Changes in freezing tolerance, plasma membrane H+-ATPase activity and fatty acid composition in Pinus resinosa needles during cold acclimation and de-acclimation

It has previously been suggested that plasma membrane ATPase (PM H+-ATPase, EC 3.6.1.3.) is a site of incipient freezing injury because activity increases following cold acclimation and there are published data indicating that activity of PM H+-ATPase is modulated by changes in lipids associated with the enzyme. To test and extend these findings in a tree species, we analyzed PM H+-ATPase activity and the fatty acid (FA) composition of glycerolipids in purified plasma membranes (PMs) prepared by the two-phase partition method from current-year needles of adult red pine (Pinus resinosa Ait.) trees. Freezing tolerance of the needles decreased from -56 degrees C in March to -9 degrees C in May, and increased from -15 degrees C in September to -148 degrees C in January. Specific activity of vanadate-sensitive PM H+-ATPase increased more than two-fold following cold acclimation, despite a concurrent increase in protein concentration. During de-acclimation, decreases in PM H+-ATPase activity and freezing tolerance were accompanied by decreases in the proportions of oleic (18:1) and linoleic (18:2) acids and increases in the proportions of palmitic (16:0) and linolenic (18:3) acids in total glycerolipids extracted from the plasma membrane fraction. This pattern of changes in PM H+-ATPase activity and the 18:1, 18:2 and 18:3 fatty acids was reversed during cold acclimation. In the PM fractions, changes in FA unsaturation, expressed as the double bond index (1 x 18:1 + 2 x 18:2 + 3 x 18:3), were closely correlated with changes in H+-ATPase specific activity (r2 = 0.995). Changes in freezing tolerance were well correlated with DBI (r2 = 0.877) and ATPase specific activity (r2 = 0.833) in the PM fraction. Total ATPase activity in microsomal fractions also closely followed changes in freezing tolerance (r2 = 0.969). We conclude that, as in herbaceous plants, simultaneous seasonal changes in PM H+-ATPase activity and fatty acid composition occur during cold acclimation and de-acclimation in an extremely winter hardy tree species under natural conditions, lending support to the hypothesis that FA-regulated PM H+-ATPase activity is involved in the cellular response underlying cold acclimation and de-acclimation.     

Seasonal water stress and the resistance of Pinus yunnanensis to a bark-beetle-associated fungus

We examined the influence of seasonal water stress on the resistance of Pinus yunnanensis (Franch.) to inoculation with Leptographium yunnanense, a pathogenic fungus associated with the aggressive bark beetle, Tomicus n. sp. Experiments took place between October 1997 and November 1999 in two plots located at the top and at the foot of a hill in Shaogiu, China, a region characterized by dry winters and wet summers. Following isolated and mass fungal inoculations, we observed the reaction zone length, fungal growth in the phloem, and the occlusion, blue-staining and specific hydraulic conductivity of the sapwood. Measurements of soil and needle water contents and predawn needle water potentials confirmed that trees were subject to mild water stress during winter, especially at the drier hilltop site. Measures of tree resistance to fungal infection of phloem and sapwood were congruent and indicated that trees were most susceptible to inoculation during the wet summer, especially at the lower-elevation plot. Specific hydraulic conductivity decreased after inoculation in summer. The results indicate that mild seasonal water stress is not likely responsible for the recent extensive damage to young P. yunnanensis stands by Tomicus n. sp. in the vicinity of our study plots. Rather, the results suggest that mild water stress enhances tree resistance to fungal pathogens associated with Tomicus n. sp.     

Physiological responses of needles of Pinus massoniana elite families to phosphorus stress in acid soil

Pinus massoniana Lamb. is a major timber species widely planted in the South China, where the soil is acidic and deficient in phosphorus (P) due to fixation by aluminum and iron. Understanding the physiological responses to rhizospheric insoluble P is essential for enhancing plantation productivity. Thus, a sand culture experiment was conducted with four levels of P treatment (0, 5, 20 g insoluble P and 10 g soluble P), and 11 P. massoniana elite families. Physiological responses were measured after two months of stress. Compared to the normal soluble P treatment, the insoluble P treatment significantly reduced the proline content and the APase activity in the needles, while it significantly increased the catalase activity by 1.3-fold and malondialdehyde content by 1.2-fold. Soluble protein content was unaffected by the treatments, but chlorophyll content was significantly lower in P-deprived treatment compared with soluble and insoluble P treatments. These physiological responses also exhibited highly significant variation among families (p < 0.01). The findings suggest that increased catalase activities in the presence of insoluble P might be involved in the activation of an anti-oxidation defense mechanism that scavenges the reactive oxygen species elicited by the stress. And this response has a strong genetic control that can be exploited to identify desirable genotypes.     

Ice nucleation and frost resistance of Pinus canariensis seedlings bearing needles in three different developmental states

Frost resistance and ice formation in different developmental states of needles of P. canariensis seedlings were assessed. Regrowth after frost damage was used to determine the overall frost survival capacity. Two distinct freezing exotherms (E1, E2) were registered. E1 was between ?1.7 and ?2.0 °C. Initial frost damage (LT₁₀) was 1.5–2.7 °C below E1. E2 was between ?5.6 and ?6.0 °C, and either corresponded with LT₅₀ or occurred in between LT₁₀ and LT₅₀. Current year needles were less frost resistant than 1-year-old needles. The overall recuperation capacity of seedlings revealed that frost survival may be underestimated when only needle damage is assessed. Freezing of seedlings with or without roots had no effect on the frost resistance of needles but recuperation capacity was significantly affected. Seedlings survived ?10 °C during summer indicating that they withstand the lowest naturally occurring frosts in Tenerife.     

Steady-state nutrition of Pinus resinosa seedlings: response to nutrient loading, irrigation and hardening regimes

To test effects of fertilization on late-season growth and nutrient content of container-grown red pine (Pinus resinosa Ait.), seedlings were subjected to a factorial combination of two fertilization regimes (exponentially increasing concentrations providing 25 (1N) and 75 (3N) mg N seedling(-1), respectively) and two irrigation regimes (well-watered and water-stressed) followed by drought-hardening or nonhardening. The fertilization treatments gave rise to steady-state nutrition in the seedlings during the prehardening phase. The 3N treatment increased macronutrient uptake significantly more than dry matter production, particularly in the roots, giving rise to what has been called luxury consumption of nutrients, or nutrient loading. Nutrient loading was higher in well-watered seedlings than in water-stressed seedlings. Among well-watered seedlings, relative growth rate of nutrient-loaded seedlings was more sensitive to drought during the hardening phase than that of nonloaded seedlings. However, when watering was resumed at the end of the hardening treatment, the relative growth rate of the nutrient-loaded seedlings (421%) exceeded that of the nonloaded seedlings (213%). Nitrogen accumulation was also higher in nutrient-loaded seedlings than in nonloaded seedlings during the post-hardening phase.     

Organ-dependent induction of systemic resistance and systemic susceptibility in Pinus nigra inoculated with Sphaeropsis sapinea and Diplodia scrobiculata

Systemic induced resistance (SIR) is a well-known host defense mechanism against pathogen attack in herbaceous plants, but SIR has only recently been documented in conifers. We tested if inoculation of Austrian pine (Pinus nigra Arnold) with Sphaeropsis sapinea (Fr.:Fr.) Dyko and Sutton or Diplodia scrobiculata de Wet, Slippers and Wingfield results in SIR or systemic induced susceptibility (SIS) to subsequent colonization by S. sapinea. Induction at the stem base resulted in significant (P < 0.01) SIR in the upper stem, and induction in the upper stem resulted in significant (P < 0.05) SIR at the stem base, indicating that SIR is bidirectional in Austrian pine. However, inoculation at the stem base resulted in significant (P < 0.01) SIS in shoot tips, demonstrating that, in the same host species, the expression of resistance can be organ-dependent, resulting in either SIR or SIS depending on the site of challenge infection. Systemic induced resistance in the stem was associated with induced lignification, supporting a potential role for this defense mechanism in disease resistance. Systemic induced susceptibility has been documented before, but this is the first demonstration of organ-dependent expression of both SIR and SIS in a tree or any other plant.     

Cold hardiness of interspecific hybrids between Pinus strobus and P. wallichiana measured by post-freezing needle electrolyte leakage

Interspecific hybrids between eastern white pine (Pinus strobus L.) and Himalayan blue pine (P. wallichiana A. B. Jacks.) were developed in Ontario, Canada, to introduce blister rust (Cronartium ribicola Fisch.) resistance genes to P. strobus. There is concern that introducing blister rust resistance has resulted in reduced cold hardiness of the progeny compared with non-hybridized eastern white pine. To test the efficacy of backcrossing with P. strobus to improve cold hardiness, 1-year-old seedlings from hybrid crosses differing in P. strobus genome composition were artificially freeze-tested. In Experiment 1, unhardened seedlings were allowed to acclimate to progressively lower temperatures in a growth room, whereas in Experiment 2, seedlings were hardened outdoors under natural weather conditions in Sault Ste Marie, Ontario. Needle cold injury was determined by calculating relative electrical conductivity based on post-freezing electrolyte leakage. Results indicated that needle fascicles from unhardened seedlings of all genotypes in the greenhouse tolerated -5 degrees C for 3 hours with little or no injury. Cold hardiness increased in parallel with declining growth room minimum temperature over the 7-week period of hardening. Cold hardiness was improved for hybrid crosses with increased Pinus strobus genome composition in Experiment 2, but the results were less conclusive in Experiment 1.     

水分胁迫对油松和樟子松抗旱性影响

以油松和樟子松2 a生苗木为材料,通过不同梯度水分胁迫处理,研究辽西地区油松和樟子松抗旱性生理特征变化,结果表明:在各水分条件处理下,油松2 a生苗木MDA含量均小于樟子松,但CAT和Pro含量均高于樟子松,说明油松苗期抗旱性更强。在40%FMC处理时,油松MDA、CAT和Pro含量都小于对照,说明该水分水平未能使苗木出现干旱状态;而樟子松MDA、CAT和Pro含量都高于对照,苗木已经出现干旱状态。在30%FMC和20%FMC处理时,油松和樟子松苗木MDA、CAT和Pro含量都随水分胁迫程度加剧而增加。在干旱半干旱辽西地区培育油松和樟子松苗木时,土壤水分临界值应分别大于田间最大持水量40%和50%。     

Fall fertilization of Pinus resinosa seedlings: nutrient uptake, cold hardiness, and morphological development

? Fall fertilization may increase plant nutrient reserves, yet associated impacts on seedling cold hardiness are relatively unexplored.

? Bareroot red pine (Pinus resinosa Ait.) seedlings in north-central Minnesota, USA were fall fertilized at the end of the first growing season with ammonium nitrate (NH₄NO₃) at 0, 11, 22, 44, or 89 kg N ha^?1. Seedling morphology and cold hardiness [assessed by freeze induced electrolyte leakage (FIEL)] were evaluated six weeks after fertilization and following the second growing season.

? Seedling height and number of needle primordia increased with fertilizer rate for both sampling years. Seedlings fertilized with 44 and 89 kg N ha^?1 attained target height (15 cm) after the second growing season. Shoot and root N concentration increased after the first growing season in fall fertilized seedlings compared to controls. Fall fertilized seedlings had lower FIEL (i.e., increased cold hardiness) compared to controls when tested at ?40 °C after the first growing season, but no significant differences in FIEL of control and fertilized seedlings were observed after the second growing season.

? Results suggest that fall fertilization of red pine seedlings can help render desired target height in the nursery, while maintaining or increasing cold hardiness levels.

     

油松针叶枯黄原因调查研究

调查结果表明，承德避署山庄及其周边地区的油松针叶大量枯黄，侧枝枯死或整树枯死是由于松大蚜的严重危害所致。     

Influence of drought stress and low irradiance on plant water relations and structural constituents in needles of Pinus ponderosa seedlings

The influence of low light on tolerance to prolonged drought was tested on unshaded and shaded seedlings of ponderosa pine (Pinus ponderosa var. scopulorum Dougl. ex Laws.). Unshaded seedlings of P. ponderosa var. ponderosa were also drought stressed to compare varietal responses to drought. The maximum irradiance received by shaded seedlings was 10% of full light. Seedlings were progressively drought stressed until predawn water potentials (Psi(x)) were -5.0 MPa. Relative water content (RWC) and the reciprocal of Psi(x) were analyzed by means of an unusual application of the pressure-volume relationship for determination of RWC of the apoplast (RWC(a)), osmotic potential at full turgor (Psi(oft)), and ratio of fully turgid weight to dry weight. Major varietal differences in drought response were in RWC(a) and needle cellulose content. The shaded seedlings showed tissue damage at relative water contents < 60%, and were killed by water deficits from which unshaded seedlings recovered. Correspondingly, shaded plants had significantly higher cell volume/cell mass ratio, Psi(oft), less cellulose in needle tissue, and lower RWC(a) than unshaded plants. These differences suggest that low irradiance restricts drought adaptation in ponderosa pine.     

Modelling the diameter distribution of Pinus sylvestris, Pinus nigra and Pinus halepensis forest stands in Catalonia using the truncated Weibull function

Parameter prediction models for the diameter distribution ofPinus sylvestris L., Pinus nigra Arn. and Pinus halepensis Mill.in Catalonia were developed using the truncated Weibull functionas the theoretical distribution. The parameter models allowone to use individual-tree models in the simulation of standdevelopment when only stand-level data are collected in forestinventories. Parameter models for the diameter distributionof stand basal area were developed. The data consisted of permanentsample plots from the Spanish National Forest Inventory in Catalonia.A total of 1780 empirical distributions of P. sylvestris, 1204distributions of P. nigra and 1535 distributions of P. halepensiswere used as modelling data. The empirical data represent left-truncateddistributions, as the smallest diameter measured in the fieldwas 7.5 cm. Two different approaches, namely, regression (two-stepmethod) and optimization approach (one-step method), were usedto find the coefficients of the parameter models. In the two-stepmodelling method, the Weibull parameters were first estimatedseparately for every empirical distribution by maximizing thelog-likelihood function of the Weibull density function. Inthe second-step, regression analysis was used to find the relationshipbetween Weibull parameters and stand basal area, number of treesper hectare and elevation of the site. The one-step method usedoptimization to find such coefficients for the parameter models,which minimized the mean of the squared differences betweenempirical and predicted cumulative tree frequencies in the wholemodelling data. The one-step optimization method performed betterthan the two-step regression method for all tree species. Theparameter prediction models developed in this study enable theprediction of the diameter distribution of P. sylvestris, P.nigra and P. halepensis in Catalonia from limited stand information.     

Micropropagation of Pinus densiflora and the evaluation of nematode resistance of regenerated microshoots in vitro

To accelerate the breeding and selection of Pinus densiflora Siebold and Zucc. resistance to pine wilt disease, a micropropagation system was established and nematode resistance evaluated in vitro. Cotyledon-hypocotyl explants from 28-day-old seedlings were first cultured on Gresshoff and Doy medium supplemented with 4.0 mg L^-1 6-benzyladenine and 0.2 mg L^-1 a-naphthaleneacetic acid(NAA) to stimulate the formation of buds. Induced buds were subsequently subcultured on Gupta and Durzan medium supplemented with 0.1%(w/v)activated charcoal for elongation. Stem sections derived from shoots were used as explants for the further multiplication. Roots were formed from shoots transferred to woody plant medium containing 0.2 mg L^-1 NAA for4 weeks. The nematode resistance test showed that symptoms in micropropagated shoots after infection with pine wood nematode(PWN) were similar to those in plants infected in the field. The wilting rate varied from 20 to100% among different clones 18 days after inoculation.The most susceptible clone was Clone 6-4 with a 100%wilting rate, while Clone 8-4 showed a relatively high resistance with a 20% wilting rate. The number of nematodes recovered from Clone 8-4 shoots was significantly lower(P = 0.05) than from Clones 5-10 and 16-4. This work contributes to the breeding of PWN resistance in P.densiflora.     

Responses of Pinus massoniana and Pinus taeda to freezing in temperate forests in central China

Abstract

Masson pine (Pinus massoniana Lamb.), a native species widely distributed in temperate forests in central China, and Loblolly pine (Pinus taeda L.), an exotic tree species introduced to China from southeastern United States, are dominant evergreen conifers that play a pivotal role in maintaining forest structure and functions for the region. We examined the effects of freezing on these species with chlorophyll fluorescence and electrolyte leakage using both field- and laboratory-based experiments in September 2009 and January 2010, respectively. We found that freezing could cause a greater impact on the Loblolly pine than the Masson pine. Although the two species showed similar values of F _v /F _m and electrolyte leakage before freezing, the Masson pine needles showed lower F _v /F _m and higher electrolyte leakage ratios than those of the Loblolly pine when treated in low temperatures (?15 to 0°C). We also found that cold-acclimation was crucial for both species to adapt to low temperatures with the F _v /F _m ratio decreased approximately by 80% in the first freezing hour for the non-acclimated needles of both species while the cold-acclimated needles showed little changes in the F _v /F _m ratio. This finding is also supported by our measurements of electrolyte leakage. These results suggest that the Loblolly pine could be more susceptible to freezing damages than the Masson pine in central China.     

Variations in the content of stress proteins in the needles of common pine (Pinus sylvestris L.) within an annual cycle

This investigation has focused on the content of heat shock proteins (Hsps) and dehydrins (dhn) in the needles of Pinus sylvestris L. (common pine) depending on environmental conditions and on adaptation of photosynthetic activity. Unlike the case of photosynthetic activity, no relationships have been revealed between variations in environmental conditions over 2 years of observations and variations in Hsps content. It has been discovered that constitutive and stress forms of Hsp70, Hsp60 and dhn are accumulated in the cold season of the year, whereas for Hsp17.6 and Hsp101 this accumulation occurs in the warm season. Seasonal air temperature variations influenced the content of Hsps; variations of temperature and moisture conditions influenced the content of dhn. The supposition is made that accumulation of Hsp70, Hsp60 and dhn in the needles may be the factor and/or the marker of the autumn and spring adaptation of the photosynthetic apparatus, while accumulation of Hsp17.6 and Hsp101 is the marker of summer “heat” adaptation.     

Theoretical modeling of the effects of temperature and moisture content on the acoustic velocity of Pinus resinosa wood

To investigate the effects of temperature and moisture content(MC) on acoustic wave velocity(AWV)in wood,the relationships between wood temperature,MC,and AWV were theoretically analyzed.According to the theoretical propagation characteristics of the acoustic waves in the wood mixture and the differences in velocity among various media(including ice,water,pure wood or oven-dried wood),theoretical relationships of temperature,MC,and AWV were established,assuming that the samples in question were composed of a simple mixture of wood and water or of wood and ice.Using the theoretical model,the phase transition of AWV in green wood near the freezing point(as derived from previous experimental results) was plausibly described.By comparative analysis between theoretical and experimental models for American red pine(Pinus resinosa) samples,it was established that the theoretically predicted AWV values matched the experiment results when the temperature of the wood was below the freezing point of water,with an averageprediction error of 1.66%.The theoretically predicted AWV increased quickly in green wood as temperature decreased and changed suddenly near 0 °C,consistent with the experimental observations.The prediction error of the model was relatively large when the temperature of the wood was above the freezing point,probably due to an overestimation of the effect of the liquid water content on the acoustic velocity and the limited variables of the model.The high correlation between the predicted and measured acoustic velocity values in frozen wood samples revealed the mechanisms of temperature,MC,and water status and how these affected the wood(particularly its acoustic velocity below freezing point of water).This result also verified the reliability of a previous experimental model used to adjust for the effect of temperature during field testing of trees.     

Diversity and identification of fungi associated with needles of Pinus radiata in Tasmania

Needle fungi have been extensively studied in conifers but rarely in Pinus radiata. Previous studies of P. radiata have been based on fungal isolation and not direct PCR detection from needles. This research was a component of a study examining factors linked to spring needle cast (SNC) in Tasmanian P. radiata plantations and aimed to identify as many as possible of the fungal species commonly associated with P. radiata needles in Tasmanian plantations. Needle samples were collected from 13 sites representative of the range of sites in which P. radiata is grown in Tasmania. Fungi were detected by a direct PCR approach and identified using barcode sequences from a reference collection of isolates from Tasmania, mainland Australia and New Zealand as well as sequences from the international nucleotide sequence databases. The total number of molecular operational taxonomic units (MOTUs) was 152, with 127 detected by direct PCR and sequencing, and only 35 operational taxonomic units (OTUs) isolated by culture-based methods. Dothideomycetes was the most diverse class detected in this study, with many MOTUs detected by direct PCR and not isolated. Leotiomycetes was the second-most diverse class and Sordariomycetes third, with several OTUs frequently isolated but rarely or not at all detected by direct PCR. DNA sequence data facilitated the discrimination and identification of OTUs, but some effort was required to avoid confusion caused by poorly identified isolates in public DNA databases. This is the most comprehensive study yet of fungi associated with pine needles in Australia and highlights the hitherto unrecognised diversity of Teratosphaeriaceae in P. radiata.     

Histology of Diplodia pinea in diseased and latently infected Pinus nigra shoots

Diplodia tip blight is a serious disease of >30 conifer species worldwide. Symptoms are particularly severe on non‐native, two‐needled Pinus species, and typically include stunted, necrotic needles and shoots and a general decline of the tree. Latent Diplodia pinea infections occur in current‐year shoots of some symptomless pines, and in some apparently healthy current‐year shoots of diseased pines. Latent infections also occur in symptomless terminal buds. A histological approach was used to investigate the nature of latent infections in shoot stems and terminal buds of landscape Pinus nigra. Fungal colonization was compared in healthy, diseased and latently infected tissues. A nested‐PCR technique that is specific for D. pinea was used to differentiate latently infected tissues from uninfected ones. Latent D. pinea infections were localized in the outer stem cortex, usually in the vicinity of needle scales at leaf axes. In contrast, pathogenic D. pinea infections were characterized by fungal colonization throughout the shoot stem tissues, even very early in symptom development. The presence of necrophylatic periderms in two of the latently infected samples suggests that host defences play a role in the production and maintenance of latent stem infections. Latent infections of terminal buds appeared to originate from the distal bud scales of axillary buds in the terminal bud cluster, and not from the subtending shoot. Fungal tissues were never observed inside asymptomatic, PCR‐negative shoots.     

Differential effects of nutrient availability on the secondary metabolism of Austrian pine (Pinus nigra) phloem and resistance to Diplodia pinea

Nutrient availability can modulate the incidence and severity of plant diseases. We examined the effects of fertility on (i) host resistance and (ii) secondary metabolite production in the Pinus nigra Arnold –Diplodia pinea (Desm.) Kickx pathosystem. Following preconditioning of trees to one of three fertility levels for one growing season, phloem tissue of one branch was harvested for chemical analyses while another branch on the same whorl was inoculated with the fungus to assess resistance. Nutrient availability had concave and convex quadratic effects on concentrations of total monoterpenes and total phenolics respectively, but had no effect on fungal lesion lengths. Lesion lengths were negatively correlated with lignin, suggesting that this compound is an important factor in resistance. Counteracting effects of nutrient availability on phenolic and terpenoid pathways may explain why fertility had no effect on expression of disease resistance.     

Morphology of epicuticular waxes in Pinus pinea needles in relation to season and pollution-climate

Micromorphological change of the epicuticular wax structure in Pinus pinea needles was analysed by scanning electron microscope and the relationships between the rates of wax weathering over seasons and the pollution-climate at one urban and two rural sites arediscussed.