Extent of colonization by Raffaelea quercivora of artificially inoculated living and gamma‐ray‐sterilized seedlings of two Japanese and three American oak species

Mass mortality of Fagacean tree species caused by Raffaelea quercivora has occurred widely in Japan. Because conidia or other propagules of the pathogen have not been found in infected trees, pathogen spread is assumed to occur primarily by hyphae. To clarify the relationship between hyphal growth of the pathogen within trees and their vessel arrangements, we examined two native Japanese oaks, Quercus crispula and Quercus glauca, and three exotic American oaks, Quercus coccinea, Quercus palustris and Quercus rubra. Quercus glauca is a radial‐porous species, whereas the other four species have a ring‐porous wood structure. Hyphal growth within inoculated potted living seedlings and in cut, sterilized stem segments of these species was examined microscopically after fungal inoculation. Water conductance in the seedlings was examined using transverse stem sections. The proportion of non‐conductive sapwood in Q. crispula, Q. coccinea and Q. palustris differed between inoculation and control treatment, being much higher in inoculated seedlings. The proportions were positively correlated with the extent of the hyphal growth. In sterilized stem segments, the extent of fungal colonization varied among the foreign ring‐porous species Q. coccinea, Q. palustris and Q. rubra. It is hypothesized that the extent of colonization by R. quercivora reflects the extent of non‐conductive sapwood irrespective of tree species, but is little affected by vessel arrangements.     

Changes in water status in seedlings of six species in the Fagaceae after inoculation with Raffaelea quercivora Kubono et Shin-Ito

To clarify the differences in susceptibility of six species in the Fagaceae (Quercus crispula, Quercus serrata, Quercus acutissima, Quercus phillyraeoides, Quercus glauca, and Castanopsis cuspidata var. sieboldii) to Raffaelea quercivora, we inoculated this fungus on seven potted 5-year-old seedlings of each species, observed symptom development, and measured xylem pressure potential (XPP) after inoculation. The first death was observed on the 11th day in Q. crispula and on the 56th day in Q. serrata. The number of dead seedlings of Q. crispula and Q. serrata were five and one, respectively, whereas no mortality was observed in the other four species. The XPP of inoculated seedlings in both Q. crispula and Q. serrata decreased after inoculation. In contrast, the XPP of inoculated seedlings of the other four species remained almost the same as in the control seedlings. These results indicate that R. quercivora is pathogenic to Q. crispula and Q. serrata and that the susceptibility of the six Fagaceae species in our study differed among species.     

Differential spread of discoloured and non‐conductive sapwood among four Fagaceae species inoculated with Raffaelea quercivora

To reveal the relationship between the susceptibility of Fagaceae species to Raffaelea quercivora Kubono et Shin‐Ito and the tangential expansion of regions of discoloured and non‐conductive sapwood among the species, we inoculated branches of three Quercus species and one Castanopsis species with the fungus. The sapwood around the inoculation hole in all four species became non‐conductive in response to the infection before the discolouration. The expansion of the region of non‐conductive sapwood ceased within 2 weeks after the inoculation. The region of non‐conductive sapwood in Q. crispula and Q. serrata was larger than that in Q. glauca and C. cuspidata var. sieboldii. These results suggest that the region of non‐conductive sapwood expanded soon after the infection by R. quercivora. It was also clear that the transverse non‐conductive sapwood has close relationship with the susceptibility among Fagaceae species to R. quercivora.     

Variations in virulence and hyphal growth of four Raffaelea quercivora isolates within Quercus crispula

Mass mortality of fagaceous trees caused by Japanese oak wilt has occurred widely in Japan. Although virulence of the causal fungus, Raffaelea quercivora, appeared to differ among isolates, its relation to the fungal growth within trees was unknown. To clarify the differences in fungal virulence against susceptible Quercus crispula, we examined fungal growth of four R. quercivora isolates within trees and the resulting virulence. In our study, the isolates were multiple‐inoculated in seedlings and single‐inoculated in twigs of mature trees. In the multiple‐inoculation test, mortality rates were examined by the observation of external symptoms. In the single‐inoculation test, water conductance and hyphal growth within the trees were examined by applying aqueous dyes and fluorescence microscopy, respectively. Mortality rates, the proportion of the cross‐sectional area comprising non‐conductive sapwood and horizontal hyphal growth differed significantly among the isolates. Univariate logistic regression analyses showed that both the proportion of non‐conductive sapwood and hyphal growth were significantly positively related to mortality rates. For three isolates, hyphal growth was significantly positively correlated with the proportion of non‐conductive sapwood. These results suggested that the virulence against Q. crispula varies among R. quercivora isolates and that the extent of fungal colonization of the tree determines fungal virulence.     

Discoloured and non‐conductive sapwood among six Fagaceae species inoculated with Raffaelea quercivora

Mass mortality of oak trees has been occurring in Japan since the late 1980s. The fungus Raffaelea quercivora has been frequently isolated from discoloured sapwood in dead or wilting trees and inoculation experiments have shown it to be capable of causing wilting and xylem discoloration in several oak species, notably Quercus crispula and Q. serrata. In this study, we inoculated seedlings of six Fagaceae species with R. quercivora and, after 56 days, measured the vertical length of the discoloration and the areas of discoloured and non‐conducting sapwood on stem cross‐sections. The sapwood discoloration and the water non‐conduction areas were larger in Q. crispula and Q. serrata than in the other species.     

Comparisons of hyphal growth of Raffaelea quercivora among four Japanese Fagaceae species

Japanese oak wilt causes widespread oak mortality in Japan. Possible differences in susceptibility to the causal fungus, Raffaelea quercivora, may be due to vessel arrangements in host trees. To clarify whether constitutive defence mechanisms including vessel arrangements or induced defence mechanisms are the main determinants of host susceptibility, we inoculated the fungus into living seedlings or sterilized stem segments of four Japanese fagaceous species. In seedlings, water conductance was assessed with dye. In both seedlings and stem segments, hyphal growth was examined by fluorescence microscopy. In seedlings, the area of non‐conductive sapwood in stem cross sections and hyphal growth differed significantly among species. In the susceptible species Quercus crispula and Quercus serrata, hyphal growth was significantly and positively correlated with the proportion of non‐conductive sapwood. In stem segments, hyphal growth was not significantly different among species or between vessel arrangements and was similar to or greater than that in seedlings. These results suggest that the extent of sapwood colonization by R. quercivora could be used as a marker for susceptibility and that susceptibility is determined mainly by induced defence responses.     

Virulence of Raffaelea quercivora isolates inoculated into Quercus serrata logs and Q. crispula saplings

This study was conducted to compare the virulences of various isolates of Raffaelea quercivora—a fungus that causes Japanese oak wilt disease—towards Japanese oak trees. Five isolates were collected from a wide range of Japan and inoculated into Quercus serrata logs and Q. crispula saplings. The tangential length of the discolored sapwood in the Q. serrata logs differed significantly among the isolates. The trend in isolate virulence was similar for the Q. serrata logs and the Q. crispula saplings. This is the first report suggesting that there is variability in virulence among isolates of R. quercivora.     

Extractives of <Emphasis Type="Italic">Quercus crispula</Emphasis> sapwood infected by the pathogenic fungi <Emphasis Type="Italic">Raffaelea quercivora</Emphasis> I: comparison of sapwood extractives from noninfected and infected samples

The extracts of Quercus crispula infected by the ambrosia fungus, Raffaelea quercivora, were investigated. Phenol and tannin analyses indicated that normal sapwood (NS) contained a considerable amount of hydrolysable tannins, while infected colored sapwood (IS) contained less hydrolysable tannins and more phenols than NS. In treating pentagalloyl glucose (PGG), which is a model compound of hydrolysable tannins, with a culture medium of R. quercivora, PGG was rapidly hydrolyzed to produce gallic acid. The resulting gallic acid decreased in concentration over the subsequent cultivation period eventually disappeared. Measuring tannase and laccase activities of the culture medium of R. quercivora, tannase activity increased gradually from the beginning, while laccase activity increased rapidly at 5 days of incubation and disappeared at 8 days. An oxidative product from gallic acid treated with laccase was isolated by preparative high performance liquid chromatography, and was identified as purprogallincarboxylic acid (PGCA) by nuclear magnetic resonance spectroscopy and electron-impact mass spectrometry. PGCA was present in a 70% aqueous acetone extract of IS, and showed slight growth inhibition against R. quercivora. Part of this study was presented at the 57th Annual Meeting of the Japan Wood Research Society, Hiroshima, Japan, 2007     

Extractives of Quercus crispula sapwood infected by the pathogenic fungus Raffaelea quercivora II: isolation and identification of phenolic compounds from infected sapwood

There has been a mass mortality of oak trees in the area along the coast of the Japan Sea. This phenomenon is caused by the ambrosia beetle Platypus quercivorus, which carries the ambrosia fungus Raffaelea quercivora. Extractives of a necrotic brownish coloration formed in the infected sapwood of Quercus crispula were investigated. The methanol extract of the damaged sapwood of Q. crispula was concentrated in vacuo and centrifuged to yield precipitates and the supernatant. The precipitates were subjected to Sephadex LH-20 column chromatography and preparative HPLC to describe a novel ellagic acid derivative (1). The 10 % methanol water-soluble part of the supernatant was subjected to medium-pressure ODS column chromatography and preparative HPLC, respectively, to analyze a known lignan (2). Sulfuric acid hydrolysis of (1) yielded an ellagic acid and a gallic acid. NMR and LC-TOF/MS indicated that an ellagic acid and a gallic acid bonded to a xylose with glycosidic and ester bonds, respectively. Compound (1) was identified as 4,5-dihydroxy-6-(3,7,8–trihydroxy-5,10-dihydro–chromeno[5,4,3-cde]chromen-2-yloxy)-tetrahydro-pyran-3-yl ester, and compound (2) was identified as (–)-lyoniresinol. The presence of (–)-lyoniresinol from damaged sapwood indicated that infection of R. quercivora may cause the formation of a pseudo-heartwood in the sapwood of Q. crispula.     

Differences in leafminer (Phyllonorycter, Gracillariidae, Lepidoptera) and aphid (Tuberculatus, Aphididae, Hemiptera) composition among Quercusdentata, Q. crispula, Q. serrata, and their hybrids

Leafminer (Phyllonorycter, Gracillariidae, Lepidoptera) and aphid (Tuberculatus, Aphididae, Hemiptera) composition were studied in three deciduous oak species, Quercus dentata, Q. crispula, and Q. serrata, and their hybrids in Tomakomai Experimental Forest of Hokkaido University, Hokkaido, northern Japan. Identification of trees in this forest was done mainly on the basis of discriminant analysis on leaf morphology with reference to trees in pure Q. dentata and Q. crispula stands and a Q. serrata stand mixed with Q. crispula. The results suggested that hybridization occurred in all combinations (i.e. Q. dentata–Q. crispula, Q. crispula–Q. serrata, and Q. serrata–Q. dentata) and the frequency of hybrids was approximately 10%. The composition of Phyllonorycter and Tuberculatus species differed between Q. dentata and Q. crispula or Q. serrata, but did not differ between Q. crispula and Q. serrata. Thus, Q. dentata could differ from Q. crispula and Q. serrata in chemical properties that determine herbivore host selection, survival, and performance, possibly reflecting eco-physiological differences or phylogenetic distances. The study insects were divided into three groups: species specialized to Q. dentata (three Phyllonorycter and one Tuberculatus species), those to Q. crispula and Q. serrata (six Phyllonorycter and two Tuberculatus species), and a species collected at least from Q. dentata and Q. crispula (one Tuberculatus species). Putative hybrid trees of Q. dentata and Q. crispula harbored both Q. dentata-specific and Q. crispula-specific insects.     

A comparison of seedling emergence and survival between Quercus glauca and Symplocos prunifolia

Seedling emergence and initial survival were compared for two evergreen broad-leaved species, Quercus glauca and Symplocos prunifolia. The relationships between the two seedling dynamics variables and environmental factors for the two species were also investigated. The number of seedlings that emerged in the study period was larger for S. prunifolia than for Q. glauca, while the survival rate was lower for S. prunifolia, presumably due to the closed canopy of this site. Models were selected for each species to determine the combination of variables explaining the most variation in emergence and survival of seedlings. The model selected for seedling emergence of Q. glauca showed that more seedlings emerged in lower hillslope positions and where the canopy in winter was more open. The model selected for S. prunifolia showed that fewer seedlings emerged on steeper slopes and that more emerged under a more open canopy in winter. With respect to seedling survival, models with only the proportion of open canopy in summer were selected for both species. These models showed that the survival rate was higher where the canopy in summer was more open. This is to be expected as both species are regarded as pioneer or mid-successional species. Q. glauca seemed to have the ability to persist as a dominant in the secondary forest at this site for longer than S. prunifolia due to the former’s better seedling survival rates and the rarity of climax species such as Castanopsis cuspidata.     

Genotype distribution of Raffaelea quercivora in the oak galleries and its composition in the mycangia of Platypus quercivorus

Raffaelea quercivora is the pathogenic fungus that causes Japanese oak wilt. The female monogynous ambrosia beetle, Platypus quercivorus, carries this fungus in mycangia on the pronotum. These beetles bore galleries in oak trees with their partners to produce offspring, and they deposit fungus on the gallery walls from their mycangia. The offspring mature in the gallery, before loading the fungal pathogen and flying from the gallery to other healthy trees. To investigate the unloading and loading modes of the fungus within the gallery, we developed four polymorphic microsatellite markers for R. quercivora and identified the fungal genotypes in the galleries and mycangia of the beetles. Small wood chips were sampled at 5–10‐mm intervals from the walls of five galleries in a dead Quercus serrata tree. The pronota were also sampled from five female adult beetles. The genotypes of the R. quercivora isolates from the wood chips and pronota were identified using the microsatellite makers. The genotypic analysis showed that each gallery was inhabited patchily by 5–10 genotypes of R. quercivora, and the mycangia of each beetle contained 3–6 genotypes. These results indicate that diverse R. quercivora genotypes are unloaded repeatedly from the mycangia of female beetles onto the gallery wall, which results in their patchy distribution on the walls. When the offspring leave the host tree, the fungal clones that proliferate in the walls are also loaded repeatedly into the mycangia of the mature beetles.     

Individual-scale responses of five dominant tree species to single-tree selection harvesting in a mixed forest in Hokkaido, northern Japan

We investigated the individual-scale responses of five dominant species (Abies sachalinensis, Acer mono, Tilia japonica, Quercus crispula, and Betula ermanii) to single-tree selection harvesting in a conifer–broadleaved mixed forest in Hokkaido, northern Japan. Using data from stems with a diameter at breast height of ≥12.5 cm, collected during 20 years of monitoring a 6.7-ha stand, we analyzed the effects of harvesting in the neighborhood on tree recruitment and the growth (diameter class transition) and mortality of the residual trees. The effects of harvesting varied considerably among tree sizes and species. Harvesting improved the recruitment of A. mono and B. ermanii, and moderated the negative effect of the initial basal area of the surrounding canopy trees on the recruitment of Q. crispula. Conversely, harvesting limited the recruitment of A. sachalinensis by offsetting the positive effect of the initial basal area of the surrounding canopy trees. The growth of A. sachalinensis and Q. crispula decreased with the initial basal area of the surrounding canopy trees. Harvesting in the neighborhood resulted in an improvement in the growth of the trees of these species only in the smaller size classes. With increasing local harvesting intensity, the mortality of smaller A. sachalinensis trees decreased, whereas the mortality of larger trees increased. These results suggest that differences in the local harvesting intensity, spatial patterns of harvesting, and initial stand structures influence the stand-scale dynamics in response to partial harvesting in the mixed forests of this region.     

A low <Emphasis Type="Italic">Platypus quercivorus</Emphasis> hole density does not necessarily indicate a small flying population

The ambrosia beetle Platypus quercivorus causes mass mortality of Fagaceae trees in Japan, and tree species differ in their susceptibility to P. quercivorus. We hypothesized that interspecific differences in susceptibility are caused by differences in beetle infestation patterns, that is, how many beetles fly to a tree and how many of those arriving bore holes. To examine how tree susceptibility is related to these parameters, two tree species with different degrees of susceptibility were studied (highly susceptible Quercus crispula and less susceptible Q. salicina). Specifically, we measured the number of male beetles per unit area that flew to the host trees (NFM) and the density of holes bored by male beetles (DH). From these two values, we calculated the proportion of male beetles flying to a host tree that bored holes (PBM). These parameters were compared for the two Quercus species. Although the two species did not differ in NFM in 2003, PBM was markedly lower for Q. salicina than for Q. crispula and DH was significantly lower for Q. salicina than for Q. crispula. Thus, the lower susceptibility of Q. salicina is partly explained by its low PBM.     

Discoloration induced by Raffaelea quercivora isolates in Quercus serrata logs and its relation to phylogeny: a comparison among isolates with and without the Japanese oak wilt incidence including outside of Japan

Japanese oak wilt (JOW) has been prevalent in Japan since the late 1980s. Infections of the fungus, Raffaelea quercivora Kubono et Shin. Ito, which is transmitted by an ambrosia beetle [Platypus quercivorus (Murayama)], can cause JOW. Although R. quercivora, P. quercivorus, and oak trees are distributed in other Asia–Pacific countries, the incidence of JOW has not been reported outside Japan. In this study, we collected R. quercivora isolates from 5 Asian countries, including Japan, and compared their ability to induce sapwood discoloration by inoculating Q. serrata logs. The tangential widths of the discoloration in sapwoods inoculated with non-Japanese isolates were equivalent to or greater than those of the 2 Japanese isolates. This indicates that a lack of JOW incidence outside Japan is not because of the lowered ability of R. quercivora to spread discoloration compared with the Japanese isolates. Statistical analyses of the relationship between discoloration and phylogeny based on DNA sequences of actin and chitin synthase showed that the discoloration width was independent of phylogenetic relatedness among the isolates. To discuss why the occurrence of JOW has not been reported outside Japan, further studies (e.g., on host susceptibility and P. quercivorus aggression) throughout Asia are needed.     

Individual growing conditions that affect diameter increment of tree saplings after selection harvesting in a mixed forest in northern Japan

We analyzed temporal patterns in diameter growth of saplings following selection harvesting in an uneven-aged mixed stand dominated by Abies sachalinensis, Acer mono, Quercus crispula, and Betula ermanii in Hokkaido, northern Japan. We examined interspecific differences in growth responses to local growing conditions including harvesting intensity, crowding, stem size, and past duration of the small growth period. Consistent with expectations based on shade tolerance of the species, the age at which the individual reached a diameter at breast height (DBH) of 12.5 cm was highest for A. sachalinensis and lowest for B. ermanii. The interspecific growth differences between saplings that had or had not experienced local harvesting increased gradually for A. sachalinensis and B. ermanii, but peaked at around 4–6 years after harvesting for Q. crispula. Generalized linear mixed model analysis clearly suggested that individual growth conditions required to enhance diameter growth of saplings differed considerably among species. For Q. crispula and B. ermanii, local harvesting intensity was most strongly and positively associated with diameter growth rate, whereas for A. sachalinensis and A. mono, stem size had the strongest negative effect. Abies sachalinensis saplings responded more to surrounding harvesting when they were relatively small, whereas A. mono showed a weak opposite response. The duration of the small growth period before harvesting had negative effect for A. sachalinensis, but not for the other species. Our study indicated that the influence of selection harvesting on growth of shade-tolerant species depends upon pre- and post-harvest growing conditions.     

Variation in leaf morphology of Quercus crispula and Quercus dentata assemblages among contact zones: a method for detection of probable hybridization

I describe variation in leaf morphology of Quercus crispula and Quercus dentata assemblages within and among their contact zones. Such relationships provide a relatively fast and accurate method for detecting hybridization. Using discriminant function (DF) analysis based on leaf characteristics, I was able to differentiate between these two species in pure zones. In three of five contact zones, the DF scores showed broad, continuous distributions with three or more peaks. The trees showing intermediate leaf morphology are considered to be hybrids. In the other two contact zones, the DF scores showed distinct bimodal distributions, suggesting that Q. crispula and Q. dentata rarely hybridize in these sites. Thus, the method described in this study should be effective for detecting probable hybridization between these two species in contact zones, and the relative frequency of hybridization among contact zones. However, there was likely to be intraspecific variation of Q. crispula between geographic ranges, restricting interpretations to patterns occurring within sites or limited geographic ranges.     

A new record of Rhizoctonia butinii associated with Picea glauca ‘Conica’ in Slovenia

In 2013, the death of needles of dwarf Alberta spruce (Picea glauca ‘Conica’) seedlings was observed in an arboretum in Slovenia. Needles on infected P. glauca ‘Conica’ twigs were covered with thick, yellowish brown hyphal cushions. The outgrowing mycelium was white and had completely overgrown some needles and connected them to each other. To definitively identify the fungus affecting P. glauca ‘Conica’, morphological examinations of isolated pure cultures were performed, and the internal transcribed spacer (ITS) region of the rDNA was sequenced. Based on morphological and molecular phylogenetic analyses together with the performed pathogenicity trial, we identified the causal agent of the observed symptoms as Rhizoctonia butinii – a recently described species. This report connects this fungus to a new locality and a new host plant.     

Effects of crown release on basal area growth rates of some broad-leaved tree species with different shade-tolerance

We compare three or four years effects of crown release on stem growth rates among four deciduous broad-leaved tree species,Fagus crenata, Quercus crispula, Magnolia obovata andAcer mono, in an even-aged coppice forest, central Japan. The crown release significantly improved relative growth rates for basal-area (RGR_BA) of the four species. However the effects of neighboring gap area (GAPA) on the RGR_BA differed among the species. The effects were significant forQ. crispula andM. obovata, indicating that growth rates of these two species increase with intensity of the crown release. In contrast, the RGR_BA ofF. crenata andA. mono were not correlated with the GAPA, suggesting that their growth rates are unrelated to the intensity. We considered that such differences among species were closely related to their photosynthetic characteristics of light use; less-tolerant species (Q. crispula andM. obovata) exhibit greater growth rates relative to the intensity of crown release than tolerant species (F. crenata andA. mono). Based on these results, we proposed implications for thinning practices in mixed forests of species with different shade-tolerance.     

Effects of previous forest types and site conditions on species composition and abundance of naturally regenerated trees in young <Emphasis Type="Italic">Cryptomeria japonica</Emphasis> plantations in northern Japan

Naturally regenerated trees in young Cryptomeria japonica plantations were investigated in 141 quadrats of 10 × 50 m within a watershed of 1,000 ha, and factors affecting their composition and abundance were examined. The species composition of naturally regenerated trees was classified into four types. Dominant species were Swida controversa, Magnolia hypoleuca and Pterocarya rhoifolia in Type A1, Betula maximowicziana, M. hypoleuca, Quercus crispula and Castanea crenata in Type A2, Q. serrata and C. crenata in Type B1, and Pinus densiflora in Type B2. The results of path analysis showed significant influences of previous forest type and geology among the factors that correlated with the species composition of naturally regenerated trees; previous forest type in particular showed a higher absolute path coefficient value. Species composition types of naturally regenerated trees corresponded to the previous forest types: Types (A1 and A2) corresponded to the natural forests composed of Thujopsis dolabrata var. hondai, Fagus crenata, Aesculus turbinata, etc., Type B1 corresponded to the secondary Q. crispula and Q. serrata forest, and Type B2 corresponded to the secondary P. densiflora forest, respectively. The abundance of naturally regenerated trees was strongly affected by geology; i.e., plantations on soft-sedimentary dacitic tuff contained great amounts of colonizing P. densiflora trees.