Relative dominance of the mold fungus,<Emphasis Type="Italic">Raffaelea</Emphasis> sp., in the mycangium and proventriculus in relation to adult stages of the oak platypodid beetle,<Emphasis Type="Italic">Platypus quercivorus</Emphasis> (Coleoptera; Platypodidae)

Microorganisms associated with the oak platypodid beetle,Platypus quercivorus (Murayama), were isolated from the mycangium of adult females, from the proventriculus of adults of both sexes, and from galleries in the period before dispersal. Fungi isolated from female mycangia were separated into three groups:Raffaelea sp., yeasts (mainlyCandida spp.), and other species.Raffaelea sp. was isolated predominantly from the teneral stage to the dispersal stage, but was not isolated from the mycangia of mother beetles in new galleries. Yeasts were isolated in every beetle stage tested and their isolation rate was over 80% from the teneral stage to the dispersal stage. All three fungal groups were found in the female proventriculus.Raffaelea sp. was isolated only in the dispersal stage at a rate of 40%, whereas the isolation rate of yeasts gradually increased beginning in the mature stage and reached 100% in the rearing stage. In contrast, in the male proventriculus, onlyRaffaelea sp. and yeasts were isolated in the dispersal and rearing stages. The isolation rate in the rearing stage ofRaffaelea sp. was less than 20%, but that of yeasts was 100%, although these rates were almost the same in the dispersal stage.Raffaelea sp. and yeasts were also isolated from cradles containing eclosing pupae. These results suggest thatP. quercivorus acquiresRaffaelea sp. and yeasts in their mycangia from the cradles immediately after eclosion, and maintain them to the dispersal stage. Then, they transmit these fungi from old to new galleries by way of mycangia, and possibly the digestive system.     

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.     

Isolation and characterization of 10 new microsatellite loci in the ambrosia beetle <Emphasis Type="Italic">Platypus quercivorus</Emphasis>

The ambrosia beetle, Platypus quercivorus, is an insect vector for the causal agent of Japanese oak wilt disease, Raffaelea quercivora. We have developed 10 new polymorphic microsatellite markers from Type 1, one of the genetic types of the beetle, using a biotin-enrichment procedure. Fifty-three adult beetles were used for characterization of the markers. The number of alleles per locus ranged from two to nine (average 4.9). The observed and expected heterozygosity ranged from 0.49 to 0.77 and from 0.44 to 0.80, respectively. None of the loci deviated significantly from Hardy–Weinberg equilibrium. No linkage disequilibrium was detected in any pairwise comparisons of loci. Most of the markers successfully amplified in other genetic types of the beetle. These markers could become useful tools for population genetic studies and for parentage and sibship analysis for this beetle.     

Associations between Pityogenes bidentatus and fungi in young managed Scots pine stands in Poland

The association between Pityogenes bidentatus and fungi was studied in young, managed Pinus sylvestris stands in Poland. Fungi were isolated from emerged adults and their galleries collected from four populations. In total, 2089 fungal isolates including 42 species, were obtained. Penicillium sp. 1 and Geosmithia sp. 1 were the most commonly isolated fungi from beetles (49% and 41% of beetles respectively). Geosmithia sp. 1 species was the dominant species in P. bidentatus galleries with a frequency of occurrence of 57.9%. Hormonema dematioides was the second most abundant fungus in gallery systems (17.1% of wood samples). Two of the isolated Geosmithia species were previously undescribed. Pityogenes bidentatus also vectored three ophiostomatoid species: Ophiostoma minus, O. piceae and Graphium sp. ‘W’. These species were occasionally isolated from beetles and their galleries, suggesting a non-specific relationship.     

Über die Variabilität der Brutbilder von Kiefernborkenkäfern (Coleoptera,Ipidae)

Summary Studies on the variability of galleries of pine bark beetles (Col., Ipidae) The variability of the galleries of bark beetles haven't been considered enough till now. Examples of anomalies are given and reduced to their causes. The great pine bast beetle,Myelophilus piniperda, builds the mother-gallery under the bark of stumps downwards instead of above. This can be fed back to the conditions of humidity in the stumps. The little pine bast beetle,Myelophilus minor, when attacking lying stems, often lays eggs only into the higher part of the gallery which is kept free from bore meal. Under the influence of different environment factors also other species of Hylesinini and Ipini show anomalies in building up their galleries.     

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.     

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.     

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.     

Responses ofQuercus sapwood to infection with the pathogenic fungus of a new wilt disease vectored by the ambrosia beetlePlatypus quercivorus

Quercus serrata andQ. crispula wilt during the summer in wide areas along the Sea of Japan. Mass attacks of trees by an ambrosia beetle (Platypus quercivorus) are characteristic before appearance of the wilting symptoms. This study investigated the pathogenic effects of a fungus detected specifically in the wilting trees. This hyphomycete fungus,Raffaelea sp., has a distribution that correlates with the discolored xylem area called wound heartwood in which vessels are dysfunctional. Tylosis formation around the hyphae indicates vessel dysfunction. In areas with discoloration, the fungal hyphae were invading living ray parenchyma cells from the vessel lumen. As a protective reaction the ray cells exuded yellow substances into the vessels, but these substances seemed ineffective against the fungal activity, probably because the fungus disperses along the beetle's gallery before enough substance can accumulate. It should allow wide discoloration in sapwood. Cambium was not necrotic around the fungus. The cytological process in the host was as follows: (1) synthesis of secondary metabolites by the stimuli of oak fungus; (2) exudation of yellow substances into vessels; and (3) dysfunction of vessels and wound heartwood formation. In regard to wilting of trees, the pathogenicity of the fungus should be assessed by its ability to stop sap flow.     

Inducing infection of oak logs by a pathogenic fungus carried byPlatypus quercivorus (Murayama) (Coleoptera: Platypodidae)

This study examined the frequency of infection by an unidentified pathogenic fungus of oak logs bored into by males alone or by both males and females ofPlatypus quercivorus (Murayama) in the field. The fungus, which is associated with mass mortality of oak trees in Japan, was not isolated from logs bored into by males alone, which bored only short entrance galleries. However, it was isolated from logs bored into by both males and females, which together bored longer galleries and reproduced successfully. This suggests that it is difficult for the fungus to colonize logs bored into by males alone. The fungus was not isolated from a log in which the beetles failed to reproduce, and in which the galleries were significantly shorter than in logs where reproduction succeeded, but it was unclear whether the shortness of the galleries prevented colonization of the log by the fungus. The study also revealed that some gallery-initiating males survived for at least 2 months in the absence of females, and that females elongated entrance galleries that had been bored by the gallery-initiating males.     

Response of swamp bay,Persea palustris,and redbay,P. borbonia,to Raffaelea spp. isolated from Xyleborus glabratus

Laurel wilt is a devastating invasive disease of members of the Lauraceae plant family. It is caused by the fungus Raffaelea lauricola, which is a nutritional symbiont of its ambrosia beetle vector, Xyleborus glabratus. In the United States, six Raffaelea spp., in addition to R. lauricola, have been recovered from mycangia of X. glabratus. We compared the response of two laurel wilt suspects, swamp bay (Persea palustris) and redbay (Persea borbonia), to five of these species, another undescribed Raffaelea sp., and R. lauricola. Six weeks after inoculation, only R. lauricola caused significantly greater symptoms than water inoculations. The fungi varied in their ability to move systemically and be recovered from the host at the end of the experiment. Stem hydraulic conductivity was decreased by R. lauricola, but none of the other taxa. Although the roles these fungi play in the life cycle of X. glabratus are not known, they do not appear to be pathogens on these host tree species.     

The Pine Shoot Beetle Tomicus piniperda as a Vector of Blue Stain Fungi to Windblown Pine

After the great gale of 1987, the role of the pine shoot beetle,Tomicus piniperda, as a vector of blue stain fungi to windblownpine in southern England was examined by macerating adult beetlesand culturing the macerate on various agar media. Isolationswere also made from pine tissue associated with beetle galleriesand tunnels. Using data from between three and nine sites, it was found that17 per cent of the overwintered adult beetles were carryingfast-growing Leptographium spp. as they began to construct breedinggalleries in spring 1988. More than half the new generationof adult beetles were contaminated with these fungi when theyemerged in June-July 1988, but this proportion dropped as thelife cycle of shoot-feeding and overwintering progressed. Itaveraged 26 per cent at the time of brood gallery constructionin spring 1989. Isolations made from pine tissue around the galleries also showedchanges in the frequency of blue stain fungi. Of early broodgalleries 25 per cent yielded Leptographium, while the figurefor late galleries was 51 per cent. These results were consistentwith the direct introduction of Leptographium by the parentbeetles into some galleries, and the subsequent rapid hyphalgrowth of the fungus within the tree to colonize tissue adjacentto other galleries. The principal species identified was L. wingfieldii. However,L. lundbergii, L. huntii, L. procerum and an unidentified Leptographiumspecies were occasionally recorded, both on the beetles andin the trees. Graphium species were quite common also. In addition,the black yeasts, Hormonema dematioides and Aureobasidium pullulans,were frequently present, particularly in the pine shoot samples.     

Blue stain fungi associated withTomicus piniperda (Coleoptera: Scolytidae) on Japanese red pine

A number of various species of blue-stain fungi were isolated fromTomicus piniperda adults at various stages of development, as well as from the galleries, pupal chambers and sapwood underneath galleries on Japanese red pine. This study was an attempt to identify the species, composition of blue-stain fungi associated withT. piniperda, the frequency of occurrence of the fungi, and their role in the sapwood-staining of Japanese red pine in Tsukuba City, central Japan. Among the seven species of blue-stain fungi isolated, an undescribed species ofOphiostoma together withO. minus were the dominant species and closely associated withT. piniperda. These two species occurred on newly emerging adults more frequently than the overwintered adults.Hormonema dematioides was also associated with the beetle, however, its frequency of occurrence from the emerged new adults was very low. Although the two other species,O. ips andGraphium sp. were also isolated from emerged beetles, the frequency of these fungi from gallery systems suggested that they were accidentally carried byT. piniperda. Leptographium wingfieldii, known to be associated with the beetle in Europe, was also isolated at a very low frequency and the fungus seemed not to be closely associated with the beetle.Ophiostoma sp. andO. minus appear to be the most important causes of blue-stain of Japanese red pine sapwood after infestation byT. piniperda.     

Stimulation of tree defenses by Ophiostomatoid fungi can explain attack success of bark beetles on conifers

? Our aim is to present why the hypothesis, that Ophiostomatoid fungi play an important role in the establishment of most bark beetle species on living conifers, is valuable.

? After summarizing knowledge about the relationships of bark beetles with conifers and fungi, we conclude that controversy results from misinterpretations when using fungal pathogenicity to demonstrate the role of Ophiostomatoid fungi in beetle establishment on host trees.

? We demonstrate that fungal pathogenicity is not the right parameter to appreciate the role of fungus in beetle establishment on host trees. We argue that artificial low density inoculations that allow the appreciation of fungus ability to stimulate tree defenses and thus to help beetles in overcoming tree resistance must be used in complement to mass inoculations. In both cases, results must be expressed in terms of tree defense stimulation rather than in terms of tree killing.

1. Fungal species stimulating tree defenses are generally not those that grow the best in the sapwood.
2. We argue that beetle development in the phloem, fungal invasion of the sapwood and phloem, and tree death, occur after tree defenses are exhausted, and that any fungus present in the beetle gallery could thus potentially invade the sapwood after defense exhaustion.

? We conclude that stimulation of the tree defense reactions in both the phloem and the superficial sapwood is a real benefit brought by fungi to the beetles during the first phase of establishment (overcoming tree resistance).

? Considering the origin of the bark beetle fungus associations attacking living trees and their general functioning based on stimulation of tree defenses, we develop three hypotheses:

1. any beetle species would be helped in its establishment in a given tree species by developing an association, even loosely, with a fungus species belonging to the Ophiostomatoid flora of that tree species;
2. the necessity of a considerably low level of tree resistance for fungus extension into the tree is the selection pressure that has led fungi to develop their intrinsic ability to stimulate tree defenses, through their ability to grow into the phloem. This association can be completed by antagonistic fungal species controlling extension of the previous fungal species in the tree tissues;
3. Beetle species using the strategy of overcoming tree resistance are associated with a fungal complex, of which species could assume three roles regarding relationships between beetles and trees: 1- to stimulate tree defenses in the phloem and superficial sapwood, 2- to grow into the sapwood after tree resistance is overcome, and 3- to control phloem extension of the first other two categories. Bringing nutrients to the beetle progeny can be a fourth role.

? We propose that bark beetle — Ophiostomatoid associations can be categorized, based on associations’ frequency and complexity while taking into account beetle aggressiveness. We show that a close correspondence exists between beetles’ aggressiveness and the ability of their main associated fungal species to stimulate the defenses of their host tree.

? We conclude with suggesting that most sapwood invading fungi might be “cheaters” which have taken advantage of the efficiency of the relationship between beetles and fungi that stimulate tree defenses.

     

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.     

Die Wirkungsbreite des Ausstoβreflexes bei Borkenkäfernreflexes bei Borkenkäfern

Summary The thrust out-reflex of the back beetles is useful to the breeding galleries of bore-meal and other substances. The beetles also avert with this reflex other insects, who try to invade the breeding galleries. Then the bark beetle thrust out its abdomen and often that means the ruin of the beetle. The enemy — f. i.Thanasimus formicarius L. — has the opportunity to kill the bark beetle when he reaches its abdomen.     

Fungi isolated from Picea abies infested by the bark beetle Ips typographus in the Białowieża forest in north‐eastern Poland

The assemblage of fungi occurring in the sapwood of Norway spruce (Picea abies) and in bark beetle galleries following attack by the Eurasian spruce bark beetle Ips typographus was investigated in the Bia?owie?a forest in north‐eastern Poland. Fungi were isolated from blue‐stained sapwood of beetle‐infested spruce trees in June 2002, and a few isolates were also obtained from ascospores and conidia taken from perithecia and asexual structures occurring in the gallery systems of the insects. The mycobiota of I. typographus in the Bia?owie?a forest was dominated by ophiostomatoid fungi, which were represented by seven species. Four species, including Ceratocystis polonica, Grosmannia penicillata, Ophiostoma ainoae and Ophiostoma bicolor were isolated at high frequencies, whereas three other taxa, Ceratocystiopsis minuta, Ceratocystiopsis alba and a Pesotum sp. were rare. The anamorphic fungus Graphium fimbriisporum and yeasts also occurred occasionally. In addition, the basidiomycete Gloeocystidium ipidophilum was relatively common. The pathogenic blue‐stain fungus C. polonica was the dominant fungal associate of I. typographus in the Bia?owie?a forest, which is consistent with a previous study at this area in the 1930s. Ceratocystis polonica was the most frequently isolated species at the leading edge of fungal colonization in the sapwood and had on an average penetrated deeper into the wood than other fungal associates. This suggests that it acts as a primary invader into the sapwood after attack by I. typographus in the Bia?owie?a forest, followed by O. bicolor, O. ainoae, G. ipidophilum and G. penicillata. Thus far, the Bia?owie?a forest is one of the few areas in Europe, where C. polonica has been reported as a dominate fungal associate of I. typographus.     

Genetic diversity and population structure of Raffaelea quercus‐mongolicae,a fungus associated with oak mortality in South Korea

Raffaelea quercus‐mongolicae is a fungus associated with oak wilt and deemed to cause extensive oak mortality in South Korea. Since the discovery of this fungus on a dead Mongolian oak (Quercus mongolica) in 2004, the mortality continued to spread southwards in South Korea. Despite continued expansion of the disease and associated significant impacts on forest ecosystems, information is lacking about the origin and genetic diversity of R. quercus‐mongolicae. Restriction‐site‐associated DNA (RAD) sequencing was used to assess genetic diversity and population structure among five populations (provinces) of R. quercus‐mongolicae in South Korea. In total, 179 single nucleotide polymorphisms (SNPs) were identified among 2,639 RAD loci across the nuclear genome of the 54 R. quercus‐mongolicae isolates (0.0012 SNPs per bp), which displayed an overall low expected heterozygosity and no apparent population structure. The low genetic diversity and no apparent population structure among South Korean populations of this ambrosia beetle‐vectored fungus support the hypothesis that this fungus was introduced to South Korea.     

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.     

Mountain pine beetle‐associated blue‐stain fungi in lodgepole × jack pine hybrids near Grande Prairie,Alberta (Canada)

The mountain pine beetle (MPB), the most serious pest of lodgepole pine in mountainous western Canada, spread northeastward into lodgepole × jack pine hybrids in the boreal forest of Alberta in 2006. The MPB vectors three species of blue‐stain fungi, which contribute to the success of the beetles. These fungi were isolated from MPB larvae and galleries in several lodgepole × jack pine stands in the Grande Prairie region of northwestern Alberta in autumn 2006 and winter and spring 2007. Fungi were recovered from more than 95% of gallery systems. The three fungi were similarly prevalent but Ophiostoma montium was the most frequently isolated fungus at each sampling point, isolated from 72% to 90% of gallery systems compared with 63% to 78% for Grosmannia clavigera, and 61% to 86% for Leptographium longiclavatum. Ophiostoma montium and G. clavigera were isolated from more larvae than gallery samples, with the opposite true for L. longiclavatum. Most gallery systems contained multiple fungi with three fungi per gallery system being more common in autumn and winter and two fungi more common in the spring. The combination of G. clavigera and L. longiclavatum was less common among gallery systems with two fungi than either of the pairwise combinations containing O. montium. Fungal prevalence was the same above and below snow level. The prevalence of the three fungi did not differ significantly among stands sampled in the spring but stands with more G. clavigera tended to have less L. longiclavatum. The winter of 2006–2007 was colder than average throughout Alberta with temperatures below ?30°C in November, January and February, and all three fungi were present after the cold winter while most larvae had died, suggesting that overwintering mortality of the fungi will not limit persistence and spread of MPB in the boreal forest.