Using an evapo-transpiration model (ETpN) to predict the risk and expression of symptoms of pine wilt disease (PWD) across Europe

The pine wood nematode (PWN) Bursaphelenchus xylophilus is the causal agent of pine wilt disease (PWD), a xylem restricting disease of pine trees. PWN, a native of North America where it very rarely kills native pine trees, has spread internationally killing host trees in China, Japan, Korea, Taiwan and Portugal, with isolated incursions into Spain. Based on the locations where tree mortality has been recorded, it appears that pine trees growing in hot, dry conditions are more susceptible to pine wilt disease. This paper describes the ETpN model, an evapo-transpiration model (previously developed by Forest Research), which has been modified to incorporate the presence of PWN inside a tree and which predicts the regions of Europe that are likely to succumb to PWD. ETpN acts independently of the vector beetle (Monochamus spp.), predicting the likelihood of PWD on the assumption that a tree in a particular region has already been infested by the pine wood nematode. Different regions across Europe are included to investigate and demonstrate how different climates affect PWD incidence significantly. Simplified, “lite” and latency models have been developed to allow a non-specialist user to determine respectively the risk of PWD at a particular location and the likelihood of delays (latency) in expression of wilt symptoms.     

Daily dynamics in xylem cell radial growth of Scots pine (Pinus sylvestris L.)

Daily dynamics of radial cell expansion during wood formation within the stems of 25-year-old Scots pine trees (Pinus sylvestris L.), growing in field conditions, were studied. The samples of forming wood layers were extracted 4 times per day for 3 days. Possible variations in the growth on different sides of the stem, duration of cell development in radial cell expansion phase and dynamics of cell growth in this phase were taken into account. The perimeters of tracheid cross-sections as a reflection of primary cell wall growth were the criterion of growth in a radial direction. For the evaluation of growing cell perimeters a special system for digital processing and image analysis of tracheid cross-sections of the forming wood was used. Growth rate for certain time intervals was estimated by the change in the relation of the perimeter of each observed cell in each of ten tracheid rows in each of 12 trees to the perimeter of the xylem cell of the same row before the expansion. Temporal differences in average values of the relations were estimated by Analyses of Variance. The existence of daily dynamics of Scots pine xylem cell radial growth has been proved. Intensive growth of pine tracheids has been shown to occur at any time of the day and to depend on the temperature regime of the day and the night as well as water supply of stem tissues. Moreover, reliable differences (P = 0.95) in the increment of cell walls during tracheid radial expansion have been found. Pulsing changes of the water potentials both of the cell and the apoplast, as the reason for the fluctuations of radial cell growth rate, were discussed.     

Evolutionary change in a pine wilt system following the invasion of Japan by the pinewood nematode, <Emphasis Type="Italic">Bursaphelenchus</Emphasis><Emphasis Type="Italic">xylophilus</Emphasis>

Pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causative agent of pine wilt disease (PWD) of pine trees and is transmitted by cerambycid beetles belonging to the genus Monochamus. PWN is believed to have been introduced into Japan from North America at the beginning of the 20th century. In this article, we first provide an outline of the PWD system and the range expansion of PWN in Japan and then review the literature, focusing on the virulence of PWN. Virulence is a heritable trait in PWN, with high virulence being closely related to a high rate of reproduction and within-tree dispersal. When two PWN isolates with different virulence levels are inoculated into pine seedlings, the more virulent nematodes always dominate in dead seedlings. In a laboratory setting, many more virulent nematodes board the insect vectors than avirulent ones. The age at which vectors transmit the most abundant PWNs to pine twigs changes during the course of a PWD epidemic. However, the relation between virulence and transmission of PWN remains as yet relatively unknown. Such information would enable ecologists to predict the evolution of the PWD system. In this review we also compare ecological traits between the PWN and the avirulent congener, B. mucronatus.     

华山松大小蠹共生真菌对华山松木质部危害的解剖学研究

通过对野外和人工接种条件下,华山松大小蠹（Dendroctonus armandi)共生直菌（Leptographium terebrantis)对寄主华山松木质部危害的解剖学研究。结果表明,华山松大小蠹共生真菌随华山松大小蠹入侵健康寄主华山松木质部组织,真菌菌丝首先在其木质部树脂道内发育,分解木质部树脂道泌脂细胞,堵塞树脂道;通过菌丝在木质部交叉场薄壁细胞内及管胞细胞间和管胞细胞内的扩展,使寄主华山松树脂代谢,抗性物质代谢和水分代谢紊乱,木质部边材组织蓝变。     

Xylem wall collapse in water-stressed pine needles

Wall reinforcement in xylem conduits is thought to prevent wall implosion by negative pressures, but direct observations of xylem geometry during water stress are still largely lacking. In this study, we have analyzed the changes in xylem geometry during water stress in needles of four pine species (Pinus spp.). Dehydrated needles were frozen with liquid nitrogen, and xylem cross sections were observed, still frozen, with a cryo-scanning electron microscope and an epifluorescent microscope. Decrease in xylem pressure during drought provoked a progressive collapse of tracheids below a specific threshold pressure (P(collapse)) that correlates with the onset of cavitation in the stems. P(collapse) was more negative for species with smaller tracheid diameter and thicker walls, suggesting a tradeoff between xylem efficiency, xylem vulnerability to collapse, and the cost of wall stiffening. Upon severe dehydration, tracheid walls were completely collapsed, but lumens still appeared filled with sap. When dehydration proceeded further, tracheids embolized and walls relaxed. Wall collapse in dehydrated needles was rapidly reversed upon rehydration. We discuss the implications of this novel hydraulic trait on the xylem function and on the understanding of pine water relations.     

Effects of spring-killed pine trees on the epidemics of pine wilt disease

Pine wilt disease (PWD) is caused by the pinewood nematode (PWN), Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle, and is transmitted by cerambycid beetles. In some pine trees infected with the PWNs in Japan, foliage changes from green to brown in summer to autumn of a nematode infection year (summer- autumn-killed trees) and the others in the following spring of a nematode infection year (spring-killed trees). The vector beetles require 1 or 2?years for development in cool summer areas and 1?year in warm summer areas. To evaluate the effects of the spring-killed trees and vectors with a long developmental time on the PWD epidemics, we presented simple mechanistic mathematical models. The models showed that it was possible for spring-killed trees to cause PWD epidemics when the transmission rate was high, and the efficacy of spring-killed trees as infection source was similar to that of summer?C autumn-killed trees. Spring-killed trees and vector beetles with a developmental time of 2?years harbored in summer- autumn-killed trees delayed epidemic timing by 3?C10?years or actually suppressed epidemics.     

Gradients and dynamics of inner bark and needle osmotic potentials in Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies L. Karst)

Preconditions of phloem transport in conifers are relatively unknown. We studied the variation of needle and inner bark axial osmotic gradients and xylem water potential in Scots pine and Norway spruce by measuring needle and inner bark osmolality in saplings and mature trees over several periods within a growing season. The needle and inner bark osmolality was strongly related to xylem water potential in all studied trees. Sugar concentrations were measured in Scots pine, and they had similar dynamics to inner bark osmolality. The sucrose quantity remained fairly constant over time and position, whereas the other sugars exhibited a larger change with time and position. A small osmotic gradient existed from branch to stem base under pre‐dawn conditions, and the osmotic gradient between upper stem and stem base was close to zero. The turgor in branches was significantly driven by xylem water potential, and the turgor loss point in branches was relatively close to daily minimum needle water potentials typically reported for Scots pine. Our results imply that xylem water potential considerably impacts the turgor pressure gradient driving phloem transport and that gravitation has a relatively large role in phloem transport in the stems of mature Scots pine trees.     

Water transport properties of seven woody species from the semi-arid Mu Us Sandy Land,China

Maintenance of water transport is very important for plant growth and survival. We studied seven woody species that inhabit the semi-arid Mu Us Sandy Land, China, to understand their strategies for maintaining hydraulic function. We evaluated water transport properties, including cavitation resistance, hydraulic recovery, and water loss regulation by stomatal control, which are associated with xylem structural and leaf physiological traits. We also discussed the water-use characteristics of these species by comparing them with those of species in other regions. Species with tracheids had higher levels of xylem resistance to cavitation and a smaller midday transpiration rate than the other species studied. Although species with vessels were less resistant to cavitation, some recovered hydraulic conductivity within 12 h of rehydration. Species with xylem tracheids could maintain their hydraulic function through resistance to cavitation and by relaxing xylem tension. Although species with vessels had less resistant xylem, they could maintain hydraulic function through hydraulic recovery even when xylem dysfunction occurred. Additionally, the species studied here were less resistant to cavitation than species in semi-arid environments, and equally or less resistant than species in humid environments. Rather than allow hydraulic dysfunction due to drought-induced dehydration, they may develop water absorption systems to avoid or recover quickly from hydraulic dysfunction. Thus, not only stem cavitation resistance to drought but also stem–root coordination should be considered when selecting plants for the revegetation of arid regions.     

Induction of air embolism in xylem conduits of pre-defined diameter.

A new method is presented that enables the induction of embolisms in a fraction of all xylem vessels, based on diameter, at one cut end of a stem segment. The method is based on the different capillary characteristic of xylem vessels of different cross-sectional size. To verify the method, air embolisms were induced in cut xylem vessels of chrysanthemum (Dendranthemaxgrandiflorum Tzvelev cv. Cassa) stem segments at different xylem tensions and compared with the distribution of gas-filled vessels as visualized by cryo-scanning electron microscopy (Cryo-SEM). At -6 kPa xylem pressure, air-entrance was only induced in large diameter vessels (>30 microm), while at -24 kPa embolisms were induced in almost all xylem vessels (>10 microm). Although the principle of the embolization method worked well, smaller diameter vessels were observed to be embolized than was expected according to the calculations. The role of cross-sectional shape and contact angle between xylem sap and vessel wall at the menisci are discussed. After correction for the observed (diameter independent) deviation from circularity of the cross-sectional vessel shape the contact angle was calculated to be approximately 55 degrees. Hydraulic resistance (Rh) measurements before and after embolization showed that the effect of embolizing only large diameter cut xylem vessels had only a small influence on overall Rh of a stem segment. Embolizing all cut xylem vessels at one cut end almost trebled overall Rh. The difference was discussed in the light of the networking capacity of the xylem system.     

Pathogenicity of aseptic Bursaphelenchus xylophilus

Pine wilt is a disease of pine (Pinus spp.) caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus. However, the pathogenic mechanism of pine wilt disease (PWD) remains unclear. Although the PWN was thought to be the only pathogenic agent associated with this disease, a potential role for bacterial symbionts in the disease process was recently proposed. Studies have indicated that aseptic PWNs do not cause PWD in aseptic pine trees, while PWNs associated with bacteria cause wilting symptoms. To investigate the pathogenicity of the PWN and its associated bacteria, 3-month-old microcuttings derived from certain clones of Pinus densiflora Siebold & Zucc. produced in vitro were inoculated under aseptic conditions with aseptic PWNs, non-aseptic PWNs and bacteria isolated from the nematodes. Six-month-old aseptic P. densiflora microcuttings and 7-month-old P. massoniana seedlings were also inoculated under aseptic conditions with aseptic PWNs and non-aseptic PWNs. The results showed that the aseptic microcuttings and seedlings inoculated with aseptic PWNs or non-aseptic PWNs wilted, while those inoculated with bacterial isolates did not wilt. Nematodes were recovered from wilted microcuttings and seedlings inoculated with aseptic PWNs and non-aseptic PWNs, and the asepsis of nematodes recovered from aseptic PWN-inoculated microcuttings and seedlings was reconfirmed by culturing them in NB liquid medium at 30°C for more than 7 days. Taken together, the results indicate that the asepsis of PWN did not cause the loss of pathogenicity.     

Structural and systematic study of an unusual tracheid type in cacti

Wide-band tracheids are a specialized tracheid type in which an annular or helical secondary wall projects deeply into the cell lumen. They are short, wide and spindle-shaped, and their bandlike secondary walls cover little of the primary wall, leaving most of it available for water diffusion. Wide-band tracheids appear to store and conduct water while preventing the spread of embolisms. They may be the most abundant tracheary element in the xylem, but they are always accompanied by at least a few vessels. Typically, fibers are absent wherever wide-band tracheids are present. Wide-band tracheids occur in the primary and secondary xylem of succulent stems, leaves and roots in genera of all three subfamilies of Cactaceae but were not found in the relictual genusPereskia, which lacks succulent tissues. In the large subfamily Cactoideae, wide-band tracheids occur only in derived members, and wide-band tracheids of North American Cactoideae are narrower and are aligned in a more orderly radial pattern than those of South American Cactoideae. Wide-band tracheids probably arose at least three times in Cactaceae.     

Impact of long-term drought on xylem embolism and growth in Pinus halepensis Mill.

 The present study was carried out to elucidate the response mechanisms of 50-year-old Pinus halepensis Mill. trees to a long-term and severe drought. The amount of water available to trees was artificially restricted for 12 months by covering the soil with a plastic roof. Over the short term a direct and rapid impact of drought was evident on the water relations and gas exchanges of trees: as the soil dried out in the Spring, there was a concurrent decrease of predawn water potential; transpiration was strongly reduced by stomatal closure. Seasonal changes in the water volume fractions of twig and stem xylem were observed and interpreted as the result of cavitation and refilling in the xylem. When droughted trees recovered to a more favourable water status, refilling of embolized xylem was observed; twig predawn water potentials were still negative in the period when the embolism was reversed in the twig xylem. A few months after the removal of the covering, no differences in whole plant hydraulic resistance were observed between droughted and control trees. Needle and shoot elongation and stem radial growth were considerably reduced in droughted trees; no strategy of trees to allocate carbon preferentially to the stem conducting tissues was apparent throughout the experiment. An after-effect of the drought on growth was observed. Received: 4 August 1997 / Accepted: 1 October 1997     

A survey of root pressures in vines of a tropical lowland forest

Pre-dawn xylem pressures were measured with bubble manometers attached near the stem bases of 32 species of vines on Barro Colorado Island, Panama, to determine if pressures were sufficient to allow for possible refilling of embolized vessels. Of 29 dicotyledonous species 26 exhibited only negative xylem pressures, even pre-dawn during the wet season. In contrast, three members of the Dilleniaceae exhibited positive pre-dawn xylem pressures, with a maximum of 64 kPa in Doliocarpusmajor. A pressure of 64 kPa is sufficient to push water to a height of 6.4 m against gravity, but the specimens reached heights of 18 m. Thus, in all 29 dicotyledons examined, the xylem pressures were not sufficient to refill embolized vessels in the upper stems. In contrast, two of the smaller, non-dicotyledonous vines, the climbing fern Lygodiumvenustrum and the viny bamboo Rhipidocladumracemiflorum, had xylem pressures sufficient to push water to the apex of the plants. Therefore, a root pressure mechanism to reverse embolisms in stem xylem could apply to some but not to most of the climbing plants that were studied. Received: 18 March 1996 / Accepted: 24 October 1996     

Drought alters timing, quantity, and quality of wood formation in Scots pine

Drought has been frequently discussed as a trigger for forest decline. Today, large-scale Scots pine decline is observed in many dry inner-Alpine valleys, with drought discussed as the main causative factor. This study aimed to analyse the impact of drought on wood formation and wood structure. To study tree growth under contrasting water supply, an irrigation experiment was installed in a mature Scots pine (Pinus sylvestris L.) forest at a xeric site in a dry inner-Alpine valley. Inter- and intra-annual radial increments as well as intra-annual variations in wood structure of pine trees were studied. It was found that non-irrigated trees had a noticeably shorter period of wood formation and showed a significantly lower increment. The water conduction cells were significantly enlarged and had significantly thinner cell walls compared with irrigated trees. It is concluded that pine trees under drought stress build a more effective water-conducting system (larger tracheids) at the cost of a probably higher vulnerability to cavitation (larger tracheids with thinner cell walls) but without losing their capability to recover. The significant shortening of the growth period in control trees indicated that the period where wood formation actually takes place can be much shorter under drought than the 'potential' period, meaning the phenological growth period.     

Water relations, xylem embolism and histological features of Pinus thunbergii inoculated with virulent or avirulent pine wood nematode, Bursaphelenchus xylophilus

The development process of pine wilt disease caused by Bursaphelenchusxylophilus (Steiner and Buhrer) Nickle, pine wood nematode,was studied ecophysiologically and histologically in relationto pathogenicity of B. xylophilus. Judging from the predawnxylem pressure potential of needles, the heat pulse velocity,and the soil water potential, the control Pinus thunbergii Parl.,used for the study, was not water-stressed. Virulent B. xylophilusisolate can kill non-waterstressed pines. In virulent B. xylophilusisolateinoculated pines, the predawn xylem pressure potentialof needles abruptly decreased when the colour of 1-year-oldneedles changed to brown and then the water conducting functionof the xylem was lost completely. Avirulent B. xylophilus isolatedoes not affect the needle colour and the xylem pressure potentialof pines. Avirulent B. xylophilus isolate-inoculated pines,however, responded to nematode invasion by decreasing hydraulicconductance of stem and root xylems. In addition, oleoresinexudation slightly decreased. The decreased hydraulic conductanceresults from embolism of tracheids caused by cavitation in thecentral part of the xylem. From histological observation, allof the parenchyma cells in virulent B. xylophilus isolateinoculatedpines died. In contrast, the parenchyma cells, degenerated inavirulent B. xylophilus isolateinoculated pines, were limitedin the embolized region of the xylem. The difference betweenthe response of pine to the virulent B. xylophilus isolate invasionand that to avirulent B. xylophilus isolate invasion indicatesthat nematode-induced death of pine relates to the death ofparenchyma cells, as well as the decrease in xylem hydraulicconductance. Key words: Embolism, hydraulic conductance, parenchyma cells, pathogenicity of pine wood nematode, pine wilt disease.     

The role of plant hormones in higher plant cellular differentiation. II. Experiments with the vascular cambium, and sclereid and tracheid differentiation in the pine, Pinus contorta

In sterile-cultured explants of stems of the pine Pinus contorta Dougl., fusiform cambial cells differentiated entirely into axial parenchyma cells when exogenous indol-3yl-acetic acid (IAA) was omitted. The normal appearance of the cambial zone was maintained when IAA was included in the medium. The IAA-maintained stability of cambial structure suggests physiological rather than epigenetic control over vascular cambium structure. IAA was essential for the occurrence of callus growth in stem explants. Callus growth was similar in appearance and extent in winter- and summer-explanted material. Tracheids differentiated in explants only when actively differentiating tracheids were already present at the moment of explanting, suggesting the absence of factors necessary for tracheid differentiation in over-wintering tissues. Sclereid differentiation, which normally does not occur in phloem or xylem development in P. contorta, occurred in callus derived from active cambial explants. The sclereids were identical to sclereids which differentiated in pith of intact stems. The possibility that sclereid and tracheid differentiation may be fundamentally similar types of gene expression is discussed. Growth of P. contorta trees in continuous darkness resulted in extensive compression-wood tracheid differentiation in the upright main stem. Normal-wood tracheids differentiated in similar trees grown in light. More tracheids differentiated in light than in darkness. This apparently is the first report of induction of compression-wood tracheid differentiation in the absence of hormone treatment or tilting of trees. Different types and numbers of tracheids differentiated at different position in two-year-old disbudded defoliated stem cuttings of P. contorta in response to apically supplied IAA. No evidence for new tracheid differentiation was seen in control cuttings; however, the results suggest that neither cambial cell division nor tracheid differentiation were actually initiated by IAA. Directed transport of additional regulatory factors toward areas of high IAA concentration is formulated as a hypothesis to explain these observations. Gibberellic acid, (S)-abscisic acid and IAA inhibited tracheid differentiation when individually supplied to basal ends of P. contorta cuttings predisposed to differentiate new tracheids. Experiments with single intact needles on Pinus cembroides var. monophylla cuttings confirmed a previous interpretation that the mature pine needle, rather than the short-shoot apical meristem at its base, promotes tracheid differentiation in the stem.     

Diversity of bacteria associated with Bursaphelenchus xylophilus and other nematodes isolated from Pinus pinaster trees with pine wilt disease

The pinewood nematode (PWN), Bursaphelenchus xylophilus, has been thought to be the only causal agent of pine wilt disease (PWD), however, since bacteria have been suggested to play a role in PWD, it is important to know the diversity of the microbial community associated to it. This study aimed to assess the microbial community associated with B. xylophilus and with other nematodes isolated from pine trees, Pinus pinaster, with PWD from three different affected forest areas in Portugal. One hundred and twenty three bacteria strains were isolated from PWN and other nematodes collected from 14 P. pinaster. The bacteria strains were identified by comparative analysis of the 16S rRNA gene partial sequence. All except one gram-positive strain (Actinobacteria) belonged to the gram-negative Beta and Gammaproteobacteria. Most isolates belonged to the genus Pseudomonas, Burkholderia and to the family Enterobacteriaceae. Species isolated in higher percentage were Pseudomonas lutea, Yersinia intermedia and Burkholderia tuberum. The major bacterial population associated to the nematodes differed according to the forest area and none of the isolated bacterial species was found in all different forest areas. For each of the sampled areas, 60 to 100% of the isolates produced siderophores and at least 40% produced lipases. The ability to produce siderophores and lipases by most isolates enables these bacteria to have a role in plant physiological response. This research showed a high diversity of the microbial community associated with B. xylophilus and other nematodes isolated from P. pinaster with PWD.     

Vulnerability to cavitation differs between current‐year and older xylem: non‐destructive observation with a compact magnetic resonance imaging system of two deciduous diffuse‐porous species

Development of xylem embolism during water stress in two diffuse‐porous hardwoods, Katsura (Cercidiphyllum japonicum) and Japanese white birch (Betula platyphylla var. japonica), was observed non‐destructively under a compact magnetic resonance imaging (MRI) system in addition to conventional quantitation of hydraulic vulnerability to cavitation from excised stem segments. Distribution of white and dark areas in MR images corresponded well to the distribution of water‐filled/embolized vessels observed by cryo‐scanning electron microscopy in both species. Water‐filled vessels were observed in MR images as white areas in Katsura and as white dots in Japanese white birch, respectively, and embolisms could be detected as a change to dark areas. The increase in the relative embolized area (REA: %) in the cross‐sectional area of total xylem during water stress, which was estimated from the binarized MR images, was consistent with the hydraulic vulnerability curves of these species. From the non‐destructive MRI observations, cavitation induced by water stress was shown to develop earlier in 1‐ or 2‐year‐old xylem than in the current‐year xylem in both species; that is, the vulnerability to cavitation differs between vessels in the current‐year xylem and those in older annual rings.