Ethylene production during symptom development of pine-wilt disease

Ethylene release from Pinus thunbergii Parl. seedlings was examined after inoculation with virulent and avirulent isolates of Bursaphelenchus xylophilus. Enhanced ethylene release was observed only in seedlings inoculated with the virulent isolate. The avirulent isolate induced necrosis of xylem ray cells and embolism in some tracheids, however, the cambium and outermost xylem was intact where water conduction was maintained. The ethylene increase coincided with cambial death in seedlings inoculated with the virulent isolate.     

Mechanism of cavitation development in the pine wilt disease

Volatile terpenes increase in xylem tissue after infection of Pinus thunbergii with the pine wood nematode (Bursaphelenchus xylophilus). The role of these terpenes in traeheid cavitation, which blocks xylem-sap ascent and leads to water deficit in pine trees, was assessed. Volatile terpene concentration increased long before initiation of tracheid cavitation. After the volatile terpenes reached the highest concentration, severe cavitation developed. Direct injection of α-pinene into healthy pine trunks formed artificial cavitation in xylem. These observations support the hypothesis that excessively produced volatiles, which are hydrophobic and have lower surface tension than water can promote tracheid cavitation in pine wilt disease.     

Responses of water-stressedPinus thunbergii to inoculation with avirulent pine wood nematode (Bursaphelenchus xylophilus): Water relations and xylem histology

The effect of water-stress conditioning on water relations and histological features ofPinus thunbergii Parl. inoculated with avirulent isolate ofBursaphelenchus xylophilus (Steiner and Buhrer) Nickle, pine wood nematode, were investigated. Pines were kept under 8 days cycle of severe water stress. One-half of the water-stressed pines died as a result of infection by avirulent pine wood nematode and water stress tended to induce increased susceptibility and/or decreased resistance of pines to avirulent pine wood nematode. In dead pines, the water conducting function of xylem was lost, and all of the parenchyma cells died. In surviving pines, the xylem hydraulic conductivity and the xylem water content were significantly reduced (12 to 23% and 77 to 83%, respectively) compared to controls. Safranin dye perfusion of excised axis stem segments indicated that the water conductance was limited to the very narrow peripheral area of xylem. Embolism caused by cavitation in the tracheids occurred in the central part of xylem and in that dysfunctional region of the xylem the axial parenchyma cells surrounding the epithelial cells, and ray parenchyma cells partly degenerated but the epithelial cells survived. The disruption of tracheid shape observed in surviving pines indicates that avirulent pine wood nematode temporarily disturbed cell division of the cambium. Considering the differences in responses between dead pines and surviving pines after inoculation with avirulent pine wood nematode, the death of water-stressed pines apparently resulted from death of cells, in particular the vascular cambium and the loss of xylem hydraulic function by cavitation.     

Correlation between acoustic emission, water status and xylem embolism in pine wilt disease

The occurrence of cavitation events and embolism during the latent, early stage and the late developmental stages of pine wilt disease was monitored nondestructively by acoustic emission (AE) and high-resolution magnetic resonance microscopy, respectively, and the results were compared with changes in leaf water potential and stem thickness. In the latent stage of the disease, when no embolisms were observed, cavitation events were detected by AE during the daytime in water-stressed Japanese black pine (Pinus thunbergii Parl.) seedlings, indicating that cavitation occurred at the individual tracheid level. In the early stage of the disease, an increase in the frequency of AE events occurred coincidentally with the occurrence of patchy embolisms at the mass tracheid level. The threshold water potential for such mass cavitation was higher than that causing cavitation of individual tracheids during the latent stage of the disease. In the advanced stage of the disease, explosive AE events were observed coincidentally with drastic enlargement of embolized areas and decreases in water potential. The AE events in the latent stage occurred only during the daytime, whereas, in the early and advanced stages of the disease, they also occurred at night. The explosive occurrence of cavitation in the advanced stage was thought to be a case of "runaway embolism."     

Physiological process of the symptom development and resistance mechanism in pine wilt disease

Pathophysiological changes during the symptom development of pine wilt disease are reconsidered from recent investigations. The symptom development is divided into two stages: the early and the advanced stages. In the early stage, small number of nematodes migrate in cortex, then in xylem of the stem, and induce denaturation and necrosis of parenchyma cells. These changes in parenchyma are regarded as defense reactions of pines which result in terpene synthesis in xylem cells and embolism in tracheids. Such changes in the early stage can be induced in both susceptible and resistant pine species by either virulent or avirulent isolates of pinewood nematode (Bursaphelenchus xylophilus), or byB. mucronatus. No change occur in physiological status of leaves, and nematode reproduction is suppressed during this stage. Pine trees can survive if symptom does not progress from this stage. The symptoms of the advanced stage usually occur only in susceptible pines infected by virulent nematode isolates. At the beginning of the advanced stage, enhanced ethylene production by stem which coincides with cambial destruction occurs, and results in embolism of the outermost xylem in the portion. The embolism causes decrease in leaf water potential and cessation of photosynthesis. After cessation of photosynthesis, symptoms develop drastically with a burst of nematode population. There seems to be some unknown mechanism which suppress nematode reproduction and invasion to the cambial zone. This mechanism is thought to be photosynthesis-dependent, so that in photosynthesis-decrased conditions, even avirulent nematodes can multiply and invade cambium to induce tree death. Water stress in hot and dry summer should accelerates symptom development from the early to the advanced stage through such decrease of photosynthesis-dependent “cambial resistance”.     

The effects of long‐term exposure to simulated acid rain on the development of pine wilt disease caused by Bursaphelenchus xylophilus

To elucidate the synergetic effects of acid rain on the development of pine wilt disease, we measured the sap flow rate in the stems and the chlorophyll content in the needles of 10‐year‐old Japanese black pine trees, Pinus thunbergii and 12‐year‐old Japanese red pine trees, Pinus densiflora, after exposure to simulated acid rain (SAR, pH 3) or tap water (TW, pH 6.3) as a control. The heat pulse method was used for the estimation of the sap flow rate. No apparent difference was found in the sap flow rate between the trees exposed to SAR and TW, but the chlorophyll content of needles at the end of the treatment was significantly higher in the trees exposed to SAR than in those exposed to TW. When the pinewood nematode, Bursaphelenchus xylophilus, the causal agent of pine wilt disease, was inoculated onto the Japanese black pines that had been exposed to SAR repeatedly for 1 year, the period to death was shortened. Japanese red pines that had been exposed to SAR for 2 years, however, did not show any development of symptoms after the nematode inoculation, suggesting that acid rain only affects pine wilt disease slightly, if at all.     

Migration of Bursaphelenchus xylophilus in cortical and xylem axial resin canals of resistant pines

Migration of the pine wood nematode (PWN), Bursaphelenchus xylophilus, in susceptible and resistant pines was investigated at the tissue level. PWN was inoculated onto the top cross‐cut surface of 20‐cm stem cuttings of susceptible Pinus thunbergii and resistant pines (P. strobus, P. rigida and P. thunbergii of a resistant family Namikata‐(t)‐73 (half‐sib)). PWNs were mainly distributed in cortical resin canals of susceptible P. thunbergii down to 15 cm from the inoculated surface by 6 h after inoculation (HAI) and all tissues (including cortical and xylem resin canals) down to the bottom at 192 HAI. In P. strobus, P. rigida and P. thunbergii family Namikata‐(t)‐73 (half‐sib), PWN was distributed in cortical resin canals down to 5 cm by 6 HAI and down to the base at 192 HAI. However, the distribution of PWN in xylem resin canals of the resistant pines was restricted near inoculated surfaces down to 5 cm, even at 192 HAI. These results demonstrated that migration of PWN in resistant pines was slowed in cortical resin canals and restricted in xylem axial resin canals, features which may be associated with the resistance.     

Avirulent isolate of the pinewood nematode Bursaphelenchus xylophilus,survives 7 months in asymptomatic host seedlings

An avirulent isolate (C14‐5) of the pinewood nematode, Bursaphelenchus xylophilus, was inoculated onto 45 seedlings of the susceptible host Japanese black pine (Pinus thunbergii) and its viability was investigated. The nematode survived inside host seedlings for approximately 7 months even when the host seemed to overcome the infection based on lack of foliar wilting and the observation of normal oleoresin flow.     

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.     

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.     

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.     

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.     

The effect of periderm formation in the cortex of Pinus thunbergii on early invasion by the pinewood nematode

The distribution of cortical resin canals and periderm formation in the cortex of Pinus thunbergii was studied in relation to early invasion of the pinewood nematode, Bursaphelenchus xylophilus. Nematode invasion was restricted in stem cuttings of P. thunbergii in which periderm closed cortical resin canals. Early invasion of the nematodes was also restricted in stem cuttings where wound periderm had formed in response to prior nematode inoculation. It was concluded that early invasion of pinewood nematodes in living bark tissue is restricted by periderm in mature stems and by wound periderm that had formed as the result of previous nematode infection.     

Suppression of ectomycorrhizal development in young Pinus thunbergii trees inoculated with Bursaphelenchus xylophilus

In order to study the changes in ectomycorrhizal development during symptom expression of pine wilt disease, root window observations were conducted concurrent with measurements of leaf water potential as well as photosynthetic and transpiration rates of 5‐year‐old Pinus thunbergii trees that were inoculated with the pinewood nematode (PWN) Bursaphelenchus xylophilus. Infected trees were compared with girdled and uninfected control trees. Ectomycorrhizas developed constantly during the experimental period in control trees but did not develop in the girdled trees. Ectomycorrhizal development ceased within 2 weeks in those trees that finally died after PWN infection. In the trees that survived PWN infection, ectomycorrhizal development ceased within 1–4 weeks of inoculation but was resumed thereafter within 3–6 weeks. Ectomycorrhizal development ceased prior to a decrease in both photosynthetic rate and leaf water potential in the inoculated trees.     

Mortality of planted <Emphasis Type="Italic">Pinus thunbergii</Emphasis> Parlat. saplings subject to coldness during winter and soil types in region of seasonal soil frost

To assess and improve methods for the development of coastal forests in Hokkaido, northern Japan, this study examined the factors affecting mortality of planted Pinus thunbergii Parlat. saplings in a region with seasonal soil frost. The needles of pine saplings at the study site turn red in late spring, suggesting xylem embolism, which can lead to death. Sapling mortality was strongly correlated with the degree of cold just after planting and with the occurrence of repeated severe winters. Volcanic ash soil had been supplied to improve the soil nutrients, and the volcanic ash soil always thawed later than the original coastal sand. Saplings in volcanic ash were significantly more damaged than those in original coastal sand. This suggests that hydraulic stress caused by cuticular transpiration under frozen soil conditions during spring may also accelerate damage due to embolisms. Hence, volcanic ash soil should not be applied to soils in regions with seasonal soil frost. A covering of woodchips can help prevent frost from reaching greater soil depths, and, indeed, fewer saplings at the study site died in areas with such coverage. Planting location can also affect pine sapling mortality. For example, shading caused by wood fencing may affect the soil freezing profile during winter; therefore, planting close to a fence should also be avoided in regions with seasonal soil frost. These findings should be used to improve the development of forests in regions with seasonal soil frost.     

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).     

Haustorial morphology of Cronartium conigenum in naturally infected cones of three Pinus species from Guatemala1

Haustorial morphology of the cone-rust pathogen, C. conigenum, was investigated among naturally infected female strobili of three Guatemalan pines, P. maximinoi, P. pseudostrobus, and P. oocarpa. Among the three pine species, haustorial shapes and sizes were more variable in P. maximinoi and P. oocarpa than in P. pseudostrobus. The haustorial shapes and sizes were more variable in parenchyma cells of the cortex, xylem, and pith than in the cells in the phloem, xylem rays, and tracheids. The haustoria were also present in larger numbers in the parenchyma cells of the cortex, xylem, and pith than in phloem parenchyma cells and tracheids. In living cells, the tips of some haustoria were appressed to the host nuclei.     

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.     

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.     

Effects of inoculum density of pinewood nematode on the development of pine wilt disease in Japanese black pine seedlings pretreated with simulated acid rain

Six‐month‐old Japanese black pine seedlings (Pinus thunbergii) were exposed to simulated acid rain (SAR) at pH 3 and 2 three times a week. After treatment for 2 months, the seedlings were inoculated with a virulent isolate (S10) of the pinewood nematode (Bursaphelenchusxylophilus), at three inoculum levels (P_i = 50, 160 or 500 nematodes per seedling). In seedlings inoculated with 500 nematodes, both population growth of nematodes and disease development were accelerated by pretreatment with SAR at pH 3 or 2. In seedlings inoculated with 50 nematodes, population growth of the nematodes was suppressed and more time was needed for seedlings to die when pretreated with pH 3 SAR. This suggests that exposure to pH 3 SAR increased not only the progress of mortality, but also simultaneously enhanced the tolerance limit of the seedlings to the pinewood nematode – the critical value of physiological burden (represented as a product of time and initial nematode population) necessary to kill a seedling. Exposure to pH 2 SAR accelerated nematode reproduction in seedlings and increased seedling mortality irrespective of the number of nematodes inoculated.