Changes in phenolic metabolites of Scots-pine phloem induced by Ophiostoma brunneo-ciliatum,a bark-beetle-associated fungus

Changes in soluble phenol contents of Scots-pine (Pinus sylvestris) phloem were studied after inoculation with Ophiostoma brunneo-ciliatum, a fungus associated with Ips sexdentatus. They were compared to the length of the fungus-induced reaction zone of the phloem, taken as an estimation for the trees' response efficiency against aggression. Five clones consisting of 14 trees were studied, and eight soluble phenolic metabolites were analysed in detail in reaction zones and in unwounded phloem sampled 3 weeks after inoculation. The stilbenes pinosylvin and pinosylvin monomethylether, and the flavonoid pinocembrin, were detected in reaction zones only. Concentrations of two p-coumaric-acid esters and an acetophenone glycoside decreased after inoculation. Variations of the flavonoids taxifolin and 3' taxifolin glucoside were inconsistent. One tree had longer reaction zones and a phenolic content different from the others. In unwounded phloem, concentrations of some phenolic compounds and in particular the ratio between the two p-coumaric-acid esters could be markers for trees' response efficiency. In the reaction zone, markers could be the ratios between concentrations of certain preformed compounds and concentrations of newly synthesized pinosylvin and pinocembrin.     

Hymenoscyphus fraxineus can directly infect intact current‐year shoots of Fraxinus excelsior and artificially exposed leaf scars

Hymenoscyphus fraxineus, the causal agent of ash dieback, was inoculated onto intact, unwounded current‐year shoots and leaf scars of 4‐year‐old, potted Fraxinus excelsior seedlings. Pieces of ash wood colonized by the fungus were used as inoculum. Three of 25 (12%) of the inoculated intact shoots and nine of 25 (36%) of the inoculated leaf scars were infected by H. fraxineus and developed typical symptoms of ash dieback, including necrotic lesions on the shoot surface and wood discoloration as well as shoot and leaf wilting distal to the inoculation site. No symptoms occurred on control seedlings, which had been inoculated in the same way but with sterile wood pieces. Visible necrotic lesions on shoots and wood discoloration were statistically significantly longer in proximal than in distal direction from the inoculation site, a pattern which resembles symptoms after natural infection. The ash dieback pathogen was re‐isolated from nine of 12 (75%) of the symptomatic seedlings. These results provide indirect supportive evidence that the fungus infects shoots via leaves and shows that it is able, under experimental conditions using a massive mycelial inoculum, to directly infect intact, unwounded current‐year shoots of its main host in Europe.     

Induced resistance of Norway spruce,variation of phenolic compounds and their effects on fungal pathogens

Three clones of Norway spruce (Picea abies) were studied for their response to mass‐inoculation with the blue‐stain fungus Ceratocystis polonica. The effect of different pretreatments (fungal inoculation and wounding) before mass‐inoculation was investigated for their possible role in an acquired resistance reaction. Pretreated trees showed enhanced resistance to the subsequent mass‐inoculation relative to control trees that received no pretreatment. Furthermore, the fungal colonization of inoculated trees was less than that of wounded trees. The phenolic content of the bark, analysed by RP‐HPLC, was compared in trees receiving different treatments. Trees inoculated with C. polonica had higher average concentration of (+)‐catechin, taxifolin and trans‐resveratrol than wounded trees. Both inoculated and wounded trees had higher average concentrations of these compounds than control trees. The effect of the phenolic extract of Norway spruce bark on the growth of the root rot fungus Heterobasidion annosum and the blue‐stain fungi C. polonica and Ophiostoma penicillatum were investigated in vitro. Heterobasidion annosum was not negatively affected, and the extracts had fungistatic effects on the blue‐stain fungi. The growth of O. penicillatum was more inhibited than the growth of the more aggressive C. polonica.     

Ceratocystis smalleyi colonization of bitternut hickory and host responses in the xylem

Colonization of Carya cordiformis sapwood by Ceratocystis smalleyi and subsequent host defence responses following artificial inoculation were investigated using anatomical and histological techniques. Hyphae of C. smalleyi were observed in all sapwood xylem features confirming the ability of the pathogen to invade and colonize the xylem tissues of the host species. The fungus was isolated from within and at the margins of discoloured sapwood areas at 2 and 12 months after inoculation. General host defence responses that included vessel occlusion with gels or tyloses, lipid accumulation, and production of phenolic compounds were observed in xylem tissues of inoculated C. cordiformis stems. Pectic substances, lipids, and to a rare extent, phenolic compounds were detected in vascular gels. The lipid‐rich barriers observed likely prevent lateral expansion of the fungus in the sapwood. Furthermore, lack of fungus sporulation within vessels may restrict axial spread of the fungus. C. smalleyi appears to be a limited vascular wilt pathogen of bitternut hickory based on these observations and previously reported sap flow reduction correlated with multiple infections in artificially inoculated trees.     

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.     

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.     

Pathogenicity of Ceratocystis resinifera to Norway spruce

The blue‐stain fungus Ceratocystis resinifera colonizes wounds on living Picea spp. and other conifers in Europe and North America. Little is known regarding the pathogenicity of this fungus and consequently, four Norwegian C. resinifera isolates were inoculated on to Norway spruce (Picea abies) using two different techniques. These included single‐point inoculations on young trees (two inoculations per tree on 14‐year‐old trees) and mass‐inoculations on older trees (～200 inoculations per tree on 34‐year‐old trees). In both experiments, C. resinifera induced minor symptoms that in most cases did not differ significantly from inoculation with sterile agar. The virulent blue‐stain fungus C. polonica, which was inoculated for comparative purposes, induced extensive symptoms, causing 83% dead cambium circumference and 82% blue‐stained sapwood, and long necrotic lesions in the phloem. The results suggest that C. resinifera is non‐pathogenic or only mildly pathogenic to Norway spruce and does not present a threat to these trees.     

Histology of current shoots of Japanese cedar (Cryptomeria japonica D. don) inoculated withCercospora sequoiae Ellis et Everhart

The histological reaction of current shoots of the Japanese cedar,Cryptomeria japonica, inoculated withCercospora sequoiae was investigated. Wound periderm was formed on current shoots of the cedar on and after the 4th day after inoculation. In the cells of the cork layer, lignin and suberin were deposited, and a phenolic substance was accumulated. In other inoculated samples, plasmolysis, contraction of the cells and necrosis of the tissues were also observed. On the 32nd day, wound perioderm and phellogen were observed in samples of the tissue inside the necrotic tissues in response to hyphal invasion. Occurrence of wound periderm and phellogen in tissues of the needles was confirmed in the inoculated samples although no hyphae were observed in and around the tissues. These results suggest that this fungus causes necrosis of host tissues and induces defense responses. A part of this paper was presented at the 106th Annual Meeting of the Japanese Forestry Society (1995).     

Ulocladium chartarum as the causal agent of a leaf necrosis on Quercus pubescens

Summary A necrosis of leaves of Quercus pubescens caused by the fungus Ulocladium chartarum is reported. This fungus causes leaf spots both under natural conditions and following artificial inoculation of unwounded attached leaves. A high incidence of leaf spotting by U. chartarum seems to be associated with wet summers.     

Symptom development of the resinous stem canker caused by inoculation withCistella japonica ontoChamaecyparis obtusa

Cistella japonica was inoculated onto the stems of youngChamaecyparis obtusa trees, and the development of external and internal symptoms was investigated for five years. Most lesions started exuding resin from May to July during the first growing season after inoculation, while a few lesions started exuding resin during the second growing season. Resin exudation lasted for two successive years on most lesions, and during successive three, four, or five years on some lesions. Resin exuded excessively in the secondary phloem of the lesions and resin cysts frequently developed there. The lesions where cambial tissue was necrosed occupied 18% of all the lesions and the resinous areas expanded to a larger size than those where resin simply exuded in the phloem.Ci. japonica was reisolated from some inoculated lesion tissues at a high frequency. The isolation frequency of the fungus from some lesion tissues two and more years later was zero or low and that ofCryptosporiopsis abietina was high. This work was supported by a grant from the Forest Agency of the Japanese Government.     

Xylem dysfunction in Yezo spruce (Picea jezoensis) after inoculation with the blue‐stain fungus Ceratocystis polonica

The blue‐stain fungus Ceratocystis polonica is pathogenic to Norway spruce (Picea abies) in Europe, as well as to Yezo spruce (Picea jezoensis) and Sachalin spruce (Picea glehnii) in Japan. The wilting mechanism in P. jezoensis saplings after inoculation with C. polonica was examined based on anatomical studies of the phloem and xylem of periodically harvested trees. In addition, the course of sap ascent in the trunks was traced by injection of acid fuchsin solution at harvest. As an initial external symptom, needle discolouration was observed. In dye conduction tests, xylem dysfunction in the xylem of inoculated trees became obvious. The dehydrated xylem area (dry zone) had extended more than 20 cm above the inoculation wounds, within 1 month after inoculation. When the sap flow to the branches had nearly stopped, the leaves began to discolour. Hyphae of C. polonica colonized the ray tissue around the inoculation wounds, but were absent at the front of the dry zones. Defence reactions occurred in ray parenchyma cells adjacent to the penetrating hyphae. It is suggested that secondary metabolites, which are formed by the ray cells and epithelial cells of resin canals, are involved in the obstruction of sap flow. Limited necrotic lesions of the phloem and cambium were not associated with foliar symptoms. It is proposed that the dry zone formation caused by C. polonica is the main mechanism leading to tree death.     

Interaction of root-knot nematode and mycorrhizal fungi on common beanPhaseolus vulgaris L.

The interactions of root-knot nematode and VAM fungus on common bean plants (Phaseolus vulgaris L.), and the sequence of nematode infection were studied in the greenhouse. The inoculation with VAM fungus caused a significant increase in plant height and fresh weight compared with non treated plants.Meloidogyne infection unsignificantly decreased plant height and dry weight. When fungus was inoculated at 15 and 30 days prior toM. incognita infection, a significant increase in fresh weight was observed. There were no significant differences in total nitrogen (mg/plant) between plants inoculated withM. incognita alone or those inoculated with bothM. incognita and VAM fungus at the same time or 15 days after the fungus inoculation. Plants preinoculated with VAM fungus 30 days prior to nematode infection had a significant increase in total nitrogen. The inoculation with VAM fungus caused a significant increase in phosphorus content. However, it was significantly decreased in plants inoculated with nematode alone and in plants inoculated with VAM fungus andM. incognita at the same time. Gall index and final nematode population were significantly increased when nematodes were treated at the same time with fungus, although there were significant decrease in nematode final population and gall index when the plants were treated with nematodes at 15 and 30 days after mycorrhizal infection. A decrease in percentage of fungal colonization was observed when nematodes were inoculated with fungus at the same time.     

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.     

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

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

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.     

Etiology of the resinous stem canker ofChamaecyparis obtusa: Cistella japonica as the causal agent

In order to confirm the pathogenicity ofCistella japonica, inoculation experiments were conducted onto several coniferous trees. Resinous lesions similar to those of the resinous stem canker developed by the inoculation withCi. japonica only onChamaecyparis obtusa. Chamaecyparis trees were heavily affected when inoculated with the fungus in November, but slightly in May and August. Resin flows occurred when inocula ofCi. japonica were put into the holes made on stem barks and on pruning scars. Isolates ofCi. japonica varied greatly in the pathogenicity. On the lesions caused by the fungus, resinous and necrotic areas expanded in fusiform or oblong shape, and resin cysts were formed in inner barks. Beneath some necrotic inner barks, sapwood was suppressed in growth for the damages in cambial cells and stained.Ci. japonica was reisolated from the inoculated lesion tissues, though isolation frequency of the fungus became lower in some lesions two or more years after the inoculation. The results of these inoculations proved thatCi. japonica was the causal agent of the resinous stem canker ofCh. obtusa.     

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.     

Anatomical and cellular responses of Pinus monticola stem tissues to invasion by Cronartium ribicola

White pine blister rust (Cronartium ribicola) causes extensive damage to white pines and their associated ecosystems across North America. The anatomical and cellular characteristics of C. ribicola colonization in Pinus monticola branch and stem tissues were studied as a basis for understanding host tree reactions that may be related to resistance. Samples examined showed typical fusiform swelling and some had produced aecia. The reaction of phloem and xylem tissues was compared with non-infected tissue using light and electron microscopy. Cortical parenchyma and phloem polyphenolic parenchyma cells underwent mitotic division, cell swelling, and ca sixfold greater accumulation of phenolic compounds in colonized vs. control stems. In the cortex and secondary phloem, haustoria were common in parenchymatous cells, and hyphae were abundant in the intercellular spaces, but cell death was rare, unless aecia had ruptured the stem cortex. Hyphae were also common in xylem rays, tracheids and between tracheids. Disease-induced changes in the cambial zone included development of cambium-derived xylem traumatic resin ducts. Results demonstrate that diverse host defence responses were activated in the bark of apparently susceptible trees, but lack of mechanical damage by C. ribicola to the phenol-containing host cells and the resin duct system allowed extensive colonization and development of aecia despite elicitation of these stem defences. Interactions between P. monticola and C. ribicola are discussed and compared with other conifer–fungus pathosystems.     

Growth response and nutrient utilization of <Emphasis Type="Italic">Casuarina equisetifolia</Emphasis> seedlings inoculated with bioinoculants under tropical nursery conditions

We investigated the role of tetrapartite associations between an arbuscular mycorrhizal (AM) fungus (Glomus geosporum), phosphate solubilizing bacteria (Paenibacillus polymyxa), Frankia and Casuarina equisetifolia on growth, nutrient acquisition, nutrient utilization and seedling quality of C. equisetifolia. Seedlings of C. equisetifolia were grown in an Alfisol soil and inoculated with G. geosporum, P. polymyxa and Frankia either individually or in combinations. Inoculation of bioinoculants stimulated seedling growth, the efficiency of nutrient uptake and improved seedling quality. However, microbial inoculation generally reduced the efficiency of nutrient utilization in dry matter production (nutrient use efficiency). Inoculation of P. polymyxa or Frankia increased the extent of AM colonization, which resulted in the accumulation of the nutrients. Seedlings inoculated with Frankia and G. geosporum had more, and heavier nodules compared to seedlings inoculated with Frankia alone. Dual inoculation of microbes was more effective than individual inoculations. The growth response of seedlings to inoculation involving all the microbes was greater than the response to either individual or dual inoculations. The results of this study showed that the tetrapartite association could improve the growth, nutrient acquisition and seedling quality of C. equisetifolia under tropical nursery conditions.     

Penetration of Picea sitchensis root bark by Armillaria mellea,Armillaria ostoyae and Heterobasidion annosum

Penetration of root bark tissues of Picea sitchensis by Armillaria ostoyae, Armillaria mellea and Heterobasidion annosum was examined in the absence of wounds, in superficial wounds (rhytidome tissues removed to expose the secondary phloem) and in wounds to the depth of the vascular cambium (deep wounding). Both species of Armillaria penetrated bark without prior wounding, but neither species formed rhizomorphs in this treatment. Armillaria ostoyae penetrated to 39 cell layers in depth by 48 days after inoculation of unwounded bark, whereas A. mellea penetrated 25 cell layers in the same time. Armillaria mellea penetrated superficial wounds significantly more rapidly than did A. ostoyae. Both species produced rhizomorphs within wounded host tissues. Inoculation of deep wounds with Armillaria resulted in a greater depth of bark necrosis with A. mellea than with A. ostoyae. In the absence of wounding, H. annosum failed to penetrate root bark tissues, but in both superficial and deep wounds hyphae penetrated beyond the ligno–suberized boundary zone (LSZ) by 12 days after inoculation. Where no inoculations were made, superficial or deep wounding led within 25 days to the restoration of a structurally continuous LSZ, and by day 48 the wound periderm (WP) was fully differentiated. In inoculated wounds, however, formation of the LSZ and WP was delayed or inhibited in most trees, particularly following inoculation with A. ostoyae or A. mellea. Suberization in the LSZ and WP remained diffuse and discontinuous 48 days after inoculation. Moreover, the presence of WP did not prevent further penetration of the tissues by the pathogens. Variations between trees in the depth of pathogen penetration were noted, possibly indicating differing susceptibilities of individual host genotypes. The possible host factors involved in resistance to penetration of root bark tissues by Armillaria and Heterobasidion are discussed.