanced within the same time frame as the induction of these defense responses.     

Effects of mass inoculation on induced oleoresin response in intensively managed loblolly pine

Oleoresin flow is an important factor in the resistance of pines to attack by southern pine beetle, Dendroctonus frontalis Zimm., and its associated fungi. Abiotic factors, such as nutrient supply and water relations, have the potential to modify this plant-insect-fungus interaction; however, little is known of the effects of inoculation with beetle-associated fungi on oleoresin flow. We observed that constitutive and induced resin yield in loblolly pine, Pinus taeda L., were affected by either fungal inoculation (with the southern pine beetle-associated fungus Ophiostoma minus (Hedgcock) H. & P. Sydow) or silvicultural treatment. The effects of mass wounding (400 wounds m(-2)) and mass wounding and inoculation with O. minus were assessed by comparison with untreated (control) trees. The treatments were applied to trees in a 2 x 2 factorial combination of fertilizer and irrigation treatments. Fertilization did not significantly affect constitutive resin yield. Even as long as 105 days post-treatment, however, mass-inoculated trees produced higher induced resin yields than control or wounded-only trees, indicating a localized induced response to fungal inoculation. We noted no systemic induction of host defenses against fungal colonization. Although beetles attacking previously attacked trees face a greater resinous response from their host than beetles attacking trees that had not been previously attacked, the effect of an earlier attack may not last more than one flight season. Despite mass inoculations, O. minus did not kill the host trees, suggesting that this fungus is not a virulent plant pathogen.     

Loblolly pine and slash pine responses to acute aluminum and acid exposures

In response to concerns about aluminum and HCl exposure associated with rocket motor testing and launches, survival and growth of full-sib families of loblolly pine (Pinus taeda L.) and slash pine (Pinus elliottii Engelm.) were evaluated in a nursery bed experiment. Each species was exposed to a single soil application of aluminum chloride (0.33 M AlCl(3), pH 2.5), hydrochloric acid (0.39 M HCl, pH 0.6) or water, with or without mycorrhizal inoculation with Pisolithus tinctorius (Coker and Couch). After 20 weeks without inoculation, survival in AlCl(3) and HCl treatments averaged 52% for loblolly pine and 72% for slash pine. Inoculation improved survival of loblolly pine, receiving HCl from 49 to 73%, and of those receiving AlCl3, from 55 to 90%. Inoculation also resulted in improved survival and growth of individual families in AlCl(3), but not in HCl treatments. Results illustrate the relative resistance of both pine species to the acute treatments supplied, the improvement in resistance associated with mycorrhizal inoculation and the importance of field testing, following hydroponic screening, to verify the resistance to soil-supplied stresses.     

Differential effects of plant ontogeny and damage type on phloem and foliage monoterpenes in jack pine (Pinus banksiana)

Coniferous trees have both constitutive and inducible defences that deter or kill herbivores and pathogens. We investigated constitutive and induced monoterpene responses of jack pine (Pinus banksiana Lamb.) to a number of damage types: a fungal associate of the mountain pine beetle (Dendroctonus ponderosae Hopkins), Grosmannia clavigera (Robinson-Jeffrey & R.W. Davidson); two phytohormones, methyl jasmonate (MJ) and methyl salicylate (MS); simulated herbivory; and mechanical wounding. We only included the fungal, MJ and mechanical wounding treatments in the field experiments while all treatments were part of the greenhouse studies. We focused on both constitutive and induced responses between juvenile and mature jack pine trees and differences in defences between phloem and needles. We found that phytohormone applications and fungal inoculation resulted in the greatest increase in monoterpenes in both juvenile and mature trees. Additionally, damage types differentially affected the proportions of individual monoterpenes: MJ-treated mature trees had higher myrcene and β-pinene than fungal-inoculated mature trees, while needles of juveniles inoculated with the fungus contained higher limonene than MJ- or MS-treated juveniles. Although the constitutive monoterpenes were higher in the phloem of juveniles than mature jack pine trees, the phloem of mature trees had a much higher magnitude of induction. Further, induced monoterpene concentrations in juveniles were higher in phloem than in needles. There was no difference in monoterpene concentration between phytohormone applications and G. clavigera inoculation in mature trees, while in juvenile trees MJ was different from both G. clavigera and simulated herbivory in needle monoterpenes, but there was no difference between phytohormone applications and simulated herbivory in the phloem.     

Mechanical injury and fungal infection induce acquired resistance in Norway spruce

Norway spruce trees (Picea abies (L.) Karst.) pretreated by wounding and fungal infection showed highly enhanced resistance to a subsequent challenge inoculation with the pathogenic bluestain fungus Ceratocystis polonica (Siem.) C. Moreau. This is the first time the effectiveness of the constitutive and inducible defenses has been shown to depend on prior wounding and infection in conifers, although such acquired resistance has previously been found in several angiosperms. Trees that were pretreated with a combination of 12 bark wounds (1.6 x 10 cm), four fungal inoculations and four sterile inoculations 1-15 days before mass inoculation with C. polonica at 400 inoculations per square meter over a 0.8 m stem section had significantly shorter necroses in the phloem, less bluestained sapwood, and less dead cambium than untreated control trees. Pretreatment with four fungal or sterile inoculations alone did not lead to enhanced resistance. Pretreatment by bark wounding alone seemed to provide an intermediate degree of resistance compared to bark wounding, fungal inoculations and sterile inoculations combined. All trees had a marked increase in the number of resin ducts in the year of inoculation compared with previous years, suggesting that formation of traumatic resin ducts play an important role in the development and maintainance of enhanced resistance.     

蓝变真菌引起的欧洲云杉木质部解剖学特征及纤维素酶活性的变化

选择奥地利境内阿尔卑斯山健康欧洲云杉为对象,研究室内接种蓝变真菌(Ceratocystis polonica)引起的寄主树木韧皮部和木质部解剖学特征的变化,揭示蓝变真菌引起欧洲云杉枯萎的机制.结果表明:接种1周后的4株欧洲云杉的木质部组织内,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株对照欧洲云杉的木质部组织内,没有检测到蓝变区域.采用生物化学分析和组织定位技术,确定接种真菌和无菌琼脂的欧洲云杉木质部区域纤维素酶的分布和活性变化.接种2周后剥皮取样检测,接种真菌的4株欧洲云杉的木质部组织内纤维素酶含量大幅度增加,其等电聚焦电泳显示明显的纤维素酶酶带;而在接种无菌琼脂的欧洲云杉木质部区域,纤维素酶含量分布较少,其等电聚焦电泳显示微弱的纤维素酶酶带.进一步证明蓝变真菌分泌的纤维素酶能利用寄主欧洲云杉木质部的纤维素,蓝变真菌是致死阿尔卑斯山境内欧洲云杉的重要病原菌.     

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.     

Methyl jasmonate and oxalic acid treatment of Norway spruce: anatomically based defense responses and increased resistance against fungal infection

To study the effect of chemical pretreatment on conifer resistance, 13-year-old Norway spruce (Picea abies (L.) Karst.) trees were treated with methyl jasmonate (MJ) or oxalic acid (OxA) on the outer bark and inoculated with the pathogenic blue-stain fungus Ceratocystis polonica (Siem.) C. Moreau 4 weeks later. Both chemicals significantly reduced symptoms of fungal infection, but MJ was more effective than OxA (51 versus 18% reduction in length of necrotic lesions in the phloem relative to untreated control trees). Anatomical examination of treated stem tissues showed that MJ induced extensive formation of traumatic resin ducts in the xylem and extra polyphenolic parenchyma (PP) cells in the secondary phloem between the cambium and the regular annual PP cell layer. No traumatic resin ducts were formed after treatment with OxA, and the coverage of extra PP cells in OxA-treated tissues was not significantly higher than in the controls. The anatomically based defense reactions induced by MJ were similar to the reactions observed after pathogen infection, mechanical wounding and bark beetle attack. Neither MJ nor OxA had apparent phytotoxic effects on Norway spruce at the concentrations used, with needle and stem tissues of all trees appearing normal without visible symptoms of toxicity. However, trees treated with MJ had 30% less radial sapwood growth than control trees. In conclusion, MJ treatment of Norway spruce appears to have practical potential as a tool for increasing plant resistance to fungal infection, but with a modest reduction in sapwood growth.     

Seasonal effect on infection and development of lesions caused by Cryphonectria parasitica in Castanea sativa

Seasonal variation in the development of chestnut blight, caused by Cryphonectria parasitica, was investigated by inoculating in situ chestnut trees and in vitro excised chestnut segments, at either monthly or 3‐monthly intervals throughout 30 months. Inoculations were made with conidia and mycelium of a virulent isolate and with mycelium of a hypovirulent isolate. Conidial inoculations of living sprouts or excised segments between May and July resulted in the greatest incidence of infection whereas inoculations in autumn and winter, in vitro as well as in situ, did not reveal any visible disease. However, from these symptomless inoculated stems, C. parasitica was isolated 3 months after inoculation. Inoculations with the mycelium of the virulent isolate always resulted in lesions, except in January 1999, and the greatest rate of lesion development occurred for inoculations made in the spring and summer. There was a significant seasonal effect on lesion development. Lesions caused by the hypovirulent isolate, smaller than those caused by the virulent isolate, followed a similar seasonal pattern. The same seasonal variations were observed for inoculations in vitro of excised segments. Relative water content (RWC) of chestnut bark significantly varied with bark sampling date. The rate of lesion development in sprouts significantly correlated with average minimum (ATn) and maximum (ATx) temperatures and the sum of rainfall during inoculation period, with the rate of lesion development measured in excised segments 10 days after inoculation (R10d) and with RWC measured on the day of inoculation. In multiple regression models, variables ATx and R10d best explained variation in lesion development.     

Low-density Ceratocystis polonica inoculation of Norway spruce (Picea abies) triggers accumulation of monoterpenes with antifungal properties

Among the most devastating pests of Norway spruce (Picea abies) are the European spruce bark beetle (Ips typographus) and the associated pathogenic blue-stain fungus Ceratocystis polonica. Following attack and colonization, the beetle and the fungus must cope with induced host chemical defenses, such as monoterpenes that are generally thought to be toxic to both symbionts. The goal of this study was to better understand the response of Norway spruce following C. polonica inoculation at low density that does not overwhelm the tree and to identify monoterpenes mobilized toward the fungus. We inoculated healthy mature trees and monitored monoterpene profiles 2, 3, and 5 months post-inoculation. We also exposed three different C. polonica strains to the most abundant or significantly up-regulated monoterpenes to determine differences in monoterpene toxicity and resistance among strains. Total monoterpene levels, including limonene, were increased at 2 and 3 months after inoculation and had dropped after 5 months. In in vitro assays, all monoterpenes were inhibitory to C. polonica. Limonene and β-pinene were the most potent inhibitors of fungal growth. The extent of inhibition varied between the three strains tested. These results showed a defense response of Norway spruce to C. polonica, in which limonene may play a critical role in inhibiting the spread of the fungus. We also showed that differences between strains of C. polonica must be taken into account when assessing the role of the fungus in this bark beetle–symbiont system.     

海岸带湿地松木麻黄混交林营造效果研究

选用抗风力强的木麻黄优良无性系（水培苗）与湿地松进行混交造林试验,６年的试验观测表明：混交方式以带状混交效果最好,其立木蓄积量分别超过木麻黄、湿地松纯林的１３．４％和８９．８％;林分结构比较稳定;带状造林时,木麻黄带在前,湿地松带在后而形成一道道防风屏障。     

闽北山地坡位对湿地松生长和养分利用效率的影响

对闽北山地坡位对湿地松生长和养分利用效率的影响进行了研究,结果表明:坡位对湿地松幼林的生长有显著影响,中坡和下坡湿地松的树高、胸径和单株材积显著大于上坡;不同坡位上湿地松幼林的林分生物量为下坡(9 522kg hm~(-2))中坡(7 137kg hm~(-2))上坡(6 266kg hm~(-2));不同坡位上湿地松对养分的利用效率不同,其中中坡对氮、磷的利用效率显著高于上坡和下坡,下坡对钾、钙、镁、铁的利用效率高于上坡和中坡。     

Effects of nitrogen source and defoliation on growth and biological dinitrogen fixation of Gliricidia sepium seedlings

Effects of four N sources and two defoliation treatments on growth and nitrogenase activity of Gliricidia sepium (Jacq.) Walp seedlings were studied in a greenhouse. All nutrients were supplied in irrigation water to the sterile growing medium. The N sources were: (1) 100 mg l(-1) of N supplied as NO(3) (-) (high-NO(3) (-)), (2) 50 mg l(-1) of N supplied as NO(3) (-) and inoculation with Rhizobium spp. medium-NO(3) (-)), (3)100 mg l(-1) of N supplied as NH(4)NO(3), and (4) inoculation with Rhizobium spp without mineral N (N(2)). At 35 weeks after sowing, mean total biomass was 130.5, 50.5, 22.9 and 17.4 g seedling(-1) in the NH(4)NO(3), N(2), medium-NO(3) (-) and high-NO(3) (-) treatments, respectively. The root/shoot ratio was high in all of the N treatments (1.73-2.77) because the seedlings had big taproots. The medium-NO(3) (-) treatment completely inhibited nodulation, whereas seedlings in the N(2) treatment were profusely nodulated. At 32 weeks after sowing, groups of seedlings in the N(2) and high-NO(3) (-) treatments were subjected to 50 or 100% defoliation. Closed-chamber acetylene reduction assays of intact root systems were conducted to compare nitrogenase activity at 7, 14 and 28 days after defoliation (DAD). At 7 and 14 DAD, nitrogenase activity of completely and partially defoliated seedlings was about 10 and 60%, respectively, of that of undefoliated controls. At 28 DAD, nitrogenase activity of completely defoliated seedlings was twice the predefoliation value, whereas nitrogenase activity of partially defoliated seedlings was only 87% of the predefoliation value. Recovery of nitrogenase activity was strongly correlated with foliage regrowth in the completely defoliated seedlings, but not in the partially defoliated seedlings. Abundant belowground C and N reserves in the large taproot probably contributed to the rapid recovery from defoliation. Accumulation of belowground biomass may also improve defoliation tolerance of mature trees.     

Protein synthesis in slash pine callus cultures exposed to water stress

Effects of water stress on protein synthesis were investigated in heterotrophic callus cultures derived from slash pine (Pinus elliottii Engelm.) cotyledon explants. Cultures were transferred to medium containing 0-15% mannitol to provide water potentials between -0.4 and -2.5 MPa. Beginning between 0 and 22 h after transfer to the new medium, cultures were incubated for 2 h with (35)S-methionine. Both the uptake of label and the incorporation of label into protein decreased with decreasing medium water potential. Incorporation, however, was reduced to a greater extent than uptake suggesting that the reduced incorporation was at least partly the result of reduced protein synthesis. Separation of labeled proteins by one-dimensional SDS-polyacrylamide gel electrophoresis and visualization by fluorography revealed the induction of two new protein bands after incubation at -1.8 MPa for 24 h. Induction of new proteins was not observed at other water potentials or after shorter incubation times. Protein bands from cultures incubated at -1.8 MPa for 24 h and then transferred for 24 h to mannitol-free medium (-0.4 MPa) were comparable to those from control cultures maintained on mannitol-free medium throughout.     

A low-cost method of mycorrhizal inoculation improves growth of Acacia tortilis seedlings in the nursery

A pot experiment in Kenya examined the effects of mycorrhizal and rhizobial inoculation on the growth and symbiont infection of Acacia tortilis seedlings in unsterile soil, in comparison with sterile and unsterile soil controls. Three mixed, arbuscular mycorrhizal inocula, originating from A. tortilis, Terminalia brownii, T. spinosa and Prosopis juliflora (produced under nursery conditions), were used to inoculate the seedlings, which also received a standard, mixed, rhizobial inoculum. Mycorrhizal infection and plant growth were significantly affected by experimental treatment (p<0.001). Some mycorrhizal infection (presumably with indigenous soil-borne inoculum) occurred in the unsterile soil control, but not in the sterile soil control, indicating that transfer of inoculum between pots in the experiment did not occur. Mycorrhizal formation was significantly more extensive in inoculated plants, which also had better shoot and root growth. 24 weeks after inoculation, plants which had received inoculum of A. tortilis origin were 105 mm tall and their roots were 56% mycorrhizal, whereas the controls most representative of normal nursery treatment were only 77 mm tall and 29% mycorrhizal. Nodulation was erratic and poor in all treatments. The results suggest that mycorrhizal inoculation can improve both mycorrhizal infection and growth of tree seedlings in unsterile nursery soil. The methods of culture and inoculation described here could easily be adopted by nurseries at little cost.     

Limited response of ponderosa pine bole defenses to wounding and fungi

Tree defense against bark beetles (Curculionidae: Scolytinae) and their associated fungi generally comprises some combination of constitutive (primary) and induced (secondary) defenses. In pines, the primary constitutive defense against bark beetles consists of preformed resin stored in resin ducts. Induced defenses at the wound site (point of beetle entry) in pines may consist of an increase in resin flow and necrotic lesion formation. The quantity and quality of both induced and constitutive defenses can vary by species and season. The inducible defense response in ponderosa pine is not well understood. Our study examined the inducible defense response in ponderosa pine using traumatic mechanical wounding, and wounding with and without fungal inoculations with two different bark beetle-associated fungi (Ophiostoma minus and Grosmannia clavigera). Resin flow did not significantly increase in response to any treatment. In addition, necrotic lesion formation on the bole after fungal inoculation was minimal. Stand thinning, which has been shown to increase water availability, had no, or inconsistent, effects on inducible tree defense. Our results suggest that ponderosa pine bole defense against bark beetles and their associated fungi is primarily constitutive and not induced.     

杨树与栅锈菌互作的组织病理学研究

用整叶组织透明染色法对亲和性、非亲和性杨树品种与落叶松-杨栅锈菌（Melampsora larici-popu-lina Kleb）相互作用的组织病理学进行了系统的观察。夏孢子接种6h后开始萌发，12h后芽管侵入气孔，此的在寄主细胞内形成吸器。芽管常形成树杈状或根状分枝，一个芽管分别侵入两个气孔或几个芽管同时侵入一个气孔。杨树与栅锈菌相互识别 中的抗锈性的表达主要发生在接种24h后，即气孔下囊形成阶段。表现为夏孢子萌发初期受抑，气孔下囊形成较迟、数量较少、初生侵染菌丝的长度受抑，吸器出现迟、数量少以及寄主叶肉细胞坏死等，但不同组合间在发生时间和程度上有较大差异。接种12h后，感病品种的叶肉细胞中出现吸器，36h后抗病性组合中出现吸器。随着菌丝的增多，抗病品种中的吸器数量、菌落面积小于感病品种、多于近免疫品种。低反应型品种中菌落线性生长受抑和吸器形成的减少均发生在叶肉细胞坏死之后，尚未发现高感品种出现叶肉细胞坏死现象。     

湿地松针叶束水培的初步研究

作者旨在总结前期工作的基础上,找出导致湿地松水培烂根问题的原因,以1a生湿地松当年生针叶束为材料,利用单因素随机区组试验设计方法,研究了不同水培方式和不同针叶束基部处理方式对湿地松针叶束水培生根的影响,用SAS对测得数据做分析处理,试验结果表明:湿地松烂根的主要原因不是缺氧,3种培养方式中静止培养生根率极显著高于循环培养,静止培养生根量显著高于摇床培养;针叶束基部处理对其生根率和生根量的影响不显著;静止培养中,每瓶培养40根针叶束的培养效果最好。