Exogenous application of succinic acid enhances tolerance of Larix olgensis seedling to lead stress

Larix olgensis A. Henry(Changbai larch) is a productive commercial species and good candidate for afforestation in northeast China. It is widely planted in lead-stressed soils which can induce oxidative damage in this plant. Increasing tolerance to lead(Pb) stress is therefore of keen interest. A greenhouse experiment was conducted to identify the biomass, physiological responses and Pb accumulation of L. olgensis seedlings to Pb stress under succinic acid(SA) application and to explore the interaction of exogenous SA applications and stress resistance. L. olgensis seedlings were planted in Pb-stressed or unstressed haplic cambisols in pots. In Pb-contaminated soils the seedlings were treated daily with concentrations of SA solutions at a rate approximately equivalent to 0, 0.04,0.2, 1.0, or 2.0 mmol kg~(-1) of soil for 10, 20, and 30 days,respectively. Pb treatment induced damage in the seedlings and led to the inhibition of biomass accumulation in roots,stems and leaves, and a rise in Pb accumulation in fine roots and leaves. Malondialdehyde(MDA) content and electrolyte leakage in leaves significantly increased while peroxidase(POD) activities, soluble protein and photosynthetic pigment contents in leaves were all reduced.Physiological toxicity was promoted with increasing Pb treatment times. When Pb-stressed seedlings were exposed to SA(especially 10.0 mmol L-1 over 20 days), the physiological responses for Pb-only were reversed and the biomass of roots, stems, and leaves dramatically increased.SA facilitated Pb uptake in fine roots and leaves but more Pb accumulated in fine roots. The results demonstrate that exogenous SA alleviates Pb-induced oxidative injuries and improves the tolerance of L. olgensis seedlings to Pb stress.     

Nutrient retranslocation from the fine roots of <Emphasis Type="Italic">Fraxinus mandshurica</Emphasis> and <Emphasis Type="Italic">Larix olgensis</Emphasis> in northeastern China

Nutrient retranslocation in trees is important in nutrient budgets and energy flows in forest ecosystems. We investigated nutrient retranslocation in the fine roots of a Manchurian Ash (Fraxinus mandshurica) and a Larch (Larix olgensis) plantation in northeastern China. Nutrient retranslocation in the fine roots was investigated using three methods, specifically, nutrient concentration, the ratio of Ca to other elements (Ca/other elements ratio) and nutrient content. The method based on nutrient content proved most suitable when investigating nutrient retranslocation from fine roots of the two species. The nutrient-content-based method showed that there were retranslocations of N, P, K and Mg from the fine roots of Manchurian Ash, with retranslocation efficiencies of 13, 25, 65, and 38 %, respectively, whereas there were no Ca retranslocations. There were retranslocations of N, P, K, Ca and Mg from the fine roots of Larch, with retranslocation efficiencies of 31, 40, 52, 23 and 25 %, respectively.     

Aboveground biomass and nutrient allocation in an age-sequence of <Emphasis Type="Italic">Larix olgensis</Emphasis> plantations

Biomass and nutrient (N, P, K, Ca, Mg) stock in various aboveground tree components (stemwood, stembark, branches and leaves) were quantified in an age sequence of pure Larix olgensis planta- tions (20, 35, 53 and 69 years old) in Northeast China. The results show that the aboveground biomass allocation in various tree components was in the order of stemwood (62%-83%), branches (9%-21%), stembark (7%-11%) and leaves (1%-6%) for all stands. The proportion of stemwood biomass to total aboveground biomass increased whereas that of other tree components decreased consistently with stand age from 20 to 53 years old, but kept relatively constant with stand age from 53 and 69 years old. The nutrient allocation in various tree components generally followed the same pattern as the biomass allocation (i.e. stemwood > branches > stembark > leaves). The proportion of nutrient stock in leaves to total aboveground nutrient stock decreased consistently with increasing stand age, while that in stemwood increased with stand age from 20 to 53 years old but then decreased from 53 to 69 years old. The rate of nutrient removal for stands was estimated at different stand ages under different logging schemes, showing that the rate of nutrient removal would be unchanged when the rotation length was shortened to 20 years by the harvest of stem only, but greatly increased by the harvest of total aboveground biomass. The rate of nutrient removal would be a considerable reduction for all elements by debarking, especially for Ca.     

Screening and verification of the factors influencing somatic embryo maturation of <Emphasis Type="Italic">Larix olgensis</Emphasis>

With embryogenic callus of Larix olgensisis, we investigated the effects of inositol, glutamine, casein hydrolysate, carbohydrate, abscisic acid and silver nitrate concentration on the maturation of the somatic embryo. Three dominant factors emerged, and we developed a response surface model based on the Box–Behnken design. We defined the optimal conditions for the maturation of somatic embryos. The contents of abscisic acid, silver nitrate, sucrose and casein hydrolysis significantly affected the amount of maturing embryos, but inositol, maltose and glutamine had no effect. By establishing a response surface model with multiple factors, we predicted that the optimal number of L. olgensis somatic embryos was 204?±?4 g^?1 on basal medium, containing 18.28 mg L^?1 abscisic acid, 5.46 mg L^?1 silver nitrate and 82.67 g L^?1 sucrose. In the verification experiments, the addition of 20 mg L^?1 abscisic acid, 5 mg L^?1 silver nitrate and 80 g L^?1 sucrose to BM yielded an average of 202.06 somatic embryos per gram. These results should guide large-scale breeding of L. olgensis.     

Variation analyses of growth and wood properties of <Emphasis Type="Italic">Larix olgensis</Emphasis> clones in China

The growth traits (tree height, diameter at breast height, and stem straightness degree) and wood properties [wood density (WD), fiber length, fiber width, ash content (AC), lignin content, cellulose content, hemicellulose content (HEC), and holocellulose content] of 208 26-year-old Larix olgensis clones were analyzed. Except for WD and AC, there were significant differences (p < 0.01) for all traits among clones. The phenotypic coefficient of variation and repeatability of all traits were 9.34–35.33% and 0.218–0.930, respectively. Tree height and diameter at breast height showed significant positive correlation; however, the correlation coefficients among growth characteristics and wood properties were mostly not significant. Ten clones (L70, L56, L82, L90, L59, L91, L61, L92, L86, and L64) were selected as excellent clones under a selection rate of 5%, using tree height, diameter at breast height, and stem straightness degree as evaluation indexes, providing genetic gains of 28.69, 17.96, and 0.67%, respectively. Ten clones (L88, L305, L59, L66, L253, L304, L277, L298, L248, and L293) were selected as excellent clones using wood properties as an evaluation index, with a selection rate of 5%, providing genetic gains in WD, fiber length, fiber width, cellulose content, and HEC of 4.14, 3.64, 9.28, 6.77, and 9.61%, respectively. This study provides a theoretical basis for selecting excellent L. olgensis clones.     

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

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

Successful <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Populus tomentosa</Emphasis> with apple <Emphasis Type="Italic">SPDS</Emphasis> gene

The problem of salinized soils has become one of the most serious constraints to agricultural and forest productivity. With the purpose of enhancing salt stress tolerance of Populus tomentosa, we transformed this tree species with spermidine synthase （SPDS） genes derived from an apple by an Agrobacterium-mediated method. Four transgenic clones were confu＇med by PCR and Southern blot analysis. As well, the expression of introduced SPDS genes was analyzed by real-time quantitative PCR.     

Germination and seedling growth of <Emphasis Type="Italic">Dimorphandra jorgei</Emphasis> MF Silva and <Emphasis Type="Italic">Swartzia macrostachya</Emphasis> Benth. var. <Emphasis Type="Italic">riedelii</Emphasis> Cowan

Dimorphandra jorgei MF Silva (Caesalpinaceae) and Swartzia macrostachya Benth. var. riedelii Cowan (Fabaceae) are tree species from the southern Bahian Atlantic Forest, identified for the restoration of degraded areas. The objective of this research is to study their seed germination and seedling growth to develop simple methods for seedling production in small scale nurseries. Because the species have distinct dormancy and germination characteristics, the seeds were submitted to different treatments. D. jorgei seeds were immersed in hot water, scarified and stored for 12 weeks in the laboratory environment (25°C). S. macrostachya seeds were submitted to different combinations of drying and storage in the laboratory environment or refrigerator (4°C). Seedlings were submitted to two light treatments for 3 months: partial shade (52% of full sun) and full shade (12% of full sun). In D. jorgei, the highest germination occurred for scarified seeds (87%) and for seeds stored for 12 weeks and then scarified (69%). In S. macrostachya the undried control seeds had the highest germination, followed by that of the refrigeration storage treatment, without predrying (99% and 97%, respectively). The average values for the growth parameters were greater in full shade for D. jorgei, while S. macrostachya seedlings showed greater growth rates in partial shade. Two months growth is recommended before D. jorgei seedlings are of the sufficient size and leaf number for field planting. On the other hand, a period of 4 months is recommended for S. macrostachya, due to its relatively slow growth.     

Insecticidal properties of <Emphasis Type="Italic">Thymus persicus</Emphasis> essential oil against <Emphasis Type="Italic">Tribolium castaneum</Emphasis> and <Emphasis Type="Italic">Sitophilus oryzae</Emphasis>

Red flour beetle Tribolium castaneum (Herbst) and rice weevil Sitophilus oryzae (L.) are considered to be the major insect pests in storage. Essential oils from aromatic plants are recognized as proper alternatives to fumigants. Thymus persicus (Ronniger ex Rech. f.) is one of these plants that have medicinal properties and is indigenous to Iran. The essential oil was obtained from aerial parts of the plant and analyzed by GC and GC–MS. Carvacrol (44.69%) and thymol (11.05%) were the major constituents of the oil extracted. In this experiment, fumigant toxicity of the essential oil was studied against T. castaneum, S. oryzae at 27 ± 1°C and 60 ± 5% RH in dark condition. The adult insects were exposed to the concentrations of 51.9, 111.1, 207.4 and 370.4 μl/l air to estimate median lethal time (LT₅₀) values. The fumigant toxicity was increased in response to increased essential oil concentrations. The LT₅₀ values at the lowest and the highest concentrations tested were ranged from 28.09 to 13.47 h for T. castaneum, and 3.86 to 2.30 h for S. oryzae. It was found that S. oryzae adults were much more susceptible to the oil than T. castaneum. After 24 h of exposure, the LC₅₀ values (95% fiducial limit) for T. castaneum and S. oryzae were estimated to be 236.9 (186.27–292.81) and 3.34 (2.62–4.28) μl/l air, respectively. These results suggest that T. persicus essential oil merits further study as potential fumigant for the management of these stored-product insects.     

A novel cold-inducible promoter,P<Emphasis Type="Italic">ThCAP</Emphasis> from <Emphasis Type="Italic">Tamarix hispida</Emphasis>, confers cold tolerance in transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Our previous studies have revealed that the ThCAP gene plays a vital role in transgenic Populus (P. davidiana × P. bolleana) in response to cold stress. However, the regulatory mechanism of ThCAP gene expression has been unclear. In this study, the 5′ flanking region of the ThCAP promoter (PThCAP) was cloned using a genome-walking method. By analyzing cis-acting regulatory elements of PThCAP, a DRE motif and MYC and MYB elements were found to be located in the promoter. To identify the regulatory elements that control the expression of the ThCAP gene promoter, a series of deletion derivatives of PThCAP, P1–P5, from the translation start code (?1538, ?1190, ?900, ?718 and ?375 bp), were fused to the GUS reporter gene, and then each deletion was stably introduced into Arabidopsis thaliana plants. Deletion analysis of the promoter suggested that only the P2 fragment had strong GUS expression in leaves and roots of A. thaliana exposed to low temperature stress. These results suggest that this 290-bp region (?1190 to ?900 bp), as an important part in PThCAP, was associated with cold tolerance of A. thaliana. Our results provide evidence for the regulatory mechanism of ThCAP gene involved in the response to cold stress, and that the gene is promising candidate gene for genetic improvement of crops.     

Osmoregulators in <Emphasis Type="Italic">Hymenaea courbaril</Emphasis> and <Emphasis Type="Italic">Hymenaea stigonocarpa</Emphasis> under water stress and rehydration

The objective of this work was to evaluate the effect of different water deficiency and rehydration levels on the concentrations of osmoregulators in two plant species (Hymenaea courbaril and H. Stigonocarpa) in the Amazon. We adopted a 2 × 5 × 5 factorial system, referring to 2 species (H. courbaril and H. stigonocarpa) and 5 stages of hydration and rehydration. The five hydration and rehydration stages were established in: (1) Control treatment E0; (2) Plants with 13 days of stress after incubation—E13; (3) Plants with 26 days of stress E26; (4) The plants that were established after 26 days after incubation and rehydrated for two days (RD2); (5) rehydrated for two days (RD4). The plants that were established after 26 days after incubation and rehydrated for four days. The experiment totaled fifty young plants with five replicates. Biochemical measurements were performed at the beginning of the experiment (E0) at 13 (E13) and 26 (E26) days after the water stress, in which the plants were rehydrated, repeating the analyses after two (RD2) and four (RD4) days. Both species increased the sucrose concentration by 18%, with a decrease of 52% in starch content. The RD4 time presented the highest mean starch concentration (0.19 mmol g^?1 of the residue for H. courbaril and 0.27 mmol g^?1 of residue for H. stigonocarpa). Increased proline concentrations were recorded for controls until RD2 for both species. For glycine betaine, the highest increases in treatments E26 and RD2 were observed for the H. courbaril species. Our rehydration period was not sufficient for total recovery of pre-stress concentrations of all studied solutes.     

Bioremediation of CCA-treated wood by brown-rot fungi <Emphasis Type="Italic">Fomitopsis palustris</Emphasis>, <Emphasis Type="Italic">Coniophora puteana</Emphasis>, and <Emphasis Type="Italic">Laetiporus sulphureus</Emphasis>

This study evaluated oxalic acid accumulation and bioremediation of chromated copper arsenate (CCA)-treated wood by three brown-rot fungi Fomitopsis palustris, Coniophora puteana, and Laetiporus sulphureus. The fungi were first cultivated in a fermentation broth to accumulate oxalic acid. Bioremediation of CCA-treated wood was then carried out by leaching of heavy metals with oxalic acid over a 10-day fermentation period. Higher amounts of oxalic acid were produced by F. palustris and L. sulphureus compared with C. puteana. After 10-day fermentation, oxalic acid accumulation reached 4.2 g/l and 3.2 g/l for these fungi, respectively. Fomitopsis palustris and L. sulphureus exposed to CCA-treated sawdust for 10 days showed a decrease in arsenic of 100% and 85%, respectively; however, C. puteana remediation removed only 18% arsenic from CCA-treated sawdust. Likewise, chromium removal in F. palustris and L. sulphureus remediation processes was higher than those for C. puteana. This was attributed to low oxalic acid accumulation. These results suggest that F. palustris and L. sulphureus remediation processes can remove inorganic metal compounds via oxalic acid production by increasing the acidity of the substrate and increasing the solubility of the metals.An erratum to this article can be found at     

Greenhouse biofumigation with <Emphasis Type="Italic">Melia azedarach</Emphasis> controls <Emphasis Type="Italic">Meloidogyne</Emphasis> spp. and enhances soil biological activity

The need for environmentally friendly agricultural practices has led to the development of plant-based nematicides for root-knot nematode control. The efficacy of these botanicals has been tested primarily under laboratory and rarely under actual field conditions. Moreover, any side effects on non-target soil organisms that support soil biological activity are usually ignored. Herein, we evaluate the efficacy of Melia azedarach preparations against Meloidogyne spp. in a tomato greenhouse, by root gall examination and soil J2 enumeration. We also assessed side effects on soil microbes through PLFA analysis and microbivorous nematodes, and we quantified several plant growth parameters (e.g., fruit number and weight, root weight). Different treatments within the greenhouse included M. azedarach ripe fruit powder (MFP), ripe fruit water extract (MWE) and furfural, one of the principal active ingredients of M. azedarach and previously known to exhibit fumigant nematicidal activity. Results were compared to those obtained with the commercial nematicide oxamyl (Vydate^® 10 SL) and an untreated control. All treatments were repeated every 20 days throughout the cultivation period. MFP and MWE suppressed Meloidogyne spp. often at the same levels obtained by furfural and oxamyl treatments and enhanced soil biological activity, as indicated by the proliferation of soil microbes and microbial feeding nematodes. Furfural and oxamyl adversely affected the soil community, especially the free-living nematodes. Moreover, furfural was phytotoxic to tomato plants in spite of its natural origin.     

Enzymatic saccharification and ethanol production of <Emphasis Type="Italic">Acacia mangium</Emphasis> and <Emphasis Type="Italic">Paraserianthes falcataria</Emphasis> wood,and <Emphasis Type="Italic">Elaeis guineensis</Emphasis> trunk

We examined the saccharification and fermentation of meals from Acacia mangium wood, Paraserianthes falcataria wood, and Elaeis guineensis trunk. The levels of enzymatic hydrolysis of cellulose and ethanol production were highest for P. falcataria wood and lowest for A. mangium wood. Ultrasonication pretreatment of meal further increased the rates of hydrolysis and ethanol production in meal from P. falcataria wood. Through this pretreatment, hemicelluloses (xylan and xyloglucan) and cellulose were released in the meal from P. falcataria wood. Loosening of hemicellulose associations can be expected to make P. falcataria wood more useful for bioethanol production.     

<Emphasis Type="Italic">In vitro</Emphasis> morphogenic response of leaf sheath of <Emphasis Type="Italic">Phyllostachys bambusoides</Emphasis>

Nodal segments from secondary branches of saplings of Phyllostachys bambusoides were inoculated in MS medium to assess the in vitro morphogenic response of leaf sheath through the induction to callogenesis by Picloram(4-amino-3,5,6-trichloropicolinic acid) at different concentrations of carbohydrate under the same conditions with presence or absence of luminosity.In our experiment,secondary explants were kept in MS medium containing 8.0 mg·L-1 of Picloram for the callus formation.Calluses were transferred in MS medium supplemented with sucrose,fructose and glucose(control,2%,4% and 6%).Results show that Picloram induced the callogenesis in leaf sheath.The secondary embryogenesis was formed in yellow-globular callus.The sucrose as carbohydrate source in the absence of light was more efficient to induce rhizogenesis.Glucose was more efficiency in the presence of light.Callogenic induction and further embryogenesis evidenced the competence and determination of leaf sheath cells.     

Effectiveness of Neem (<Emphasis Type="Italic">Azadirachta indica</Emphasis>) insecticides against Brassica pod midge (<Emphasis Type="Italic">Dasineura brassicae</Emphasis> Winn<Emphasis Type="Italic">.</Emphasis>)

The efficiency of the botanical insecticide (BI) NeemAzal T/S (containing 1% Azadirachtin A), on the basis of azadirachtin applied in a dose of 20 g a.i. ha⁻¹ against Brassica pod midge (Dasineura brassicae), has been monitored for 4 years. The biological efficiency of BI was compared with the efficiency of some synthetic insecticides. It was ascertained that BI was very efficient in decreasing the number of damaged oilseed rape pods (ranging from 56.5 to 85.9% compared to untreated plants) and its efficiency was comparable with synthetic insecticides based on Chloronicotinyl (Thiacloprid) and Neonicotinoid (Acetamiprid). BI’s efficiency was, in some years, even significantly higher compared to pyrethroid (λ-cyhalothrin). The high biological efficiency of azadirachtin that we ascertained was significantly reflected in increasing potential crop yields. The yield increase of azadirachtin ranged between 9.3 and 19.4% compared to the control sample. Azadirachtin showed the highest yield for the whole time of experimentation, and in some years the yield increase was significantly higher compared to some synthetic agents.     

Phenotypic plasticity blurs ecotypic divergence in the response of <Emphasis Type="Italic">Quercus coccifera</Emphasis> and <Emphasis Type="Italic">Pinus halepensis</Emphasis> to water stress

The Mediterranean evergreen woody plants Quercus coccifera and Pinus halepensis grow in a range of environments where selection by drought, heat and high irradiance can drive genetic and phenotypic differentiation of populations. However, the role of these stresses in filtering out maladaptive genotypes remains unknown. We hypothesize that this filtering is an important process for woody Mediterranean species due to their low phenotypic plasticity reported in previous studies. We have studied the response of saplings of Q. coccifera and P. halepensis, originating from two contrasting populations (a rock outcrop and a garrigue formation), to water stress. Isozyme characterization of genetic diversity was done to determine whether populations were genetically distinct. Water response analysis was based on water relations, gas exchange, chlorophyll a fluorescence, pigment content, antioxidant status and morphological and structural parameters. Ecotypic differentiation was found for both Q. coccifera and P. halepensis populations, with a higher population isozyme similarity and a higher frequency of dominance of a few genotypes at the rock outcrop in both the species. P. halepensis exhibited small but significant differences between populations for plastic responses to water, with lower phenotypic plasticity in saplings from the rock outcrop. Although it was not found in Q. coccifera, this pattern suggests that ecotypic differentiation rendering stress-tolerant ecotypes involves a decreased plasticity. Phenotypic plasticity was not high but it explained over 75% of the total variability among individual plants. Thus, and although evidence for ecotypic divergence was found in both the species, saplings were plastic enough to blur ecotypic differentiation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Expression of a scorpion toxin gene <Emphasis Type="Italic">BmKit</Emphasis> enhances the virulence of <Emphasis Type="Italic">Lecanicillium lecanii</Emphasis> against aphids

Lecanicillium lecanii has been developed as biopesticides and widely used in the biological control of several insects in agricultural practice. However, the poor efficacy has blocked its application. The genetic manipulation has been proved as a useful tool to improve the virulence of entomopathogenic fungus. In this study, a scorpion toxin gene (BmKit) from Buthus martensi was cloned and transferred into L. lecanii and then its resulting activity against cotton aphids (Aphis gossypii) was assessed. The results showed that the engineered strain BmKit-12 grew significantly quicker than WT in host insects. The median lethal concentration (LC₅₀) for BmKit-12 was 7.1-fold lower than that for WT, and the median survival time (LT₅₀) for BmKit-12 was reduced by 26.5 % compared with that for WT. Although the conidial yield was reduced by 31.4 % on insect cadavers, the mycelia growth, sporulation, and conidia germination of BmKit-12 on plates were not significantly different from WT. Considering the lower amount of mycelia for BmKit-12 in host insects in late growth stage (fungal sporulation stage), we think the expression of BmKit may not affect the capability of sporulation. In conclusion, the expression of BmKit gene can significantly enhance the pathogenicity of L. lecanii against cotton aphids in a relatively cost-effective way.     

Fine root biomass and morphology of <Emphasis Type="Italic">Pinus densiflora</Emphasis> under competitive stress by <Emphasis Type="Italic">Chamaecyparis obtusa</Emphasis>

The fine root (diameter ≤2.0 mm) biomass and morphology of Japanese red pines (Pinus densiflora) grown under different aboveground conditions (i.e., high and low competitive environments) were examined in a pine–cypress mixed forest. All P. densiflora subject trees were about 40 years old, and the aboveground condition (i.e., size) of red pines appeared to be influenced by the surrounding Japanese cypress (Chamaecyparis obtusa). Smaller P. densiflora exhibited lower fine root biomasses, shorter root lengths, and lower root tip densities, but longer specific root lengths and higher specific root tip densities relative to larger pines. These results suggest that P. densiflora may adjust the morphological traits of fine roots to the different conditions in biomass allocation to fine roots of individuals with different aboveground growth.