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.     

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.     

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.     

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.     

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.     

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.     

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.     

Manipulation of tannins in oaks by galling cynipids

We measured the protein precipitation capacity of tannins in the tissues of Quercus crispula and Quercus serrata galls induced by a cynipid (Trigonaspis sp.) to examine the nutrition hypothesis on gall induction. The protein precipitation capacity was significantly lower in the nutritive tissues of galls, on which the cynipids feed, than in ungalled sound leaves and in the outer gall tissues. In addition, the protein precipitation capacities in the leaves and in the outer gall tissues were significantly larger in Q. crispula than in Q. serrata samples, whereas that of the nutritive tissues did not differ between these oak species. These results suggest that gall induction is one of the adaptations of galling cynipids to host–plant defensive compounds, and support the nutrition hypothesis.     

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.     

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.     

Spatial distribution of Raffaelea quercivora hyphae in transverse sections of seedlings of two Japanese oak species

To compare the distributions of Raffaelea quercivora hyphae within seedlings of a susceptible species, Quercus crispula, and a resistant species, Q. glauca, we examined water conductance at transverse sections. Raffaelea quercivora was inoculated into the stems of seedlings in July (summer) and October (autumn) and observed by light and fluorescence microscopy at 1 and 2 weeks after inoculation. The hyphal distribution patterns expressed as the I_δ index differed between the species at each occasion. The hyphal growth was wider in Q. crispula than in Q. glauca in July inoculation, but did not differ in October inoculation. Non‐conductive sapwood in Q. crispula in transverse section was wider than that in Q. glauca at each occasion. The differences of hyphal growth and hyphal distribution patterns between species as little as 1 week after inoculation reflect differences in host susceptibility to the fungus.     

Winter nitrate uptake by the temperate deciduous tree <Emphasis Type="Italic">Quercus serrata</Emphasis>

Winter nitrogen use in deciduous species is largely uncharacterized. We investigated nitrate uptake in the fine roots of a deciduous oak (Quercus serrata Thunb. ex. Murray). We conducted a ¹⁵N-labeling experiment using saplings of Q. serrata in the winter. During three weeks of labeled nitrate application, the concentration of ¹⁵N in the fine roots increased significantly. The amount of nitrogen absorbed, as nitrate, was 1.16 ± 1.02 mg N g DW⁻¹, equivalent to 7.6 ± 5.8% of the total nitrogen content. Our results indicate that Q. serrata saplings have significant potential for nitrate uptake in the fine roots in midwinter (i.e., in the absence of leaves). Although a significant amount of nitrogen applied as nitrate was accumulated, nitrate concentration in the fine roots remained low during the labeling period. Furthermore, significant nitrate reductase activity was detected. These data suggest that Q. serrata saplings can assimilate nitrate in the fine roots in midwinter.     

Discrimination among host tree species by the ambrosia beetle Platypus quercivorus

The ambrosia beetle Platypus quercivorus is a vector for the fungus that causes Japanese oak wilt, and susceptibility to infestation by P. quercivorus varies by tree species. We postulated that P. quercivorus discriminates among host tree species differing in susceptibility to attack. To test this postulate, we counted the number of flying male beetles (NFM), the number of holes bored by male beetles (NH), and the number of flying female beetles (NFF) per unit area of bark surface in three fagaceous tree species: Quercus crispula (with high susceptibility to infestation) and Q. serrata and Castanea crenata (both with low susceptibility). NFM and NH were used to calculate the proportion of male beetles that bored holes out of those that flew to the tree (PBM). We used generalized additive models to predict NFM, NFF, and PBM. The locations of trees, expressed as x and y coordinates, numbers of weeks after the first male beetle’s flying (WEEK), diameters of trees 130 cm above ground (DBH), and tree species (SP) were incorporated into the models as candidate explanatory variables. The best-fit models for NFM and NFF included WEEK and DBH and the effect of location; SP was not included in the models. For PBM, the best-fit model included WEEK, DBH, and SP. The results indicate that male P. quercivorus prefer Q. crispula to Q. serrata and C. crenata and that selection is made before boring holes on trees, but that P. quercivorus do not discriminate among host species when they fly to trees.     

Acorn defenses to herbivory from insects: Implications for the joint evolution of resistance,tolerance and escape

Acorn predation by insects and its effects on seedling establishment were investigated among three co-occurring oak species (Quercus variabilis, Q. serrata and Cyclobalanopsis glauca) in a subtropical evergreen broadleaved forest, Southwest China. All oak species had high tannin concentration (over 10%) but differed in acorn mass and germination schedule. We hypothesized that the defensive traits in acorns (e.g. seed mass, tannins and germination schedule) act together to reduce damage from insect seed predators. Q. variabilis had significantly lower predation (51.2%) than either Q. serrata (71.4%) or C. glauca (73.8%). Seedling establishment decreased with increasing injury for all oak species studied. As much as 43% of insect infested acorns germinated and established as viable seedlings despite extensive acorn mortality due to insect seed predators. The larger seed mass of Q. variabilis appears to tolerate insect infestation and retain enough stored reserves for seed germination and early growth of seedlings. Autumn germination also benefited Q. variabilis and Q. serrata, allowing escape from both direct and ancillary effects of insect feeding by fast reserve shifting to immediate germination of mature acorns. Our results indicate that large seed mass, tannin and autumn germination act together to ameliorate effects of insect seed predation, and the joint evolution of resistance and tolerance (as well as escape through autumn germination) in acorns may be the selective consequences driven in part by interactions with insect seed predators.     

Types of frass produced by the ambrosia beetle <Emphasis Type="Italic">Platypus quercivorus</Emphasis> during gallery construction,and host suitability of five tree species for the beetle

We studied the biology of the ambrosia beetle Platypus quercivorus in the logs of five tree species to determine: (1) the relationship between the amount of frass produced by beetles and tunnel length, (2) the relationship between frass shape and the morphological characteristics of P. quercivorus mouthparts, and (3) the suitability of five tree species for P. quercivorus reproduction. Five logs each from healthy Quercus crispula, Q. serrata, Castanea crenata, Sorbus japonica, and Cryptomeria japonica trees were used in this experiment. The results showed that there was a linear relationship between the amount of frass and tunnel length. Whenever powdery frass was produced, larvae were found in the gallery in the log, while fibrous frass was present only in galleries that contained just adults. The mouthparts of adults were completely sclerotized, which likely accounts for the fibrous frass production. Host preference of P. quercivorus was examined at two stages using five tree species. The first stage is digging initiation, which concerns male preference for digging a tunnel. More holes were made by males on S. japonica and Q. serrata logs, while fewer holes were made on C. japonica logs. The second stage is characterized by female orientation, mating, and progeny development. Platypus quercivorus could complete its lifecycle only in the two Quercus spp.     

Effects of asynchronous acorn production by co-occurring <Emphasis Type="Italic">Quercus</Emphasis> trees on resource utilization by acorn-feeding insects

To clarify the effects of asynchronous seed production among tree species on the population of seed predators, we investigated the relationship between the annual variation in production of mature acorns and the insect damage in those acorns of two sympatric oak species, Quercus variabilis Blume and Quercus serrata Thunb. ex Murray, over 4 years at two study sites. The annual variation in acorn production was noticeable, with a coefficient of variation (CV) at the two sites of 1.05 and 0.80 for Q. variabilis and 0.87 and 0.73 for Q. serrata. Annual fluctuation in acorn production by Q. serrata was synchronized between the two sites. Since annual fluctuation in acorn production was not synchronized between the two species, the CVs for the total acorn production by both oak species (0.83 and 0.62 at the two sites) were lower than those for Q. variabilis and Q. serrata alone. The rate of predation by the specialist predators (Curculio weevils) on the acorns of both species was not related to the annual acorn crop size. Prolonged diapause of Curculio weevils might stabilize their populations. The rate of acorn predation by the generalist predators (tortricid moths) was also not related to the annual crop size. Asynchronous acorn production by the two oak species would help to stabilize the population.     

Cesium adsorption from aqueous solutions onto Japanese oak charcoal III: effects of water-extraction treatment

To remove group 1 and 2 metal ions from Japanese oak (Quercus serrata Thunb. and/or Quercus crispula Blume) charcoal (OC), a water-extraction treatment was employed. The treatment was repeated 1, 4, 8, or 12 times. The amounts of four metal (sodium, potassium, magnesium, and calcium) ions eluted from non-treated and treated OC were determined using an ion chromatographic apparatus. The effects of water-extraction treatment on the ability of OC to adsorb cesium were investigated using 2.50 × 10^?5 and 2.50 × 10^?6 mol/L aqueous cesium chloride solutions. As for both aqueous solutions, an unexpected result was obtained in that the cesium-adsorption ability of OC decreased suddenly when the repetition of water-extraction treatments exceeded a certain threshold. We explain the results in terms of pH of the sample solutions and the point of zero charge for OC.     

Differences in spectral trajectory with stand volume development between Japanese larch and Japanese oak in Hokkaido,Japan

Our objective was to investigate the differences in the spectral trajectory of the LANDSAT Thematic Mapper with stand volume development between Japanese larch (Larix kaempferi) and Japanese oak (Quercus mongolica var. crispula) in the experimental forest of Kyushu University, Hokkaido, Japan. We used nonlinear regression and a categorical variable to investigate the relationship between spectral reflectance factor and stand volume for each forest type. This research showed that the reflectance factor of Japanese larch and Japanese oak reached saturation and that both forest types had different spectral trajectories with stand development. In addition, we found that middle of development stage (stand volume was between 100 and 200 m³/ha) of each forest type had similarity in reflectance factor.     

The stand dynamics of a mixed coppice forest of shade-tolerant and intermediate species

We investigated stand dynamics of a mixed coppice forest of shade-tolerant and intermediate species, Fagus crenata Bl. and Quercus crispula Bl., in five stands with canopy-stem densities from 420 to 1320 ha⁻¹ (estimated stand age: 37–62 years). The mean basal area (BA), exposed-crown area (ECA) and crown depth (CD) per stem were significantly larger in F. crenata in the three lower density plots. Also Q. crispula had considerably less overtopped stems in the two lower density plots. The mean annual BA increments of the two species were similar in the earlier 20 years; however, in the most recent 5 years, F. crenata had a higher relative growth rate for basal area (RGR_BA) than Q. crispula. The most critical variable of the RGR_BA was the ECA for F. crenata and the relative height (RH) for Q. crispula. We predict that dominance of F. crenata will gradually increase over that of Q. crispula as stands mature. This seems to be caused by lower shade-tolerance despite the similar initial growth rates of Q. crispula compared with F. crenata.