Constant association of ophiostomatoid fungi with the bark beetle <Emphasis Type="Italic">Ips subelongatus</Emphasis> invading Japanese larch logs

Ceratocystiopsis, Ceratocystis, Grosmannia, and Ophiostoma species were isolated from Ips subelongatus and beetle-infested Japanese larch logs collected at several areas in central and northern Honshu Island, Japan, to determine constant associates of I. subelongatus. Ceratocystiopsis minuta, two species of Ceratocystis, three species of Grosmannia, and four species of Ophiostoma were isolated. Of the fungi isolated in the present study, G. laricis, O. brunneociliatum, and O. piceae were constant associates of the beetles. Ceratocystis fujiensis, Ceratocystiopsis minuta, and Ophiostoma sp. F were occasionally isolated with high frequencies of occurrence but were not consistent associates. Ceratocystis fujiensis was most often isolated as the leading fungal invasion in the sapwood of Japanese larch logs invaded by I. subelongatus, confirming that the fungus acts as a primary invader of sapwood in beetle-attacked logs. Contribution No. 221, Laboratory of Plant Parasitic Mycology, Graduate School of Life and Environmental Sciences, University of Tsukuba     

Accumulation of Phenolic Compounds in Conifer Callus Cultures in Response to Wood Blue-Stain Fungi

Callus cultures of Siberian larch (Larix sibiricaLedeb.) and Siberian spruce (Picea obovataLedeb.) were used to demonstrate the elicitor activity of two ophiostomatoid fungal species, Ceratocystis laricicolaand Ceratocystis polonica, as the pioneer settlers on larch and spruce, respectively. The extract from C. laricicolamycelium stimulated the accumulation of lignin in larch cells by 37% and that of bound proanthocyanidins by 25%. In spruce callus cultures, C. laricicolaand C. polonicaincreased the bound PA content by 25 and 46%, respectively. In the callus cultures of larch and spruce, the addition of extract of C. laricicolaincreased the concentration of p-hydroxybenzoic acid 13-fold and nearly 4-fold, respectively. The metabolic characteristics of accumulation of phenolic compounds in conifer cells are discussed.     

Influence of ectomycorrhizal fungi on the response of Sitka spruce and Japanese larch to forms of phosphorus

Ectomycorrhizal fungi (Paxillus involutus, Suillus grevillei and two unidentified basidiomycetes from excised Sitka spruce mycorrhizas) were isolated from stands of Sitka spruce either in monoculture or in a mixture with Japanese larch in an Irish conifer plantation. The growth of these fungi and their mycorrhizal formation in Sitka spruce and Japanese larch were examined after incubation in modified Melin-Norkrans medium containing either KH₂PO₄, Ca₃(PO₄)₂ or Fe phytate as the phosphorus (P) source. P. involutus and S. grevillei utilized all three P sources. The unidentified basidiomycetes had limited ability to utilize Fe phytate. Basidiomycete 1 showed poor growth on KH₂PO₄ whereas growth of basidiomycete 2 was low on Ca₃(PO₄)₂. Pure culture synthesis studies confirmed that P. involutus and the two basidiomycetes formed mycorrhizas with both tree species but S. grevillei was mycorrhizal only on Japanese larch. P. involutus formed more mycorrhizas in both conifers than the other fungi. Following inoculation with each of the four fungi, shoot and root dry mass of both Sitka spruce and Japanese larch seedlings was enhanced compared with uninoculated/nonmycorrhizal controls. On Fe phytate, Paxillus-inoculated Sitka spruce seedlings had the lowest primary root length and on KH₂PO₄, Suillus-inoculated Japanese larch had the greatest number of short roots. The only differences when Sitka spruce seedlings were grown in either monoculture or in a mixture with Japanese larch mycorrhizal with S. grevillei were primary root length and number of short roots after growth on media containing Fe phytate.     

Colour of larch heartwood and relationships to extractives and brown-rot decay resistance

Larch heartwood is appreciated for its good mechanical properties, its colour and its texture, and it is often used outdoors because of its natural durability (decay resistance). In this study the colour of larch heartwood was studied in relation to extractives and decay resistance, with the aim to estimate durability of larch heartwood from its colour. On a total of 293 trees colour in the CIE L*a*b* space (L* lightness, a* red/green axis, b* yellow/blue axis), extractives content (acetone and hot-water extractives, amount of phenolics) and the brown-rot decay resistance were determined. For calculating the relative decay resistance ( x), mass loss after inoculation for 16 weeks with two fungi [ Coniophora puteana (Schum.ex.Fr.) Karst., Poria placenta (Fr.) Cke, European standard EN 113] of larch heartwood samples was compared to Scots pine ( Pinus sylvestris L) sapwood reference samples (EN 350-1). Different species [Japanese larch ( Larix kaempferi Lamb.), Hybrid larch (Larix deciduax L. kaempferi) and European larch ( L. decidua Mill.)], provenances and age classes (38-year, >150-year) were included. Japanese larch heartwood turned out to be significantly more reddish (higher a*-values) compared to the European larch provenances. Reddishness of the hybrids was intermediate. The red hue (+a*) was strongly correlated with the amount of phenols ( r =0.84) and decay resistance ( r =0.63) and therefore suitable for prediction of both parameters. The results suggest that colour measurements of larch heartwood could be of benefit in tree breeding programs and for an optimised utilization of larch timber.     

Co-inoculation of Ceratocystis polonica and Heterobasidion annosum with callus of two Norway spruce clones with different in vivo susceptibility

We have studied how callus cultures from two clones of Norway spruce influence the growth of two pathogens, Ceratocystis polonica and Heterobasidion annosum, when co-cultivated in vitro. In field experiments, trees of clone 409 were susceptible to both fungi, whereas clone 589 was less affected. Callus was cultured on medium containing cytokinins (benzylaminopurine, kinetin) and with or without auxin (2,4-dichlorophenoxyacetic acid). For co-cultivation with fungus, one piece of callus was placed towards the edge of each Petri dish. One and 14 days after inoculation with callus the dishes were co-inoculated with the fungus. Both clones strongly stimulated the initial growth of both fungi. Clone 589 inhibited the growth of both fungi when the fungi were inoculated one day after the callus. When the callus was cultured on medium without auxin for 14 days before co-inoculation clone 589 strongly inhibited the growth of both fungi, whereas clone 409 inhibited H. annosum only. This revised version was published online in June 2006 with corrections to the Cover Date.     

Pathogenicity to western larch (Larix occidentalis) of two fungi, Ophiostoma pseudotsugae and Leptographium abietinum, associated with the Douglas fir beetle (Coleoptera: Scolytidae)

1 Pole-sized, live western larch Larix occidentalis Nutt. were mass-inoculated with Ophiostoma pseudotsugae (Romb.) von Arx or Leptographium abietinum (Peck) Wingf., two blue-stain fungi associated with the Douglas fir beetle Dendroctonus pseudotsugae Hopkins, to assess their pathogenicity. 2 Inoculation with O. pseudotsugae resulted in significantly greater percentages of necrotic phloem compared with L. abietinum inoculations. 3 The percentage of occluded sapwood was significantly greater after L. abietinum inoculations compared with O. pseudotsugae inoculations. 4 Within the inoculation band, all trees had more than 60% functional sapwood 4 months after treatment. 5 The results suggest that western larch can successfully limit colonization by O. pseudotsugae and L. abietinum. 6 The inability of the fungi to thrive in live western larch may be a factor in the consistent failure of Douglas fir beetle broods in this host tree species.     

Molecular phylogenetic analyses reveal a close relationship between powdery mildew fungi on some tropical trees and Erysiphe alphitoides, an oak powdery mildew

To investigate the phylogenetic relationships among the powdery mildew fungi of some economically important tropical trees belonging to Oidium subgenus Pseudoidium, we conducted molecular phylogenetic analyses using 30 DNA sequences of the rDNA internal transcribed spacer (ITS) regions and 26 sequences of the domains D1 and D2 of the 28S rDNA obtained from the powdery mildews on Hevea brasiliensis (para rubber tree), Anacardium occidentale (cashew), Bixa orellana, Citrus spp., Mangifera indica (mango), and Acacia spp. The results indicate that the powdery mildew fungi isolated from these tropical trees are closely related to one another. These powdery mildews are also closely related to E. alphitoides (including Erysiphe sp. on Quercus phillyraeoides). Because of the obligate biotrophic nature of the powdery mildew fungi, the relationship between powdery mildews and their host plants is conservative. However, the present study suggests that a particular powdery mildew species has expanded its host ranges on a wide range of the tropical trees. This article also suggests that a powdery mildew fungus distributed in temperate regions of the Northern Hemisphere expanded its host ranges onto tropical plants and may be a good example of how geographical and host range expansion has occurred in the Erysiphales.     

Comparison of insect,fungal, and mechanically induced defoliation of larch: effects on plant productivity and subsequent host susceptibility

Summary Larch sawfly, Pristiphora erichsonii Hartig, and larch needlecast fungus, Mycosphaerella laricinia (R. Hartig) Neg., are early season defoliators restricted only to Larix host trees. Larch defoliation (100%) by either the fungus or insect, but not mechanical removal, induced systemic responses that reduced sawfly consumption and digestion rates one year later. In a feeding behavior assay, larvae quickly abandoned seedlings previously defoliated by M. laricinia. Adult female oviposition choice and egg deposition were unaffected. Seedling growth was not affected during the year of defoliation by M. laricinia, but was significantly reduced one year later. Defoliation by M. laricinia reduced stem volume, radial growth, root biomass and new shoot production. The latter tissue is the only oviposition resource for larch sawfly, and, in contrast, is not influenced by sawfly feeding. We hypothesize that M. laricinia infection may limit larch sawfly populations where both species coexist. However, this reduction is at a substantial net cost to larch productivity.     

Callus cultures and bark from Norway spruce clones show similar cellular features and relative resistance to fungal pathogens

In field experiments, clones of Norway spruce [Picea abies (L.) Karst.] showed different degrees of resistance against pathogenic fungi inoculated into the bark that correlate with differences in polyphenolic parenchyma (PP) cells of the bark. Cells of spruce callus cultures, particularly towards the callus surface, resemble PP cells and this study looks at changes in callus cells during infection and the relative resistance of cultures from clones of low (weak) or high (strong) resistance to fungal infection. Callus cultures, initiated from trees with different resistance, were co-inoculated with Ceratocystis polonica (Siem.) C. Moreau and Heterobasidion annosum (Fr.) Bref. Callus cells from strong clones resemble PP cells of bark tissue from strong clones, having more polyphenolic bodies, while callus cells from weak clones are more similar to PP cells from those clones, which have less extensive phenolic bodies. Callus cultures from trees with weak resistance were more quickly overgrown by both species of pathogenic fungi than cultures from trees with strong resistance. Callus cells of infected cultures showed changes similar to activated PP cells of bark, including enhanced accumulation of polyphenolics. Phenolic bodies were more numerous and more extensive (larger and denser) in callus cells of strong versus weak clones under all conditions. Thus, callus cells may perform similar functions in defense as PP cells in the bark. Callus from trees of varying resistance seem to reflect the relative resistance of the trees from which they are derived, and this study indicates that some mechanisms of resistance can be studied using callus from trees of different resistance.     

Heterozygosity and hybrid performance in larch

Random Amplified Polymorphic DNAs (RAPD) were used for estimating genetic distances between 12 European larches (Larix decidua) and 12 Japanese larches (L. kaempferi) that were the parents in a factorial mating design. One hundred and eleven fragments were used for establishing genetic distances based on Jaccard's coefficient between parents. Thirteen fragments differentiated the larch species. The genetic distance between individuals of the same species (D _J =0.39 in the Japanese larch and 0.45 in the European larch) was lower than the genetic distance between species (D _J =0.72). A UPGMA dendrogram based on genetic distances clearly clustered each larch species, confirming the speciation at a molecular level. Correlations between genetic distances of the parents and performances of the hybrid families were established for various quantitative traits. Significant values were found for growth characters and branch insertion angle, which suggested an effect of general heterozygosity level on hybrid traits. These correlations also evolved with tree age: the maximal correlation was noticed on 6-year-old trees for height. The lack of correlation between parental genetic distances and hybrid performances for the other quantitative traits suggested that these characters were controlled by fewer genes. The results of this study show that crosses between genetically distant parents produce hybrids with excellent growth performances; this represents a potential selection criterion of the genitors.     

Short-term effects of thinning and liming on forest soils of pitch pine and Japanese larch plantations in central Korea

The influences of thinning (50% of standing density) and liming (Ca+Mg, 2 Mg ha⁻¹) on soil chemical properties were investigated for 2 years (2001, 2002) in 40-year-old pitch pine (Pinus rigida Mill.) and 44-year-old Japanese larch (Larix leptolepis Gord.) plantations established on similar soils. In general, soil properties varied significantly among plantations and treatments. For both plantations, thinning significantly increased soil organic C (SOC) concentrations whereas there were no significant changes in soil pH and Ca and Mg concentrations. In addition, thinning increased total soil N and Na concentrations for the pitch pine plantation and available P concentration for the Japanese larch plantation in the second year after the treatment. Liming did not affect soil chemical characteristics for the pitch pine plantation except for Na concentration. However, for the Japanese larch plantation, liming significantly increased soil pH and K, Ca and Mg concentrations and decreased SOC and total soil N concentrations. For both plantations, soil Al concentration did not change after thinning and liming and decreased exponentially with increased pH values. The increases in SOC and total soil N concentrations after thinning were possibly due to increases in decomposition of organic matter and root death. Although differences were not statistically significant, soil available P concentration tended to increase at early stages of liming for both plantations. These results suggested that thinning and liming seemed to regulate soil chemical properties for pitch pine and Japanese larch plantations established on similar soils.     

Succession of microfungal flora on Rodgersia podophylla plants at the forest side of Cryptomeria plantation

Microfungal flora on aerial parts of a forest floor plant, Rodgersia podophylla, were studied at the forest side of a Japanese cedar plantation. From May to October, Nigrospora spp. were dominantly isolated from leaves, whereas Acremonium spp. and Fusarium spp. were dominant on stems, suggesting that the aerial part of the plants about 1 m height can offer two different habitats for these dominant fungi. In September and October, we could easily discern different types of tissue on the withering leaves, i.e., brown (necrotic lesion), yellowish (border tissue between brown and green areas), and green (healthy tissue). Nigrospora spp. and Pestalotiopsis spp. were continuously isolated on the brown area as well as on green and yellowish areas. Pathogenicity of N. sacchari and P. neglecta on potted plant leaves was confirmed by inoculation. From these, the fungi of these two genera seemed to have changed from quiescent to pathogenic with leaf senescence. Sporulations by fungi of the two genera were recognized on overwintered stems. These fungi may overwinter in stems that are slow to decompose, and seem to go over to the leaves in the following spring. Thus, they could be candidates for parasites that may play an important role in decomposition of the plant.     

Paternal inheritance of chloroplast DNA in Larix

Restriction enzyme analysis was used to determine the inheritance of chloroplast DNA in conifers. The plant material studied included five individual trees of European larch (Larix decidua Mill.) and Japanese larch (Larix leptolepis Sieb. & Zucc.) and six hybrids from controlled crosses between these species. The chloroplast DNA fragment patterns generated by Bam-HI and Bcl-I were species-specific. Paternal inheritance of chloroplast DNA patterns was found in most Larix crosses. One hybrid showed maternal chloroplast DNA patterns. In addition, two other hybrids had mixed Bam-HI patterns suggesting recombination between maternal and paternal chloroplast DNA. The mechanisms favoring paternal inheritance in conifers are not known. Paternal inheritance of chloroplast DNA is suggested it to be a general phenomenon in conifers.     

Biological control of crown rot of bananas with Pichia anomala strain K and Candida oleophila strain O

The antagonistic activity of two yeast strains (Pichia anomala (E.C. Hansen) Kurtzman, strain K and Candida oleophila Montrocher, strain O) against the parasitic complex responsible for banana crown rot was evaluated. The strains were applied at three different concentrations (10⁶, 10⁷, 10⁸ cfu/ml) and their efficacy tested in vivo on three separate fungi (Colletotrichum musae (Berk. & Curt.) Arx, Fusarium moniliforme Sheldon, and Cephalosporium sp.) and on a parasitic complex formed by association of these three fungi. At the concentrations used C. musae appeared to be the most pathogenic. The complex showed intermediate aggressiveness between C. musae and both other fungi.Statistically significant antagonistic effects were observed on C. musae, F. moniliforme, and the fungal complex. The highest protection level (54.4%) was observed with strain O added at 10⁸ cfu/ml on crowns previously inoculated with the fungal complex. The level was lower when the fungi were inoculated separately.Furthermore, the antagonistic effect was strongly reinforced when strain O at 10⁸ cfu/ml was applied 24 h before fungal complex inoculation (59.9%), as compared to its application 15 min (24.3%) or 3 h (27.3%) after fungal complex inoculation. Bananas showed increased susceptibility to the fungal complex from March to June, and this influenced the level of protection by yeast, which decreased over the same period. A strict negative correlation (R² = 0.83) was highlighted between susceptibility of banana to crown rot and protection provided by yeast.     

The influence of micrografting in vitro on tissue culture behavior and vegetative propagation of old European larch trees

The influence of a new micrografting method in vitro was tested by using long shoot meristems of adult 140 year old European larch trees grafted onto larch seedling rootstocks in sterile peat pellets. Five months after grafting and transfer of grafts to ex vitro conditions, new shoots from sprouting scions were re-established into tissue culture. The propagation behavior of shoots derived from these explants was compared with shoots established via bud culture from the same donor trees. Shoot explants from micrografts multiplied more rapidly than shoots explants from the original adult donor trees. Rooting experiments with cuttings from six micrografted clones resulted in 49.9 ± 11.9% rooting. Cuttings from donor trees invariably showed no root formation. The results confirm a rejuvenating influence of micrografting in larch.     

Reduced parasitism of a leaf-mining moth on trees with high infection frequencies of an endophytic fungus

We investigated relationships between endophytic fungi and a leaf-mining moth, Phyllonorycter sp., along an elevational gradient from 2255 to 2895 m. The fungi and moth larvae inhabit leaves of Quercus gambelii. Fungal frequencies and larval densities varied with elevation. However, larval densities were not associated with the frequencies of infection by endophytic fungi. Survival of larvae was positively associated with the most dominant fungus, Gnomonia cerastis, owing to reduced parasitism of moth larvae on trees with high frequencies of Gnomonia.     

Application of Fungal Metabolites for Evaluation of Pine Stand Vigor in Conditions of Industrial Pollution

Two methods for evaluation of pine stand vigor were tested on permanent sample plots near Krasnoyarsk City: visual evaluation and exposure to fungal metabolites. In the first case, forest vigor was estimated using the six-point system of the Sanitary Regulations of the Russian Federation as the mean score for 200 trees on the sample plots. In the second case, the vigor was evaluated from the size of the necrotic spot on inner bark of the tree after inoculation of 22–25 randomly chosen pines with the extract of fungus Ceratocystis laricicola Redfern and Minter. The necrotic spot size was significantly larger in pines from polluted forest as compared to background one. This points to the decrease in infection protection of trees affected by pollution, although visual evaluation of the stand vigor could not distinguish the polluted and background stands.__________Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 4, 2005, pp. 506–512.Original Russian Text Copyright © 2005 by Polyakov, Polyakova, Pashenova, Stasova.     

Fungi associated with the southern pine beetle: avoidance of induced defense response in loblolly pine

Summary Loblolly pines (Pinus taeda) are rapidly killed by colonizing southern pine beetle (Dendroctonus frontalis). The female beetles carry two species of fungi (Ceratocystis minor var. barrasii and an unnamed basidiomycete) within a mycangium. The insects are also frequently associated with a blue-staining form of C. minor. These fungi are inoculated into the tree during colonization. The tree has an induced defensive response that involves resin soaking and necrosis of affected tissue isolating the invading organlsms. The blue-staining fungus stimulates formation of this response in the tree, but the two mycangial fungi do not. These results suggest that the beetles are closely associated with two highly pathogenic fungi that do not stimulate one of the critical components of tree defense.     

Ceratocystis species, including two new species associated with nitidulid beetles, on eucalypts in Australia

The genus Ceratocystis includes important fungal pathogens of trees, including Eucalyptus spp. Ironically, very little is known regarding the diversity or biology of Ceratocystis species on Eucalyptus species in Australia, where most of these trees are native. The aim of this study was to survey for Ceratocystis spp., and their possible insect associates, on eucalypts in Australia and thus to establish a foundation of knowledge regarding these fungi on the continent. Collections were made in three states of Australia from wounds on trees, as well as from nitidulid beetles associated with these wounds. Ceratocystis spp. were identified based on morphology and multigene sequence comparisons. Of the 54 isolates obtained, two previously unknown species of Ceratocystis were found and these are described here as Ceratocystis corymbiicola sp. nov. and Ceratocystis tyalla sp. nov. Furthermore, the distribution of Ceratocystis pirilliformis is expanded to include Eucalyptus spp. in Tasmania.