Inoculation of several Bursaphelenchus xylophilus group nematodes into adult trees of Pinus thunbergii and their survival in the trees

To clarify the pathogenicity of Bursaphelenchus nematodes to adult pine trees, inoculation experiments using six species of B. xylophilus group nematodes and ca. 10‐year‐old trees of Pinus thunbergii were conducted. Trees inoculated with an avirulent isolate (C14‐5) of B. xylophilus did not die during the survey, but showed a decline in oleoresin exudation compared with the controls. Fifteen months after the inoculation, a small number of B. xylophilus survived in a tree inoculated with B. xylophilus C14‐5. Trees inoculated with B. mucronatus, B. doui, B. luxuriosae, B. conicaudatus and Bursaphelenchus sp. NK224 (undescribed) showed no decline in oleoresin exudation and no external symptoms of wilt. However, 9 months after the inoculation, a small number of B. luxuriosae survived in a tree inoculated with the nematodes, although four other nematode species were not isolated from trees inoculated with them. These results were approximately consistent with our previous results (Kanzaki, N.; Aikawa, T.; Maehara, N.; Ichihara, Y., 2010, J. For. Res.; in press), in which an avirulent isolate (OKD‐1) of B. xylophilus and B. luxuriosae caused water flow inhibition without external symptoms in 3‐year‐old seedlings. Therefore, to examine the pathogenicity of the nematodes to pines, it is useful to use 3‐year‐old seedlings in inoculation experiments when adult trees cannot be used.     

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.     

Migration of pine wood nematodes in the tissues of <Emphasis Type="Italic">Pinus thunbergii</Emphasis>

The distribution of pine wood nematodes (Bursaphelenchus xylophilus, PWNs) in Japanese black pine (Pinus thunbergii) tissues was investigated by staining with fluorescein isothiocyanate-conjugated wheat germ agglutinin. After PWNs were inoculated to current-year stems of pine seedlings, their distribution at about 5 cm below the inoculation site was confined only to cortical resin canals 1 day after inoculation, and then spread to other tissues, including resin canals of short branches. When PWNs were inoculated onto cross or tangentially cut surfaces of stem segments, maximal PWN migration speed was estimated to be faster through cortical resin canals and xylem axial resin canals vertically (>6.7 and <2.3 mm/h, respectively) than through cortical tissues both vertically and horizontally (<1.2 and <0.2 mm/h). To examine whether PWNs in cortical resin canals could invade surrounding tissues, segments in which PWNs resided only in cortical resin canals were prepared by removing the top portion 6 h after inoculation. Additional incubation of such segments caused extended PWN distribution to xylem axial resin canals and then to other tissues. A similar experiment with top portions of girdled segments removed 12 h after inoculation also showed extended PWN distribution from xylem axial resin canals and pith to cortical resin canals and then to other tissues. These results provided direct evidence that PWNs have the ability to migrate from cortical resin canals and xylem axial resin canals to other tissues.     

Occurrence of <Emphasis Type="Italic">Bursaphelenchus mucronatus</Emphasis> (Nematoda; Aphelenchoididae) in France and association with <Emphasis Type="Italic">Monochamus galloprovincialis</Emphasis> (Coleoptera: Cerambycidae)

As a consequence of the recent introduction of the pine wood nematode Bursaphelenchus xylophilus in Portugal, nematodes of the genus Bursaphelenchus were looked for in various French pine forests, in trees attacked by Monochamus galloprovincialis, the vector insect of B. xylophilus, and in the insects themselves. Trap trees were felled in 12 localities distributed all over the country. Nematodes were extracted from transversal stem discs; insects emerging from the trap trees were studied. B. hellenicus, B. leoni, B. mucronatus and B. sexdentati were isolated, but not B. xylophilus. The presence of B. mucronatus and the absence of B. xylophilus were confirmed by molecular markers. B. mucronatus was isolated from several regions with an average prevalence of 19%. The infestation of M. galloprovincialis by B. mucronatus reached 26.7%. The wide distribution of B. mucronatus in France could have an effect on the extension of B. xylophilus in a case of an introduction.     

Recessive inheritance of morphological characteristics of utsukushimatsu,<Emphasis Type="Italic">Pinus densiflora</Emphasis> f.<Emphasis Type="Italic">umbraculifera</Emphasis>

Pinus densiflora f.umbraculifera, commonly known as utsukushimatsu, is a distinctively shaped form of Japanese red pine whose growth is restricted to a forest stand in Shiga Prefecture, Japan. The inheritance mode of morphological characteristics of utsukushimatsu was studied to preserve the genetic resource of this pine. As previously reported, F₁ trees grown from open-pollinated seeds harvested from trees inhabiting the native stand showed two phenotypes: one resembling utsukushimatsu, which produces multiple trunks, and the other resembling normalP. densiflora, which produces one or a few trunks. In the present study, controlled pollination was carried out using F₁ and normalP. densiflora trees. Segregation ratios of the two phenotypes observed in the F₂ population showed that the morphological characteristics of utsukushimatsu are inherited recessively. This suggests that the mutation of one gene or a few closely linked genes controls the morphological characteristics of utsukushimatsu. Since multiple trunk formation of utsukushimatsu might be related to a loss of lateral bud inhibition, it follows that a simple gene mutation breaks apical dominance inP. densiflora.     

Abundance-dependent transmission of the pinewood nematode, <Emphasis Type="Italic">Bursaphelenchus xylophilus</Emphasis> (Nematoda: Aphelenchoididae), to the Japanese pine sawyer, <Emphasis Type="Italic">Monochamus alternatus</Emphasis> (Coleoptera: Cerambycidae), adult in its pupal chamber

The transmission ratio of the pinewood nematode (PWN), Bursaphelenchus xylophilus, to the emerging adult Japanese pine sawyer (JPS), Monochamus alternatus, in its pupal chamber is a determinant of the number of the nematodes carried by JPS beetles. To investigate the factors affecting the transmission ratio, we counted the number of the nematodes carried by 36 newly emerged JPS beetles and the number remaining in and around their pupal chambers, and then estimated the transmission ratio (the number of nematodes carried by a JPS adult as a percentage of the total number of nematodes aggregating in and around its pupal chamber). The total number of nematodes aggregating in and around a pupal chamber ranged from 0 to 19,041, and the number of nematodes carried by a beetle ranged from 0 to 18,920. The transmission ratio correlated with neither the water content of the wood around the pupal chamber nor the degree of wood discoloration caused by blue-stain fungus. The transmission ratio varied with the abundance of the nematodes aggregating in and around the pupal chamber. In pupal chambers with more than 1,000 nematodes, almost all the nematodes were transmitted to the beetle. However, in pupal chambers with fewer than 1,000 nematodes, the transmission ratio varied greatly, from 0 to 100%. These results suggest that aggregation of many PWNs in the pupal chamber might stimulate transmission of the PWNs to the JPS adult and that this abundance-related transmission might contribute to the large variation in the number of PWNs carried by the JPS beetle.     

Inoculation with the ectomycorrhizal fungi <Emphasis Type="Italic">Pisolithus arhizus</Emphasis> and <Emphasis Type="Italic">Scleroderma</Emphasis> sp. improves early growth of <Emphasis Type="Italic">Shorea pinanga</Emphasis> nursery seedlings

Trees of the family Dipterocarpaceae are the dominant trees in Southeast Asian tropical forests where they play an important ecological role and are also important commercially. An experiment was conducted to determine the effect of ectomycorrhizal fungi on the growth of dipterocarp species in peat soils. Seedlings of Shorea pinanga were inoculated with spores of two ectomycorrhizal fungi, Pisolithus arhizus and Scleroderma sp. were grown in pots containing sterilized peat soil for 7 months. The percentage of ectomycorrhizal colonization on S. pinanga exceeded 86%. Colonization of S. pinanga roots by ectomycorrhizal fungi resulted in increased shoot height, stem diameter, number of leaves, and shoot fresh and dry weight. Survival rates of S. pinanga were greater for inoculated seedlings than control seedlings. These results suggest that inoculation of ectomycorrhizal fungi can improve the early growth of S. pinanga grown in tropical forests and that this technique will accelerate the rehabilitation of degraded dipterocarp forests.     

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.     

Suggestions on management measures of pine forest ecosystems invaded by <Emphasis Type="Italic">Bursaphelenchus xylophilus</Emphasis>

Pine wood nematode （PWN）, Bursaphelenchus xylophilus （Steiner et Buhrer） Nickle is an important invasive alien species in forests of China and has become one of the most destructive forest diseases. In order to improve the resistance and resilience of pine forest ecosystems against B. xylophilus invasion and make the pine forest ecosystem more timely responsive to PWN invasion, we made some recommendations based on five years of intensive observations. We advocate a set of management measures with the theme “Prevention is priority, but integrated with curative techniques and ecological resilience” on the pine forest ecosystem invaded by B. xylophilus; details of accurate measures are proposed. The aim is to discover the underlying problems of present pine forest ecosys-tems and to take, correspondingly, administrative measures and strategies, which will encourage the pine forest ecosystem to develop in a benign way.     

Control of a new turf pest, <Emphasis Type="Italic">Dorcadion pseudopreissi</Emphasis> (Coleoptera: Cerambycidae), with the entomopathogenic nematode <Emphasis Type="Italic">Heterorhabditis bacteriophora</Emphasis>

The use of biological control in turf has increased to avoid possible negative effects on humans. Entomopathogenic nematodes (EPNs) belonging to the families Steinernematidae and Heterorhabditidae have control potential against many economically important insect pests. In the present study, the efficacy of Heterorhabditis bacteriophora against a new pest on turf, Dorcadion pseudopreissi, was examined in the field. Cages (1 × 1 × 1 m) with female and male D. pseudopreissi were placed on two kinds of turf: Lolium perenne and Festuca arundinacea. After beetles had deposited eggs into the moist soil surface, the cages were removed and H. bacteriophora was applied at 0.5 million infective juveniles/m² to half the plots. Application of H. bacteriophora caused a statistically significant reduction in numbers of beetle larvae in L. perenne plots and a non-significant reduction in F. arundinacea plots. The area damaged by D. pseudopreissi was significantly reduced by nematode application in both turf species. The number of nematodes declined after application, but small numbers could still be detected after 6 months.     

<Emphasis Type="Italic">Ophiostoma</Emphasis> species associated with bark beetles infesting three <Emphasis Type="Italic">Abies</Emphasis> species in Nikko,Japan

Ophiostoma species were isolated from bark beetles and Abies mariesii, A. veitchii and A. homolepis attacked by the beetles in Nikko, Tochigi, central Honshu, Japan. One to two Ophiostoma species were frequently isolated from each species of bark beetle. Ophiostoma subalpinum was the most common associate of Cryphalus montanus. Ophiostoma sp. B as well as O. subalpinum was a common fungus associated with Polygraphus proximus. Ophiostoma europhioides was isolated from Dryocoetes hectographus and D. autographus as one of the common associates. Ophiostoma sp. J and Ophiostoma sp. S were frequently isolated from D. autographus and D. striatus, respectively. These fungi seem to have specific relationships with particular bark beetles. Ophiostoma sp. B, Ophiostoma sp. J and Ophiostoma sp. S have unique morphological characteristics and appear to be new species. Five trees of A. veitchii, approximately 43 years old, were inoculated with five Ophiostoma species to assess the relative virulence of the fungi. Ophiostoma subalpinum, Ophiostoma sp. B, and O. europhioides had relatively higher virulence than the other species studied.     

Aboveground biomass equations for individual trees of <Emphasis Type="Italic">Cryptomeria japonica</Emphasis>, <Emphasis Type="Italic">Chamaecyparis obtusa</Emphasis> and <Emphasis Type="Italic">Larix kaempferi</Emphasis> in Japan

Generic equations are proposed for stem, branch and foliage biomass of individual trees in even-aged pure stands of Cryptomeria japonica, Chamaecyparis obtusa and Larix kaempferi. Biomass data was collected from a total of 1,016 individual trees from 247 stands throughout Japan, and five regression models were assessed by root mean square error, mean bias, fit index (FI), and AIC. The results show that a power equation using diameter at breast height (dbh) and height is the most suitable for all species and components. This equation is more accurate than the familiar power equation that uses ‘dbh² height’, and it expresses the greater volume of branch and foliage mass of trees with a lower height/diameter ratio. A power equation using dbh is more reasonable for models with dbh as the only independent variable and more accurate than a power equation using ‘dbh² height’ for estimating branch and foliage mass. Estimating error for branch and foliage mass is larger than that for stem mass, but the entire aboveground biomass can be estimated with an error of less than 19%, except in the case of small trees with dbh less than 10 cm.     

Introduced <Emphasis Type="Italic">Tuber aestivum</Emphasis> replacing introduced <Emphasis Type="Italic">Tuber melanosporum</Emphasis>: a case study

A Tuber melanosporum plantation established in 1994/1995 on Kibbutz Bar’am (in the Upper Galilee, Israel) gradually lost its T. melanosporum mycorrhiza. In 1999, T. aestivum inoculated seedlings were inadvertently introduced into the plantation to fill the gaps between trees. A single T. melanosporum fruit body was found in 1999. Although no truffles were found after 1999 and until 2009, in that year and in 2010, truffles were collected. Morphological and molecular analyses proved these to be T. aestivum. Thus, the intentionally introduced T. melanosporum mycorrhiza was replaced by that of another introduced mycorrhizal fungus, T. aestivum. Local oak species produced higher yields compared to introduced host species known to be good T. melanosporum plant symbionts. The yield was comparable to that reported for young commercial orchards, but the fruiting season was earlier than in Europe.     

<Emphasis Type="Italic">Bursaphelenchus mucronatus</Emphasis> (Nematoda: Aphelenchoididae) vectored by <Emphasis Type="Italic">Monochamus </Emphasis><Emphasis Type="Italic">urussovi</Emphasis> (Coleoptera: Cerambycidae) in Hokkaido,Japan

Bursaphelenchus mucronatus Mamiya et Enda has been recovered for the first time from adults of the cerambycid beetle, Monochamus urussovi (Fischer), in Hokkaido, Japan. The nematode was also recovered from the inner bark of Picea jezoensis (Siebold et Zuccarini) Carrière and Abies sachalinensis (Fr Schmidt) Masters infested with M. urussovi larvae. PCR–RFLP analysis indicated that B. mucronatus in Hokkaido is the European type.     

Role of <Emphasis Type="Italic">tao</Emphasis> (<Emphasis Type="Italic">Belotia mexicana</Emphasis>) in the traditional Lacandon Maya shifting cultivation ecosystem

For centuries, the Lacandon Maya have farmed the forest while also preserving and regenerating it. The Lacandon manage their fallow by planting certain tree species, and removing volunteer trees that are not optimal for soil fertility. This study focused on tao (Belotia mexicana), one of the Lacandon tree fallow species, and its impact on the soil as it matures in the secondary forest. The effect of tao on soil fertility was evaluated using the following soil fertility parameters: phosphorus, carbon, nitrogen, earthworm density, pH, and soil moisture. Results were compared using a split-plot analysis. Soil C:N ratios decreased with age of tao, indicating an improvement of litter quality over time. Soil extractable phosphorus decreased with age of tao and increased with distance from tao, which suggests that tao is depleting phosphorus. These results provide an introduction for further analysis into how native trees enhance soil fertility in the Lacandon system.     

Difference between sprouting traits of <Emphasis Type="Italic">Cercidiphyllum japonicum</Emphasis> and <Emphasis Type="Italic">C. magnificum</Emphasis>

Cercidiphyllum japonicum and C. magnificum are deciduous tree species that produce large numbers of sprouts. They are found mainly in riparian and/or disturbed areas. C. japonicum is distributed in the montane zone in Japan, whereas C. magnificum is distributed mostly in the subalpine zone of central Japan. However, the two species sometimes coexist, e.g., on the talus slope at the valley head of the Chichibu Mountains. We investigated differences in sprouting traits in these two species by comparing class distributions of sprout diameter at breast height (DBH) and heights of individuals on the talus slope. Sprout DBH and individual height were smaller in C. magnificum as compared to C. japonicum. Moreover, the analysis of the DBH-class distribution of each species indicated that C. magnificum had numerous small sprouts and experienced high mortality, whereas C. japonicum had fewer small sprouts and low mortality. It is likely that the sprouting traits of C. magnificum make it more adapted to severe conditions in the subalpine zone than those of C. japonicum.     

Effects of Atlas cedar (<Emphasis Type="Italic">Cedrus atlantica</Emphasis>) defoliation on performance of the pine processionary moth (<Emphasis Type="Italic">Thaumetopoea pityocampa</Emphasis>)

The pine processionary moth (Thaumetopoea pityocampa) is an important defoliating insect of native cedar stands in Northern Africa. In 2002 and 2003, we studied the size of needles of Atlas cedar trees that had been defoliated or not in the previous year, and compared the oviposition preference and larval performance of processionary moth on these two types of trees in cedar stands of central Algeria. Needle length and their number per needle cluster were twice as high on non-defoliated trees than on defoliated trees. There were about ten times fewer egg batches on defoliated than on non-defoliated trees in both study years. Young larvae raised in the laboratory on the foliage of previously defoliated trees were smaller compared to those fed on the foliage from healthy trees. Larval colonies transferred to trees in the field showed two times higher mortality on defoliated trees. It was concluded that the decline in population density in defoliated stands often observed after outbreaks results both from the fact that the female moths avoid laying their eggs on defoliated trees and the lower performance of larvae which later feed on the new-grown needles.     

Insecticidal efficacy of <Emphasis Type="Italic">Isaria farinosa</Emphasis> in different life stages of <Emphasis Type="Italic">Pissodes punctatus</Emphasis> (Coleoptera: Curculionidae)

The insecticidal activity of an entomopathogenic fungus, Isaria farinosa on pine bark-weevil (Pissodes punctatus) at different life stages underwent laboratory testing. Larvae, pupae and adults of Pissodes punctatus were exposed to a range of concentrations of fungal conidia in suspension for 5 s; mean mortality and LC₅₀ values were calculated. The results showed I. farinosa can effectively infect larvae, pupae and adult pine bark-weevils, and the highest mean mortalities at each life stage can exceed 88%. The mean mortalities increased with higher concentrations of I. farinosa. Larvae were most susceptible with LC₅₀ 1.2 × 10⁶ conidia ml⁻¹ (15 days), pupae (30 days) exhibited the second largest effects, and adults (15 days) with LC₅₀ 1.72 × 10⁶ and 1.99 × 10⁶ conidia ml⁻¹, respectively, at end of the experiments. This suggests that I. farinosa could be a potential bio-control agent against the pine bark-weevil.     

Factors affecting radial growth of the invasive <Emphasis Type="Italic">Prunus serotina</Emphasis> in pine plantations in Flanders

Prunus serotina is an invasive species that causes problems mainly in pine forests on sandy soils in Western Europe. Thus far, there have been almost no studies considering the diameter growth of P. serotina in its introduced range. We analysed the radial growth of P. serotina in seven Scots pine (Pinus sylvestris) plantations in Flanders (Belgium) using multiple linear regression and a potential × modifier approach. The radial growth of P. serotina in the understory of the studied pine plantations was reduced by up to 50% when compared to potential P. serotina growth in full light conditions. Size and site effects were strongly growth-reducing; crowding effects were important only for small individuals. The actual radial growth of P. serotina below pines was determined primarily by the diameter and age of the P. serotina tree and by competition with neighbouring trees.     

Pathogenicity testing of four Bursaphelenchus species on conifer seedlings under greenhouse conditions

The pinewood nematode, Bursaphelenchus xylophilus (Steiner and Buhrer, J. Agric Res. 48, 1934, 949), Nickle (J. Nematol. 2, 1970, 375), is the causative agent of the pine wilt disease and causes serious damage to pine forests around the world. During a survey for the pinewood nematode, four other Bursaphelenchus species (Bursaphelenchus mucronatus, B. sexdentati, B. anamurius and B. vallesianus) were isolated from wilted pine trees in Turkey. To understand the effects of these Bursaphelenchus species on wilting of pine trees, a study was conducted under greenhouse conditions. Two‐year‐old seedlings of three pine species (Pinus nigra, P. brutia and P. pinea) and one cedar species (Cedrus libani) were used. Fifteen seedlings of each species were inoculated with nematodes and 10 seedlings of each species served as controls. The inoculum densities used for each seedling contained approximately 1000 (±100) nematodes of all life stages in 0.25 ml of distilled water. The first wilting symptoms were observed in the fifth week in all pine species but not in the cedar seedlings. All seeding mortality occurred between the 5th and 13th weeks of the study; no mortality was observed outside of this period. The most pathogenic nematode species was B. mucronatus, closely followed by the other species. The most susceptible seedling species was P. nigra, and C. libani was the most resistant species.