Survival ofPinus densiflora andP. thunbergii forests in Ibaraki Prefecture severely damaged byBursaphelenchus xylophilus

The total area ofPinus densiflora andP. thunbergii forests in Ibaraki Prefecture in 1978 was 65,200 ha, which decreased to 30,300 ha by 1985 mainly due to pine wilt mortality caused byBursaphelenchus xylophilus. This damage has also continued thereafter. To estimate the survivability of pine trees in Ibaraki Prefecture, pine tree mortality has been studied in eight experimental forests for over 20 years, and ground surveys throughout the Prefecture were also conducted in 1995. Survival in the experimental forests corresponded well to the results of ground surveys. Pine forests remained as pure stands if control measures were undertaken or if they were located in cool areas. In warm areas where no control was undertaken, most of the pine forests disappeared and only a few pine trees remained in mixed forests, while on dry soils no mature or old pine trees survived. Since surviving pine forests are often cut for wood utilization,P. densiflora andP. thunbergii may decrease in area to become rare species in the future unless controls are applied and/or reforestation with resistant pines is carried out. A part of this paper was orally presented at the 108th Annual Meeting of the Japanese Forestry Society (1997).     

Susceptibility of adult trees of the endangered species Pinus armandii var. amamiana to pine wilt disease in the field

Adult trees of Pinus armandii var. amamiana (PAAm) and P. thunbergii grown in the field were inoculated with 100000 or 1000 of the nematode Bursaphelenchus xylophilus to evaluate their susceptibility to pine wilt disease. PAAm trees inoculated with 100000 nematodes started to show disease symptoms 2 weeks after inoculation, and all died within 29 weeks. Although the PAAm trees inoculated with 1000 nematodes tended to show delayed disease symptoms compared with those inoculated with 100000 nematodes, all of them died within 33 weeks after inoculation. All P. thunbergii trees inoculated with 1000 nematodes had died 6 weeks after inoculation. In the nematode-inoculated PAAm trees, death of branches distal to the nematode inoculation site was the first visible symptom, followed by the systemic discoloration of needles, whereas the whole tree wilted simultaneously in P. thunbergii trees. In nematode-inoculated PAAm trees, the period from inoculation to death was longer than that in P. thunbergii. These results suggest that adult PAAm trees are susceptible to pine wilt disease, but are less vulnerable than P. thunbergii.     

Field survival of containerized red and jack pine seedlings inoculated with mycelial slurries of ectomycorrhizal fungi

Red pine and jack pine seedlings growing in styroblocks were inoculated 8 wk after sowing with mycelium/agar slurries of 3 mycorrhizal fungi (Laccaria bicolor, Scleroderma citrinum, and an unidentified basidiomycete), and one suspected mycorrhizal fungus (Cantharellula umbonata). Seedlings inoculated with L. bicolor developed mycorrhizae earlier and in greater numbers than the other inoculation treatments, with red pine out-performing jack pine in both respects. At 34 wk following sowing, seedlings were outplanted on a cleared xeric site in Baraga Co., in Michigan's Upper Peninsula. Seedlings inoculated with C. umbonata failed to form mycorrhizae and were not outplanted. Inoculation treatments did not affect shoot or root weight at outplanting. Red pine inoculated with L. bicolor averaged 21% and 19% greater survival compared with control seedlings after one and two years in the field, respectively. Other inoculation treatments failed to increase seedling survival for either tree species. Jack pine demonstrated higher overall survival than did red pine for both years in all corresponding inoculation treatments.     

Inhibition of the regeneration of Japanese black pine (<Emphasis Type="Italic">Pinus thunbergii</Emphasis>) by black locust (<Emphasis Type="Italic">Robinia pseudoacacia</Emphasis>) in coastal sand dunes

Pine forests are declining because of pine wilt disease and Robinia pseudoacacia, a nitrogen-fixing species, is dominating coastal forests along the Sea of Japan. We examined the effects of R. pseudoacacia on the regeneration of a native pine species, Pinus thunbergii. Two 200 m² plots were set up at the border of a P. thunbergii and R. pseudoacacia-dominated area in a coastal forest. We conducted monthly censuses of emergence, distribution and survival of pine seedlings in the plots from May 2003 to December 2004. Light intensity and soil properties were also measured to analyze the relationships between the survival of pine seedlings and environmental conditions using the Mantel test and the structural equation model. Pinus thunbergii seedlings emerged in spring–early summer and in late autumn. Survival of pine seedlings in the R. pseudoacacia-dominated subplots was less than half that in the pine-dominated subplots. Survival of pine seedlings emerging in May 2003 was significantly reduced by the lower light intensity and higher soil nitrogen in R. pseudoacacia-dominated subplots. The tendency was the same for seedlings emerging from April to May 2004. We concluded that R. pseudoacacia reduced the intensity of light during the growing season and increased the nitrogen content of soil, which resulted in inhibition of the natural regeneration of P. thunbergii.     

不同松树感染松材线虫后NO和核酸酶变化与其抗病性的关系

以黑松、马尾松和火炬松为研究对象,对其接种松材线虫后4,12,24,48,72,96和120 h的NO和核酸酶的活性进行测定.3种松树接种松材线虫后,针叶内NO平均含量的积累为火炬松＜马尾松＜黑松,这种差异在12 ～48 h尤为明显;48h后较其割伤处理(CK1)的增加幅度为火炬松＞马尾松＞黑松.同时3种松树接种后的核酸酶变化趋势存在差异,单链核酸酶活性在4～48 h为火炬松＞马尾松＞黑松,而火炬松双链核酸酶活性在4～96h时却一直低于马尾松和黑松,这表明不同松树体内单链或双链核酸酶的活性变化与其抗病性有关.且3种松树在接种松材线虫后,表现出症状的时间顺序为黑松最早,马尾松次之,火炬松最晚.这表明NO和核酸酶的变化与松树感染松材线虫后的病程发展密切相关.     

Inhibiting factors of symptom development in several Japanese red pine (<Emphasis Type="Italic">Pinus densiflora</Emphasis>) families selected as resistant to pine wilt

Pinus densiflora and P. thunbergii, native to Japan, are highly susceptible to pine wilt disease caused by infection with a pine wood nematode (Bursaphelenchus xylophilus). Trees of these susceptible species have occasionally been found surviving in forests that are extensively damaged by this disease. Seedlings from a part of surviving trees that were selected as resistant families indicate lower mortality rates after the infection. The factors that prevent the symptoms from developing in resistant families of a susceptible species, P. densiflora, as based on the analysis of the pathogens behavior in the tree tissue and the anatomy of the resistant families, are presented in this paper. Nematode populations remained lower in the stems of seedlings from resistant families of P. densiflora than in the stems of non-resistant families. Areas dysfunctional in water transport developed in the stems of resistant families, but did not reach a size large enough to seriously block the ascent of sap. These results suggest that there are systems within the seedlings that prevent nematode migration and reproduction. The 2-year-old seedlings from resistant families of P. densiflora, however, did not suppress the pathogen activity. Numerous branches are a visible characteristic in the seedlings of some resistant families. The arrangement of the resin canals, the only channels in the tree to the pathogen migration, was disoriented at the joints between the branches and the main stem. Such a structure may be effective as a barrier to nematode dispersal.     

Inoculation of seven pine species or hybrid seedlings with Korean isolates of pinewood nematode under greenhouse conditions

? Seedlings of seven pine species or hybrids, Pinus densiflora, Pinus thunbergii, Pinus virginiana, Pinus echinata, Pinus koraiensis, Pinus thunbergii × Pinus massoniana and Pinus × rigitaeda, were inoculated in a greenhouse with 100, 1 000 and 10 000 pathogenic nematodes, Bursaphelenchus xylophilus, to compare initial symptom development and to evaluate susceptibility to pinewood nematodes.

? Genetic types were significantly different in the starting day of needle discoloration (χ² = 43.48; P < 0.001) and the period from nematode inoculation to seedling death (χ² = 32.85; P > 0.001), but treatment groups were not statistically different in the days from inoculation to seedling death in any of the pine species.

? There was no relationship between seedling mortality and the number of nematodes recovered from infected seedlings. Only eight of the nematode-inoculated seedlings remained alive by day 83 after inoculation, including four seedlings each of P. echinata and P. × rigitaeda grown from bulked seed collections.

? Seedlings from the bulk lots were likely more genetically diverse than those from the single-source lots. A broader genetic representation of pine species or hybrids is suggested for screening for planting stocks resistant to B. xylophilus.

     

Identification of Cylindrocladium sp. causing damping-off disease of Japanese black pine (Pinus thunbergii) and factors affecting the disease severity in a black locust (Robinia pseudoacacia)-dominated area

The regeneration of Japanese black pine (Pinus thunbergii) seedlings is inhibited in a black locust (Robinia pseudoacacia)-dominated area. We examined the presence of pathogenic fungi in Japanese black pine seedlings in the area in order to determine the effect of pathogenic fungi on the inhibition of regeneration. When Japanese black pine seedlings were planted in the soil obtained from a black locust-dominated area, all of the seedlings died under low-intensity light conditions, whereas 84% of the seedlings survived in the soil obtained from a Japanese black pine-dominated area under the same light conditions. One fungus was isolated from 48.7% of the dead pine seedlings and was identified as Cylindrocladium pacificum Kang, Crous & Schoch, based on the morphological characteristics, growth, and DNA analysis. This fungus was also isolated from 50% of the dead pine seedlings in 2005 and 66.7% of the seedlings in 2006—both were planted in a black locust-dominated area. The virulence of this fungus increased under high-nitrogen and/or low-intensity light conditions. These results reveal the possibility that the soil eutrophication and shading by the black locust are conducive to a severe damping-off disease and threaten the survival and regeneration of Japanese black pine seedlings.     

Response of Japanese red pine to inoculation with a blue stain fungus,Ceratocystis piceae

To confirm the pathogenicity of a blue stain fungus,Ceratocystis piceae (Münch) Bakshi to the Japanese red pine (Pinus densiflora Sieb. et Zucc.), the responses of healthy young pine trees and stressed trees which were girdled by the half-circumferential girdling technique were investigated by the fungal inoculation test. Although neither of the pine trees inoculated withC. piceae in the non-girdled treatment nor the controls died, mortality of the trees girdled and inoculated withC. piceae was 28.6%. In the pine trees inoculated withC. piceae, the mean area of the necrotic lesion of the sapwood was significantly larger than that of the controls, and the mean of the water pressure potential of the xylem decreased, regardless of the girdling treatment. TheC. piceae was reisolated from the wood pieces near the inoculation points on the inoculated trees, but not from the controls. These results suggest that under strongly stressed conditions, the Japanese red pine trees might have been killed by heavy infestations ofC. piceae carried by bark beetles. A part of this paper was presented at the 103rd Annual Meeting of the Japanese Forestry Society (1992).     

The process of decline of an endangered tree species,Pinus armandii Franch. var.amamiana (Koidz.) Hatusima, on the southern slope of Mt. Hasa-dake in Yaku-shima Island

The process of decline of an endangered tree species,Pinus armandii var.amamiana, was monitored on the southern slope of Mt. Hasa-dake in Yaku-shima Island from 1994 to 1998. There are 163 trees ofP. armandii var.amamiana over 1.3 m in height. They are distributed on steep ridges and rocks with a thin soil layer mostly consisting of friable granite. During the monitoring period, 21 trees ofP. armandii var.amamiana died and the mortality rate was 12.9%. Dead trees were categorized into three types: standing, uprooted and landslide. The uprooted- and landslide-typed dead trees were found only after a severe typhoon struck Yaku-shima Island. This suggests that the combination of fragile site conditions and severe typhoons play an important role in the process of decline ofP. armandii var.amamiana. The standing-typed dead trees were presumed to have been killed by pine wilt disease, accounting for 71.4% of the dead trees. However,Bursaphelenchus xylophilus, the cause of pine wilt disease, was not detected from any of the wood chips or branch samples from the standing-typed dead trees ofP. armandii var.amamiana. This might indicate that some factor(s) other than pine wilt disease could be responsible for the standing-typed death ofP. armandii var.amamiana in natural habitats. A part of this paper was presented at the 109th Meeting of the Japanese Forestry Society.     

The effects of canopy gaps on seedling emergence and growth of two deciduous broad-leaved species (Cornus controversa andPrunus grayana) in aCryptomeria japonica plantations

The effects of canopy gaps on seedling emergence and growth ofCornus controversa andPrunus grayana were studied in a 21-year-oldCryptomeria japonica plantation. The seeds of the two species were sown in December 1995 and their fate was followed until March 2000.P. grayana germinated in 1996, butC. controversa germinated in 1997. In both species studied, more than 70% of the seedlings survived in the forest edge until the end of the experiment, although none survived in the forest understory in the first growing season. In the gap, the survival rate was higher inP. grayana than inC. controversa. In this experiment, some trees were cut to enlarge the area of the gap, in which the growth rate increased markedly forC. controversa seedlings, but not forP. grayana seedlings after the cutting. These two species showed substantial differences in the patterns of seedling emergence, survival and growth in aCr. japonica plantation which had a canopy gap.     

Fluorescein-labeled wheat germ agglutinin stains the pine wood nematode, <Emphasis Type="Italic">Bursaphelenchus </Emphasis><Emphasis Type="Italic">xylophilus</Emphasis>

Pine wilt disease, caused by the pine wood nematode (PWN, Bursaphelenchus xylophilus), is a major threat to pine forests throughout East Asia. Nonetheless, its mechanism of invasion has not yet been described in detail. To better understand the pathology of this disease, it is important to examine the distribution of PWNs within pine tissue during the course of disease development. We attempted to stain nematodes with fluorescein-conjugated wheat germ agglutinin (F-WGA) as a means to locate and track the spread of PWNs. Although PWNs proliferated on Botrytis cinerea fungus were successfully stained only on their vulvas and spicule holes, PWNs extracted from inoculated Pinus thunbergii seedlings were stained on their surface. Stainability, or the percentage of PWNs stained with F-WGA over more than half of their surface, was about 20% by 1 day after inoculation, but increased to 80% at 10 days. The stainability of PWNs extracted from a 5-cm main stem segment that included the inoculation site was less than that of PWNs extracted from other parts of the main stem farther away (i.e., those that had dispersed). These results suggest that stainability is related to dispersal activity in time. Thus, to raise the stainability of PWNs at shorter timeframes after inoculation, PWNs with more than 80% stainability were re-inoculated into pine seedlings. This resulted in more than 70% stainability from 1 to 6 days after inoculation. In F-WGA stained thin paraffin sections of pine tissue of re-inoculated seedlings, PWNs brightly fluoresced under epifluorescence and were easily detected against the dark background of pine tissue. This staining technique with F-WGA is an excellent tool for detecting PWNs in pine tissue.     

Impact of invasion of pine wood nematode on the growth of dominant shrub Pleioblastus amarus in Pinus massoniana communities

When pine trees are invaded by pine wilt diseases, the severely infected pine trees will die and fall down, or they will be removed when found to be damaged by the disease. It gives rise to the invasion of other species in these empty niches originally occupied by pine trees, i.e., competing surrounding trees or understory shrubs will invade the empty niches during the following years. As a result, the spatial distribution and pattern of the main tree species in a pine forest will change, and a niche variety in the main population will occur. In the end, the direction of the succession and restoration of the pine forest ecosystem will be affected. In our study, a Pinus massoniana forest with the dominant shrub, Pleioblastus amarus, was invaded by pine wood nematode and was clear cut. Selecting this community as our research object, we studied the effect of the invasion of the pine wood nematode on the growth of the dominant shrub, P. amarus, in this Pinus massoniana forest. Our results show that, after the attacked pine trees were removed, the niche was occupied by Pleioblastus amarus and other shrubs, which benefited the growth of P. amarus to its climax. Growth of P. amarus at the climax stage was greater compared with the unhealthy pine forest and the control group.     

Pathogenicity of the pine wood nematode at different developmental stages

The ability of the pine wood nematode,Bursaphelenchus xylophilus, a pathogen that causes pine wilt disease, to kill cortical cells of Japanese black pine,Pinus thunbergii, during early development of the disease was conjectured to be a function of nematode developmental stage. A tangential segment of bark was removed from a 2-cm-long current-year stem. The cortex-exposed segments with cut cortical resin canals were designated as + RC-segments and those without them as − RC-segments. When a nematode population containing many older juveniles and adults (NL) was inoculated onto the cut surface, the − RC-segments were still alive 4 d after inoculation, as were non-inoculated control segments. When cortex-exposed segments were inoculated with either a nematode population containing many younger nematodes (NS) or with nematodes isolated from inoculated pine cuttings that also contained many younger juveniles, most tissue cells in − RC-segments died 4 d after inoculation, suggesting that younger juveniles killed pine cells directly, in contrast with older juveniles and adults. When nematodes were inoculated onto + RC-segments in which they could easily enter resin canals, both NL and NS killed the segment tissues. This suggests that NL is pathogenic to pine cells while living in resin canals. Such differences in the pathogenicity of NL and NS to pine parenchymatous cells were also demonstrated in a pathogenicity assay system using bark peelings, which allowed an estimate of direct attack on the cambial cells by nematodes. Based on these results, we hypothesize that younger juveniles are pathogenic to pine parenchymatous cells, while adults and older juveniles are not pathogenic. This work was supported in part by Grants-in-Aid for Scientific Research (No.01440012 and 06454088) and for Young Scientists (to K.I.) from Ministry of Education, Science, Culture, and Sports of Japan, and by a grant from PROBRAIN.     

Pathogenitätstests mit Bursaphelenchus mucronatus an Kiefern und Fichtensämlingen in Deutschland

Pathogenicity tests with Bursaphelenchus mucronatus on pine and spruce seedlings in Germany Inoculation tests on Pinus sylvestris seedlings with a German, a Siberian and a French isolate of Bursaphelenchus mucronatus and on Picea abies seedlings using only the German isolate have clearly shown, under German field conditions, that the nematode can cause wilt symptoms on the apex of about 60% of the inoculated P. sylvestris plants. The nematodes in all these tests mostly remained near the inoculation site. However, further spread of the nematodes and wilt symptoms occured when a climate chamber, at 25°C, was used or when the French isolate was used outdoors. At 25°C, 10% of the inoculated plants with at least 1000 nematodes per seedling died. Seedlings' apices wilted if more than 50 nematodes per trunk were present, whereas smaller numbers of nematodes could cause partial wilting. Nevertheless, a few pine seedlings did tolerate high nematode-population densities, up to 966 per plant. No further progress of wilt of plants with symptoms was observed the following year. Inoculations of P. abies with a German isolate resulted in population development near the site of inoculation. Spruces largely tolerated nematode infestations without any development of wilt symptoms.     

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.     

Correlation of seedling size and branch number with disease resistance of Pinus thunbergii seedlings to Bursaphelenchus xylophilus

Japanese black pine (Pinus thunbergii) seedlings resistant to pine wood nematode (PWN; Bursaphelenchus xylophilus) are routinely selected in Japanese field inoculation trials. Correlations between morphological factors (such as height, stem diameter at ground level and number of branches on seedlings) and disease resistance were examined to improve the production efficiency of 1‐year‐old black pine seedlings for inoculation. Family relatedness and environmental conditions strongly affected seedling resistance; accordingly, logistic regression analysis was used to separate effects of these two variables. Height and stem diameter at ground level significantly correlated with disease resistance in seedlings inoculated with PWN. Because (a) interactions between stem diameter at ground level and environmental condition were significant and (b) height did not interact with any other factor, it was concluded that height of 1‐year‐old Japanese black pine seedlings independently correlated with PWN resistance. Thus, field inoculation tests should use tall seedlings to achieve enhanced survival rates.     

Comparison of histological responses and tissue damage expansion between resistant and susceptible Pinus thunbergii infected with pine wood nematode Bursaphelenchus xylophilus

Pine wilt disease caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, has been epidemic and has had disastrous impacts on pine forests and forest ecosystems in eastern Asia. Many pine species in this area are susceptible to this disease. Pinus thunbergii is particularly susceptible. In Japan, tree breeders have selected surviving trees from severely damaged forests as resistant candidates, and have finally established several resistant varieties of P. thunbergii. However, this breeding procedure requires much time and effort due to the lack of physiological and phenotypical information about resistance. To investigate the resistance mechanisms of selected P. thunbergii, we compared histochemical responses, tissue damage expansion, and PWN distribution in resistant and susceptible clones of P. thunbergii after PWN inoculation. The results suggested that the mechanisms of resistance are as follows: damage expansion in the cortex, cambium, and xylem axial resin canals are retarded in resistant trees soon after inoculation, probably due to the induction of wall protein-based defenses. Suppression of PWN reproduction was particularly caused by inhibition of damage expansion in the cambium. The slow expansion of damage in each tissue provides time for the host to complete the biosynthesis of lignin in the walls of cells that surround the damaged regions. This lignification of cell walls is assumed to effectively inhibit the migration and reproduction of the PWNs. The mechanism of initial damage retardation is presumed to be a key for resistance.     

黑松和马尾松苗受松材线虫侵染后部分化学物质的响应

为研究松材线虫侵染对寄主植物生理生化物质代谢的影响,以黑松和马尾松4 5年生苗为实验材料,测定了接种松材线虫对2种寄主植物的营养物质和次生代谢物质含量的变化及其规律。结果显示:接种松材线虫初期,黑松和马尾松的总糖含量均较高,随接种时间的延长,总糖含量呈不断下降趋势;黑松的可溶性糖含量一直降低并明显低于对照,马尾松的可溶性糖含量在侵染前期与对照相比变化不明显,而15 d后快速降低;黑松和马尾松的可溶性蛋白含量侵染前期均低于对照,后升高,再降低;黑松的单宁含量明显高于对照,马尾松的单宁含量接种第1天略低于对照,第3天后开始升高;黑松和马尾松的总酚含量均在侵染前期高于对照,而后降低。这些物质的变化趋势显示松材线虫侵染不同寄主植物的生理反应。     

Genetic improvement in pine wilt disease resistance inPinus thunbergii: The effectiveness of pre-screening with an artificial inoculation at the nursery

Open pollinated progenies from 15 clones of Japanese black pine (Pinus thunbergii), that had been selected for their tolerance to pine wilt disease, were tested by artificial inoculation with pine wood nematode for six years at a nursery of Kyushu Regional Breeding Office. Family variations in the respective year were all significant. An analysis of variance across the six years’ tests confirmed highly significant effect of year and of families; however, the year by family interaction was significant also. Phenotypic and genetic variances estimated from each year’s result were fitted to a quadratic regression using their mean survival ratio as an independent variable. The variances were greatest at around 0.5 of the mean survival ratio. Estimated heritability showed a similar trend of change, whereas the peak of the expected amount of improvement shifted toward the lower survival range: around 0.4, where a greater selection differential was anticipated. It was concluded that a useful amount of genetic improvement that may be reduced by 30% due to the interaction, would be expected by prescreening seedlings where the mean survival ratio after inoculation was around 0.25 to 0.6.