Retardation of pine wilt disease symptom development in Japanese black pine seedlings exposed to simulated acid rain and inoculated withBursaphelenchus xylophilus

Three- or 4-year-old Japanese black pine seedlings were exposed to simulated acid rain (SAR) at pH 3 for two months, then inoculated with a virulent isolate (S 10) of pinewood nematodes. The experiments were repeated three times in 1996–1998. The exposure to SAR killed no seedlings, and retarded the development of disease symptoms in the seedlings inoculated with nematodes. In the experiments in 1996 and 1997, however, cessation of resin exudation, and primary decrease in the xylem conductivity occurred earlier in the seedlings exposed to SAR than in those exposed to tap water as a control. These results imply that acid rain at pH 3 influenced an increase in some resistance of Japanese black pine seedlings to pinewood nematode, and that this improved resistance could potentially overcome damage caused by acid rain. The exposure to SAR did not have any significant effect on the water relations of the seedlings itself, suggesting that retardation of the symptom development after the exposure to SAR could not be attributed to the improvement in the water relations. This work was supported in part by a Grants-in-Aid for Scientific Research from the Japan Forest Technical Association (JAFTA).     

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.     

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.     

Spatiotemporal analysis of pine wilt disease: Relationship between pinewood nematode distribution and defence response in Pinus thunbergii seedlings

Pine wilt disease is of major concern as it has destroyed pine forests in East Asia and Europe. Several studies have suggested that invasion by the pinewood nematode (PWN) Bursaphelenchus xylophilus, which causes this disease, evokes an excessive defence response in pine trees, resulting in tree death. However, few studies have quantitatively evaluated the correlation between PWN distribution and tree defence responses. Therefore, the present study aimed to quantify the number of PWNs and expression levels of putative pathogenesis‐related (PR) genes in different positions of Japanese black pine (Pinus thunbergii) seedlings over time. To quantify the number of PWNs in the seedlings, we used TaqMan quantitative real‐time PCR (qPCR) assay. During the early phase of infection, most PWNs were distributed around the inoculated sites, with only a small number being detected at distant sites, but the expression levels of PR genes were highly upregulated throughout the seedlings. Both the number of PWNs and expression levels of PR genes then increased drastically throughout the seedlings, all of which exhibited external symptoms. Thus, it appears that the rapid migration of PWNs induces a defence response throughout the seedling; however, this may not be effective in controlling these parasites, thereby ultimately leading to plant death.     

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.     

Mutual influences in growth and reproduction between pine wood nematode Bursaphelenchus xylophilus and bacteria it carries

The interactions between pine wood nematode and three bacterium strains isolated from the nematode, Bursaphelenchus xylophilus, which are two strong pathogenic bacterium strains, Pseudomonas fluorescens GcM5-1A and Pseudomonas putida ZpB1-2A and a weak-pathogenic bacterium strain, Pantoea sp. ZM2C, were studied. The result showed that the strong-pathogenic GcM5-1A strain and ZpB1-2A strain significantly increased fecundity, reproduction rate, and the body volume of the adult nematode. Meanwhile, pine wood nematodes significantly promoted reproduction of the two strong-pathogenic bacterium strains. However, the weak-pathogenic bacterium strain, ZM2C, completely inhibited reproduction of pine wood nematodes. Aseptic pine wood nematodes significantly inhibited reproduction of the strain ZM2C. The results indicated that mutualistic symbiosis exists between pine wood nematodes and the two pathogenic bacteria it carries. The phenomenon showed that the pathogenic bacteria carried by the nematode were not accidentally contaminated, but rather had existed as symbionts of the nematode with which it had coevoluted over a long period. The role of mutualistic symbiosis in the process of pine wilt disease was also discussed. __________ Translated from Journal of Nanjing Forestry University, 2005, 29(3): 1–4 [译自: 南京林业大学学报, 2005, 29(3): 1–4]     

Effect of simulated acid rain on the growth performance of the European pine sawfly [Neodiprion sertifer)

The effect of simulated acid rain on the performance of Neodiprion sertifer larvae fed on treated Scots pine needles was tested in a subarctic area with low ambient pollution level. Acid rain treatments (pH 4 and pH 3, both with H₂SO₄ and HNO₃) did not significantly affect the relative growth rates or cocoon weights of N. sertifer 2 or 3 years after the start of the treatments. The relative growth rate of N. sertifer larvae was slightly (nonsignifi‐cantly) higher on plots receiving only ambient rain than on irrigated control plots. The quality of foliage before the start of the treatments explained a significant proportion of the variation in the performance of N. sertifer larvae in the bioassay conducted 3 years later.     

Somatic embryo production and plant regeneration of Japanese black pine (Pinus thunbergii)

Somatic embryogenesis in Pinus thunbergii was initiated from megagametophytes containing immature zygotic embryos. Embryogenic cultures were maintained and proliferated in medium supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine. High maturation frequencies of somatic embryos were obtained on maturation media containing maltose, activated charcoal, abscisic acid, and polyethylene glycol as osmotic agent. The best result among the cell lines tested was achieved with the cell line T-205-3. More than 900 somatic embryos per petri dish, on average, were obtained after about 8 weeks of culture on maturation medium. Sixty percent of somatic embryos tested germinated after transfer to plant growth regulator-free medium and then 85% of them converted into plantlets.     

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”.     

Biomass distribution in 40-year-old trees of Japanese black pine

Measurements were carried out to survey the quantity of above- and below-ground biomass and its distribution of five Japanese black pines (Pinus thunbergii Parl.) growing on a sandy soil. The roots, divided into diameter groups, were surveyed using two methods—soil coring and excavation. Average dry weight of total biomass of the trees was 176,185 g. Roots represented 13.2%, below-ground stump 6.5%, stem 70.4% and branches with needles 9.9% of total biomass. Roots made up about two thirds and stump one third of below-ground biomass. Total length of below-ground biomass (except roots with diameter < 0.1 cm) was 479.1 m/tree. Roots with diameter of 0.1–0.2 cm represented only 0.7% of below-ground biomass, however as much as 49.9% of their total length. Roots with diameter over 10.0 cm constituted as much as 21.6% of below-ground biomass, however were only 0.3% of its total length. Root systems had well developed tap roots to maximal depth of 231 cm. The results indicated that mass and length of roots with diameter 0.5–2.0 cm had a close correlation with branch mass. Mass and length of roots with diameter 2.0–10.0 cm closely correlated to stem mass. Stem mass, root mass and root length closely correlated to DBH. A rather low correlation was found between DBH and mass of branches and below-ground stump. DBH was a suitable variable for predicting total biomass.     

Impact of the invasion of pinewood nematode and the following different removal disturbance intensities on the plant diversity of Masson pine community

Reported in this paper are our findings of the study on changes in species diversity of different Masson pine communities after the invasion of Bursaphelenchus xylophilus (Steiner and Buhren) Nickle and subsequent and different removal disturbance management approaches. Based on the results, the species diversity indices for the arbor layers of the different communities can be arranged in the following order (from high to low): 1) broad-leaved stand after the removal of all infected pine trees in the pure pine stand in Fuyang; 2) lightly infected Masson pine and Schima superba mixed stand in Fuyang; 3) uninfected stand mixture of Masson pine and Castanopsis fargesii as a control; 4) lightly infested pure Masson pine stand in Fuyang; 5) Quercus variables stand formed following the selective removal of infected pine trees from a mixed Masson pine and Q. variables stand in Zhoushan Islands; 6) pure young Masson pine stand formed following the removal of all infected pine trees from a pure Masson pine stand; 7) pure Liquidambar formosana stand after the removal of infected pine trees from a pure pine stand in Zhoushan Islands; 8) a mixed stand consisting of Pinus thunbergii and the Masson pine in Zhoushan Island; and 9) moderately infected Masson pine stand in Zhoushan Islands. All the three diversity indices (R ₀ = richness, H′ = Shannon-Wiener index, and E = evenness) for the shrub layer did not show any significant differences among the various communities except for the pure pine stand in Zhoushan Island, which had the lowest diversity. The three indices for the herb layer of the pure young Masson pine, Q. variables stand, and L. formosana stand were higher than that of other stands. The integrated analysis showed that the Masson pine forest in different geographical situations and extent of damage had distinct disparity, just as in different disturbance degrees and restoring manners. We created the “index of disturbing intensity of stump and fallen woods” (IDISFW) to represent the degree of disturbance of tree removal to plant diversity. We found that the relationship between the species diversity index and the IDISFW shows both a lower and higher degree of removal disturbance that follows the “mid-altitude bulge” theory. Specifically, both excessive and insufficient removal of infected trees will cause a decline of plant species diversity to a certain degree. Covariance analysis indicated that different IDISFW had no significant effects on the species diversity of the arbor layer, but had different impacts on that of the shrub and herb layers, and therefore can be used to assess changes in species diversity of different Masson pine communities after the invasion of pine wood nematode. __________ Translated from Chinese Journal of Applied Ecology, 2006, 17(7): 1,157–1,163 [译自: 应用生态学报]     

Biomass of fine and small roots in two Japanese black pine stands of different ages

The biomass and the spatial distribution of fine and small roots were studied in two Japanese black pine (Pinus thunbergii Parl.) stands growing on a sandy soil. More biomass of fine and small roots was found in the 17-year-old than in the 40-year-old stand. There were 62 g m⁻² of fine roots and 56 g m⁻² of small roots in the older stand, which represented mean values of 608 g for fine and 552 g for small roots per tree, respectively. In the younger stand, a total of 85 g m⁻² of fine roots and 66 g m⁻² of small roots were determined, representing a mean of 238 g for fine and 186 g for small roots per tree, respectively. Fine and small root biomasses decreased linearly with a soil depth of 0–50 cm in the older stand. In the younger stand, the fine and small roots developed only up to a depth of 30 cm. Horizontal distributions (with regard to distance from a tree) of both root groups were homogeneous. A positive correlation in the amount of biomass of fine and small roots per m² relative to tree size was found. Fine and small root biomasses increased consistently from April to July in both stands. The results also indicated earlier growth activity of the fine roots than small roots at the beginning of the growing season. The seasonal increases in fine and small root biomasses were slightly higher in the younger stand than the older stand.     

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.     

Mulch and hexazinone herbicide shorten the time longleaf pine seedlings are in the grass stage and increase height growth

Herbaceous plant control with mulchor hexazinone herbicide influenced planted longleafpine (Pinus palustris Mill.) seedling totalheight on a silt loam site in central Louisiana. Thesite had been sheared and windrowed in 1991 and rotarymowed before three treatments were established in arandomized complete block design: (1) Untreatedcheck: no herbaceous plant control after planting;(2) Five mulches: on each plot, five randomlyassigned mulches were placed around seedlings; themulches were either a mat of cotton, hemlock andpolyester, pine straw, woven polypropylene, orperforated polyethylene; and (3) Hexazinone: theherbicide hexazinone at 1.12 kg active ingredient/hawas annually sprayed in the first two growing seasonsover the rows of unshielded seedlings. The longleafseedlings were planted in February 1993.After three growing seasons, seedlings on the mulchand hexazinone treatments were taller than those onthe check plots. About 59% of the mulched andhexazinone treated seedlings had grown out of thegrass stage (at least 12 cm tall) compared to 17% ofthe check seedlings. After five growing seasons, thepercentage of longleaf pine seedlings out of the grassstage was similar on all treatments and averaged 87%. However, these better growing pines were taller on themulch and hexazinone treatments (a 142-cm average)than on the checks (78 cm). Pine straw was anineffective mulch probably because the straw smotheredthe seedlings. The longleaf saplings were tallestwhen the perforated polyethylene mat was used.     

Comparison of stand dynamics after dieback caused by pine wilt disease among pine forests with different management regimes in western Japan

In pine forests damaged by pine wilt disease, in western Japan, the effect of protection regimes of pine trees on the stand dynamics were examined in the following four stands: (1) lightly damaged stand (age 30–40 years) with no procedure in operation for protecting pine trees; (2) severely damaged stand (age 30–40 years) with no procedure in place for protecting pine trees; (3) severely damaged stand (age 50 years) with a selective cutting of infected trees; (4) severely damaged stand (age 30–40 years) with a selective cutting of infected trees. All the stands had been abandoned before the pine wilt disease damage. The understory structure of the severely damaged stand with no protection procedure was similar to that of the lightly damaged stand. Frequent invasion by tree species and acceleration in the growth of understory trees occurred after the dieback in the selective cutting stand. These results suggest that a deficiency in the canopy layer caused by the dieback resulted in low disturbance intensity in the early stages after the dieback, but the selective cutting increased the intensity by the reduction in the understory as well as the canopy layer. The intensity of the disturbance in the selective cutting stands was larger in the younger stand because it had a higher density of selectively cut pine trees. The different stand structure of pine forests occurred after the dieback because the intensity of the disturbance varied as a result of the selective cutting operation and the stand age.     

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.     

Relationship between the incidence of pine wilt disease and the drainage area

We examined the relationship between the incidence of pine wilt disease and moisture conditions in the stand level ofPinus densiflora Sieb. et Zucc. forests in the warm-temperate zone of the western part of Japan. For this analysis, pine trees killed by pine wilt disease were distinguished from the suppressed trees by their position in the layer of the pine forest stand. The drainage area, which is small in the upper part and large in the lower part of the slope, was adopted for representing the moisture conditions in the soil of the slope. The percentage of the pine trees killed by pine wilt disease increased as the size of the drainage area increased. This result suggested that the incidence of pine wilt disease tended to be high in areas with moist conditions. Pine trees attacked by the pinewood nematode die from extensive water deficit due to tracheid cavitations. Pine wilt disease mainly emerges in the summer when the soil water conditions become especially severe, and the radical water stress is thought to accelerate the disease. It was assumed that pine trees in the plots with the small drainage area resisted the influence of the attack of the pinewood nematode because pine trees in the plots with the small drainage area encountered long-term water stress and acquired drought tolerance. Pine trees in the plots with the large drainage area were presumed to be well established in the moist conditions and not to have acquired drought tolerance. The drought tolerance of pine trees was thought to be an important factor in resistance to pine wilt disease.     

Relating the survival and growth of planted longleaf pine seedlings to microsite conditions altered by site preparation treatments

Pine plantations in the southeastern United States are often created using site preparation treatments to alleviate site conditions that may limit survival or growth of planted seedlings. However, little is understood about how site preparations affect longleaf pine (Pinus palustris P. Miller) seedlings planted on wet sites. In a 2-year study (2004 and 2005) on poorly drained, sandy soils of Onslow County, North Carolina, we examined the effects of common site preparation treatments on microsite conditions and quantified relationships between microsite conditions and longleaf pine seedling survival and growth. Treatments used in the study included site preparations designed to control competing vegetation (chopping and herbicide) combined with those that alter soil conditions (mounding and bedding). During both years, mounding and bedding treatments reduced the amount of moisture within the top 6 cm of soil and increased soil temperatures when compared to flat planting (p < 0.001). Soil moisture was inversely related to seedling mortality in 2004 (r² = 0.405) and inversely related to root collar diameter in 2005 (r² = 0.334), while light was positively related to root collar diameter in 2005 (r² = 0.262). Light availability at the seedling level was highest on treatments that effectively reduced surrounding vegetation. Herbicides were more effective than chopping at controlling vegetation in 2004 (p < 0.001) and 2005 (p = 0.036). Controlling competing vegetation, especially shrubs, was critical for increasing early longleaf pine seedling growth.     

Grass interference limits resource availability and reduces growth of juvenile red pine in the field

The effects of competing grasses on resource availability, growth and ecophysiological characteristics of 3-0 red pine (Pinus resinosa Ait.) seedlings were examined the first two years following outplanting in Anoka County, Minnesota, USA. Equal numbers of seedlings were planted into suppressed and undisturbed grass communities in a sandy soil. Grass suppression was maintained throughout the first growing season, but partially discontinued thereafter on the site. During the first field season interference from grass reduced pine seedling root collar diameter, needle length, number of new root tips, and lateral root length by over 40%. Mean pre-dawn needle water potential was 0.55 MPa lower in undisturbed grass plots during a brief drought in year one, but otherwise water stress was not significantly (p=0.05) influenced by grass interference. The presence of grass also reduced, up to 50%, the photosynthetically active radiation reaching the seedling canopy. At the end of year one, total biomass N, P, K, and Ca content were significantly (p=0.05) less in seedlings growing in the undisturbed grass community. Nitrogen was deficient in seedlings growing in grass. After two growing seasons, seedling shoot length (p=0.03), root collar diameter (p=0.001), and needle length (p=0.001) were significantly less (40, 54 and 20%, respectively) for seedlings growing in undisturbed grass. Seedling growth reductions induced by grass competition were associated with multiple environmental stressors in the field and not restricted to water stress as was observed in earlier studies with pine species at low and mid-latitude sites.