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.     

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.     

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.     

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.     

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.     

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.     

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 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.     

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.     

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     

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.     

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.     

Different development of pine wilt disease in artificially infectedPinus thunbergii seedlings potted together with different tree species

To evaluate the effect of adjacent tree species on the susceptibility of Japanese black pine (Pinus thunbergii) to pine wilt disease, an inoculation experiment was conducted usingP. thunbergii seedlings potted with seedlings of six tree species,i. e. Alnus sieboldiana, Eurya japonica, Lespedeza bicolor formacutifolia, Pinus thumbergii, Robinia pseudo-acacia andSarothamus scoparius. About ten months after planting, they were inoculated with the pinewood nematode (Bursaphelenchus xylophilus) in early July 1992. After that, the proportion of pine seedlings with completely discolored foliage increased more quickly when the seedlings were potted withR. pseudo-acacia, S. scoparius orA. sieboldiana than when potted withP. thunbergii, L. bicolor orE. japonica. At the end of the study period, 17 weeks after inoculation, it reached 90.6%, 90.0%, 87.5%, 72.7%, 63.3%, and 59.4% when the pine seedlings were potted withR. pseudo-acacia, S. scoparius, A. sieboldiana, P. thunbergii, L. bicolor andE. japonica, respectively. This indicated that the susceptibility ofP. thunbergii seedlings to pine wilt disease was influence by the species of adjacent trees.     

Pre- and post-dispersal damage to the acorns of two oak species (Quercus serrata Thunb. andQ. mongolica Fisher) in a species-rich deciduous forest

A comparative study of pre- and post-dispersal damage to the acrons of two oaks,Quercus serrata Thunb. andQ. mongolica Fischer, was done over 3 years in a species-rich deciduous forest in central Japan. The numbers of acorns bored into by moth larvae and byKobuzo rectirostris (Roelofs) was low and relatively uniform each year for both oak species, but the numbers bored into byCurculio spp. varied from year to year. The proportions of acorns attacked were independent of the density of acorns produced for both oak species. The proportions of the dispersed acorns of both oak species on the forest floor that disappeared between autumn and spring were less than 27%, and were lower for species in the stand that belong to family Fagaceae. However, by the next summer, most dispersed sound acorns had been infested by moths that specialized on post-dispersal acorns.     

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.     

Effects of endophytic and ectomycorrhizal basidiomycetes on Quercus virginiana seedling growth and nutrient absorption

Quercus virginiana (Fagaceae), native to the southeastern United States, has recently been introduced to the Yangtze Delta (China) and is often planted in landscaping and in coastal shelter forests. Here, we performed pot inoculation assays to evaluate the effects of two types of basidiomycetous root fungal symbionts, across four orders, on the growth and nutrient absorption of Q. virginiana seedlings. The results indicated that two isolates showed plant growth-promoting activities 2 months after inoculation. In particular, the ectomycorrhizal Sistotrema sp. (Cantharellales) had a pronounced effect on root development and morphology. With regard to nutrient absorption, the total nitrogen content in aboveground tissues was improved in inoculated seedlings, although most differences were not significant. Meanwhile, the total phosphorus contents in seedlings were either positively or negatively affected, probably depending on the symbiont type. Interestingly, the total potassium content in stems and leaves was significantly enhanced in all treatment groups. In addition, indole-3-acetic acid and abscisic acid production were determined in Sistotrema sp. and Atractiella rhizophila (Pucciniomycotina) by high-performance liquid chromatography tandem mass spectrometry. Our findings suggest that both endophytic and ectomycorrhizal fungi have beneficial effects on Q. virginiana growth, and highlight the potential for the development of mycobiome-based seedling raising techniques.     

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.     

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.     

Wood identification of JapaneseCyclobalanopsis species (Fagaceae) based on DNA polymorphism of the intergenic spacer betweentrnT andtrnL 5′ exon

DNA was extracted from wood samples of six representativeCyclobalanopsis species (Fagaceae) growing in Japan that cannot be distinguished from one another by conventional microscopy. A part of the intergenic spacer region betweentrnT andtrnT 5 exon was amplified and sequenced. The sequences obtained from wood samples were grouped into three DNA types by a single nucleotide polymorphism as reported previously in leaf samples: I (Quercus acuta, Q. sessilifolia, Q. salicina), II (Q. myrsinaefolia, Q. glauca), and III (Q. gilva). Thus,Q. gilva can be distinguished from the otherQuercus species, and the others are separated in two subgroups based on DNA polymorphism. The present findings support the possibility of wood identification based on DNA polymorphism.