Changes in host chitinase isoforms in relation to wounding and colonization by Heterobasidion annosum: early and strong defense response in 33-year-old resistant Norway spruce clone

We studied the defense reactions of 33-year-old susceptible and resistant clones of Norway spruce (Picea abies (L.) Karst.) to the major root-rot fungus Heterobasidion annosum (Fr.) Bref. and determined if tissue cultures can be used as a model system for studying defense responses of mature trees at the molecular level. Quantitative PCR analysis of genomic DNA obtained from samples taken at different times along the lesion length in living bark indicated that the fungus was present in higher amounts and extended further into the host tissue in the susceptible clone than in the resistant clone. In protein extracts from the same lesion samples, there were differences in temporal and spatial changes in host chitinase isoform profiles between the resistant and susceptible clones. Host chitinase isoforms with pI values approximately 4.8, 4.4 and 3.7 increased more during the first 7 days after wounding and inoculation and extended further along the lesion length in the resistant clone than in the susceptible clone. These results suggest that the time from wounding and infection to induction of defense-related expression is shorter in the resistant clone indicating a more efficient host defense response than in the susceptible clone. Tissue cultures from the same clones were not resistant to H. annosum and showed no difference in the timing of the increase in chitinase isoforms in response to the pathogen. However, tissue cultures from both clones showed an increase in chitinase isoforms within 6 to 24 h past inoculation, indicating that increased chitinase expression in response to the pathogen is part of a general defense response common to both mature clones and tissue cultures.     

Multiplex real-time PCR detection of pathogen colonization in the bark and wood of Picea sitchensis clones differing in resistance to Heterobasidion annosum

A quantitative multiplex real-time polymerase chain reaction (PCR) procedure was developed to assess the extent of Heterobasidion annosum (Fr.) Bref. growth in Sitka spruce (Picea sitchensis (Bong.) Carr.) bark and wood and to determine correlations between lesion length and fungal colonization. Based on lesion length and real-time PCR, the responses of four 3-year-old Sitka spruce clones to inoculation with H. annosum were characterized as showing either resistance or susceptibility to the pathogen. In susceptible clones, the extent of bark colonization did not differ from the visible length of the bark lesion, whereas lesions were longer than the extent of fungal colonization in resistant clones. The resistant clones contained considerably less fungal DNA than the susceptible clones, relative to the amount of host DNA in both the bark and the wood, indicating less resistance and more host cell death in the susceptible clones following inoculation. In both resistant and susceptible clones, fungal colonization in the wood extended beyond the visible necrotic lesion in the bark, indicating that host defense responses are weaker in wood than in bark. The spread of the pathogen in both bark and wood was less in the resistant clones than in the susceptible clones, indicating that defenses in both bark and wood of the resistant clones were superior to those in the susceptible clones.     

Intra- and inter-provenance variability in phloem phenols of Picea abies and relationship to a bark beetle-associated fungus

One hundred Norway spruce (Picea abies (L.) Karst.) clones (three ramets per clone) were analyzed for phloem phenol composition and concentration before and 10 days after wound inoculation with sterile malt agar. Fifty clones (Experiment 1) belonged to the same provenance, whereas the remaining clones (Experiment 2) belonged to five provenances from three geographic areas. In Experiment 2, two additional ramets from the same clones were mass inoculated with Ceratocystis polonica (Siem.) C. Moreau to quantify the resistance of each clone. Tree response to wound inoculations was characterized by increased catechin concentration in both experiments, accompanied by increases in astringin and decreases in piceid in Experiment 1. In both experiments, we observed a diverse group of phenolic compounds whose concentrations increased (catechin, astringin) or did not vary (taxifolin glucoside) in response to wound inoculations, whereas concentrations of a homogeneous group of stilbene compounds decreased (piceid) or did not vary (isorhapontin, unidentified stilbene). In Experiment 2, provenances from the alpine and Hercynian-Carpatic areas differed from provenances from the Baltic area with respect to the relative importance of these two groups of compounds, further indicating that the two groupings of phenolic compounds structure the Norway spruce populations. Eighty days after mass inoculation, the percentage of healthy sapwood, which was taken as a measure of tree resistance, indicated that clones from the Baltic area were less resistant to mass inoculations than clones from the alpine and Hercynian-Carpatic areas. We conclude that the degree of resistance of Norway spruce trees to mass inoculations with a bark beetle-associated fungus can be predicted based on the diversity of constitutive phloem phenols and the ability to induce phenol synthesis in response to wounding.     

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.     

Resin flow responses to fertilization, wounding and fungal inoculation in loblolly pine (Pinus taeda) in North Carolina

Resin flow is the primary means of natural defense against southern pine beetle (Dendroctonus frontalis Zimm.), the most important insect pest of Pinus spp. in the southern United States. As a result, factors affecting resin flow are of interest to researchers and forest managers. We examined the influence of fertilization, artificial wounding and fungal inoculation on resin flow in 6- and 12-year-old stands of loblolly pine (Pinus taeda L.) and determined the extent of that influence within and above the wounded stem area and through time. Fertilization increased constitutive resin flow, but only the younger trees sustained increased resin flow after wounding and inoculation treatments. An induced resin flow response occurred between 1 and 30 days after wounding and inoculation treatments. Wounding with inoculation resulted in greater resin flow than wounding alone, but increasing amounts of inoculum did not increase resin flow. Increased resin flow (relative to controls) lasted for at least 90 days after wounding and inoculation. This increase appeared to be limited to the area of treatment, at least in younger trees. The long-lasting effects of fungal inoculation on resin flow, as well as the response to fertilization, suggest that acquired resistance through induced resin flow aids in decreasing susceptibility of loblolly pine to southern pine beetle.     

Physiological and biochemical differences among Ulmus minor genotypes showing a gradient of resistance to Dutch elm disease

Dutch elm disease (DED) spread across Europe and North America in the 20th century killing most natural elm populations. Today, breeding programmes aim at identifying, propagating and studying elm clones resistant to DED. Here, we have compared the physiology and biochemistry of six genotypes of Ulmus minor of variable DED resistance. Leaf gas exchange, water potential, stem hydraulic conductivity and biochemical status were studied in 5‐year‐old trees of AB‐AM2.4, M‐DV2.3, M‐DV2 × M‐CC1.5 and M‐DV1 and 6‐year‐old trees of VA‐AP38 and BU‐FL7 before and after inoculation with Ophiostoma novo‐ulmi. Leaf water potential and net photosynthesis rates declined, while the percentage loss of hydraulic conductivity (PLC) increased after the inoculation in susceptible trees. By the 21st day, leaf predawn and midday water potential, stomatal conductance to water vapour and net photosynthesis rates were lower, and PLC was higher in trees of susceptible (S) genotypes inoculated with the pathogen than in control trees inoculated with water, whereas no significant treatment effect was observed on these variables in the resistant (R) genotypes. Fourier transform infrared spectroscopy analyses revealed a different biochemical profile for branches of R and S clones. R clones showed higher absorption peaks that could be assigned to phenolic compounds, saturated hydrocarbons, cellulose and hemicellulose than S clones. The differences were more marked at the end of the experiment than at the beginning, suggesting that R and S clones responded differently to the inevitable wounding from inoculation and repeated sampling over the experimental course. We hypothesize that a weak activation of the defence system in response to experimental wounding can contribute to the susceptibility of some genotypes to O. novo‐ulmi. In turn, the decline in shoot hydraulic conductivity and leaf carbon uptake caused by the infection further exacerbates tree susceptibility to the fungus.     

Growth,chlorophyll fluorescence and biochemical markers in clonal ramets of shisham (<Emphasis Type="Italic">Dalbergia sissoo</Emphasis> Roxb.) at nursery stage

About 19 clones of shisham (Dalbergia sissoo Roxb.) were raised by using healthy coppice shoot cuttings. After 2 year of establishment and growth, they were examined to identify suitable clone of shisham at nursery stage. Clones were studied for growth (height and basal diameter), chlorophyll fluorescence (Fv/Fm) and biochemical contents (chlorophyll a, b, total chlorophyll, free amino acids, total soluble proteins, total soluble sugars, starch and reducing sugar) in the leaves. Clone C2 and C3 (Gonda, Uttar Pradesh, India) had the highest growth and the chlorophyll fluorescence rate among examined clones. Between these two clones, C2 gave the highest response on height, basal diameter and Fv/Fm. Except free amino acids, all other biochemical contents were greater in the clones belonging to Gonda, Uttar Pradesh, India. Growth parameters, i.e. height and basal diameter increment at nursery stage had a positive correlation with all the studied parameters, except free amino acid and total soluble protein. These findings have demonstrated that there are remarkable clonal variations in growth, physiological and biochemical responses at nursery stage. Thus, physiological and biochemical markers can enhance selection efficiency, in addition to growth characteristics such as height and basal diameter as these have practical value in the tree improvement. The study suggests that clones of Gonda, Uttar Pradesh, India had the best performance and could be further tested in plantation programs.     

Nutritional and pathogenic fungi associated with the pine engraver beetle trigger comparable defenses in Scots pine

Conifer bark beetles are often associated with fungal complexes whose components have different ecological roles. Some associated species are nutritionally obligate fungi, serving as nourishment to the larvae, whereas others are pathogenic blue-stain fungi known to be involved in the interaction with host defenses. In this study we characterized the local and systemic defense responses of Scots pine (Pinus sylvestris L.) against Ophiostoma brunneo-ciliatum Math. (a blue-stain pathogen) and Hyalorhinocladiella macrospora (Franke-Grosm.) Harr. (a nutritional fungus). These fungi are the principal associates of the pine engraver beetle, Ips acuminatus (Gyll.). Host responses were studied following inoculation with the fungi, singly and as a fungal complex, and by identifying and quantifying terpenoids, phenolic compounds and lignin. Although the length of the necrotic lesions differed between control (wound) and fungal treatments, only two compounds (pinosylvin monomethyl ether and (+)-α-pinene) were significantly affected by the presence of the fungi, indicating that Scots pine has a generic, rather than specific, induced response. The fact that both nutritional and blue-stain fungi triggered comparable induced defense responses suggests that even a non-pathogenic fungus may participate in exhausting host plant defenses, indirectly assisting in the beetle establishment process. Our findings contribute to the further development of current theory on the role of associated fungal complexes in bark beetle ecology.     

Chemical and structural barriers to Microcyclus ulei,the agent of South American leaf blight,in Hevea spp.

Young leaves of different rubber-tree clones were inoculated with Microcyclus ulei under controlled conditions. Under ultraviolet light, intense blue fluorescent light was emitted from the penetration site 6 h after inoculation. The speed of the appearance of this fluorescence was related to the degree of resistance of the clones: 12–36 h in totally resistant clones, 36–120 h in marked partially resistant clones, and 120 h in weak partially resistant clones. The source of this fluorescence may have been scopoletin. Strong accumulation of this coumarin may inhibit pathogen invasion in a very early phase. Four days after inoculation, intense accumulation of lignins in only a few cells at the penetration sites was observed in totally resistant clones. Intense lignin accumulation around lesions may also stop pathogen development in some partially resistant clones     

Anatomical-based defense responses of Scots pine (Pinus sylvestris) stems to two fungal pathogens

We investigated the cellular responses of stem tissues of mature Scots pine (Pinus sylvestris L.) trees to inoculations with two fungal pathogens. The bark beetle vectored fungus, Leptographium wingfieldii Morelet, induced longer lesions in the bark, stronger swelling of polyphenolic parenchyma cells, more polyphenol accumulation and increased ray parenchyma activity compared with the root rot fungus, Heterobasidion annosum (Fr.) Bref., or mechanical wounding. Axial resin ducts in the xylem are a general feature of the preformed defenses of Scots pine, but there was no clear induction of additional traumatic axial resin ducts in response to wounding or fungal infection. The anatomical responses of Scots pine to pathogen infection were localized to the infection site and were attenuated away from bark lesions. The responses observed in Scots pine were compared with published studies on Norway spruce (Picea abies (L.) Karst.) for which anatomically based defense responses have been well characterized.     

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.     

Effect of season and different fungi on phenolics in response to xylem wounding and inoculation in Eucalyptus nitens

Pot‐grown and plantation‐grown Eucalyptus nitens trees (approximately 2 and 3 years old, respect‐ively) were experimentally wounded and inoculated with different fungi and in different seasons. Decay lesion development and defence zones were assessed. Two zones were described, a narrow brown decay interface (interface reaction zone, IRZ) and a diffuse zone beyond this being either pale brown or purple (reaction zone, RZ). The total phenol levels in the reaction zone were determined. Selected phenolics (pedunculagin, tellimagrandin 1, tetragalloylglucose, pentagalloylglucose and catechin) were quantified by liquid chromatography–mass spectrometry (LC–MS). A range of fungi (mainly decay‐causing) were used to inoculate wounds and the results indicated that more extensive decay lesions were generally associated with greater production of soluble phenols in response. Sterile inoculations and weakly aggressive fungi were associated with no or little xylem discoloration, whereas aggressive fungi elicited more discoloration and phenolic accumulation in advance of infection. This indicates that phenol accumulation is not a generalized response to wounding, but a variable response due to the interaction between microorganisms and sapwood. In plantation‐grown trees examined 6 months after wounding, purple reaction zones were commonly associated with large decay lesions. Seasonal differences in decay column area caused by Ganoderma applanatum were not significant 1 month after wounding and inoculation.     

Physiological responses of Eucalyptus spp. hybrids to infection by Ceratocystis fimbriata

In Brazil, the selection and planting of eucalyptus clones resistant to Ceratocystis fimbriata is the pre‐eminent control strategy for the wilt pathogen. However, little is known about host defence responses associated with resistance of eucalyptus to C. fimbriata infection. In this work, enzymatic activity, sugars, lignin, total phenols and phenolic compounds involved in the defence response of eucalyptus clones resistant (RC) and susceptible (SC) to the Ceratocystis wilt were evaluated. Changes were detected in the sugars produced by RC clones, with higher concentrations occurring compared with SC. A similar response occurred with lignin content at 28 days after inoculation (dai) in RC plants. SC plants had an increase in polyphenol oxidase and peroxidase activities at 7 dai. In contrast, RC plants demonstrated high activity of the same enzymes at 2 and 4 dai, decreasing afterwards. There was no difference in phenylalanine ammonia‐lyase activity between resistant and susceptible clones. Hydroxycinnamic acid concentration was higher in RC than in SC; however, there was no difference between RC and SC in flavonoid concentrations. RC had high concentrations catechin, chlorogenic acid and caffeic acid. Histochemical tests demonstrated the presence of phenolic compounds and lignin, at higher intensities, in xylem of RC plants. Inoculation with C. fimbriata induced defence responses in both resistant and susceptible eucalyptus clones, but both the intensity and speed of the responses were higher in RC. Alterations in the concentrations of sugars and lignin, as well as certain enzymes and phenolic compounds, could be used to predict the relative susceptibilities to Ceratocystis wilt of different phenotypes of eucalyptus clones.     

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.     

Genetic effects on total phenolics, condensed tannins and non-structural carbohydrates in loblolly pine (Pinus taeda L.) needles

Carbon allocation to soluble phenolics (total phenolics, proanthocyanidins (PA)) and total non-structural carbohydrates (TNC; starch and soluble sugars) in needles of widely planted, highly productive loblolly pine (Pinus taeda L.) genotypes could impact stand resistance to herbivory, and biogeochemical cycling in the southeastern USA. However, genetic and growth-related effects on loblolly pine needle chemistry are not well characterized. Therefore, we investigated genetic and growth-related effects on foliar concentrations of total phenolics, PA and TNC in two different field studies. The first study contained nine different genotypes representing a range of genetic homogeneity, growing in a 2-year-old plantation on the coastal plain of North Carolina (NC), USA. The second study contained eight clones with different growth potentials planted in a 9-year-old clonal trial replicated at two sites (Georgia (GA) and South Carolina (SC), USA). In the first study (NC), we found no genetic effects on total phenolics, PA and TNC, and there was no relationship between genotype size and foliar biochemistry. In the second study, there were no differences in height growth between sites, but the SC site showed greater diameter (diameter at breast height (DBH)) and volume, most likely due to greater tree mortality (lower stocking) which reduced competition for resources and increased growth of remaining trees. We found a significant site?×?clone effect for total phenolics with lower productivity clones showing 27-30% higher total phenolic concentrations at the GA site where DBH and volume were lower. In contrast to the predictions of growth-defense theory, clone volume was positively associated with total phenolic concentrations at the higher volume SC site, and PA concentrations at the lower volume GA site. Overall, we found no evidence of a trade-off between genotype size and defense, and genetic potential for improved growth may include increased allocation to some secondary metabolites. These results imply that deployment of more productive loblolly pine genotypes will not reduce stand resistance to herbivory, but increased production of total phenolics and PA associated with higher genotype growth potential could reduce litter decomposition rates and therefore, nutrient availability.     

J 16: An apex protein associated with juvenility of Sequoiadendron giganteum

Shoot apex proteins from 11 seedlings, two juvenile clones and three mature grafted clones of Sequoiadendron giganteum (Lindl.) Buchh. were examined throughout the year by SDS-polyacrylamide gel electrophoresis. A membrane-associated protein known as J 16, with an approximate molecular weight of 16 kD, was always detected in juvenile tissue, but was absent in tissue of the mature clones. The J 16 protein was also present in extracts of a mature clone that had been morphologically rejuvenated by micrografting. Protein extracts from the rejuvenated clone, were separated by electrophoresis and assayed for J 16 using an immunoblotting procedure with anti-J 16 serum. This test revealed two bands, J 16 and another protein with a lower molecular weight. The small molecular weight protein was not detected in juvenile clones.     

‘Defence lignin’ and hydroxycinnamyl alcohol dehydrogenase activities in wounded Eucalyptus gunnii

To learn more about lignin formation in response to wounding in trees, we adopted two complementary approaches: (1) microscopic and histochemical studies of the wound response in 3.5‐month‐old Eucalyptus gunnii plantlets and (2) biochemical investigations of hydroxycinnamyl alcohol dehydrogenase activities in wounded 6‐year‐old, field‐grown E. gunnii trees. The first approach revealed that a barrier zone was formed in response to wounding in both ground tissues (cortex barrier and pith reaction zone) and vascular tissues. The barrier zone was barely detectable after 24 h but well‐developed 7 days after wounding. Microscopic analyses indicated that the barrier zone was formed by the reinforcement of cell walls with ‘lignin‐like material’ in both ground tissues and vascular tissue, and that, in addition, the lumen of certain xylem cells (vessels and fibres) were blocked by the deposition of polymeric phenolic material. Histochemical characterization revealed that the lignin‐like material (‘defence lignin’) deposited in ground tissue cell walls and xylem cell blockages was poor in syringyl (S‐type) lignin units and therefore differed from the usual mixed guaiacyl–syringyl (G–S) lignin unit composition of E. gunnii developmental lignin. In contrast, S‐type lignin appeared to be deposited in the cell walls of immature developing secondary xylem cells at a stage when the cell walls of comparable cells from unwounded control stems contained lignin poor in syringyl units. The second approach indicated that two different types of cinnamyl alcohol dehydrogenase activity are induced, and apparently regulated differentially, in response to wounding in E. gunnii trees. Coniferyl alcohol dehydrogenase activity was induced immediately and continued to increase throughout the first 15 days of the 17‐day experimental period, while sinapyl alcohol dehydrogenase activity was first detected at 8 days after wounding and continued to increase throughout the experimental period. The biological roles of the two alcohol dehydrogenase activities are discussed in relation to the formation of defence lignin versus developmental lignin in trees.