CTAB-silica Method for DNA Extraction and Purification from Castanea mollissima and Ginkgo biloba

A new method CTAB-silica for DNA extraction and purification from the leaves and buds of Castanea mollissima and Ginkgo biloba was tested. The method is based on the silica-based purification protocol developed by Boom et al. (1990). By modifying the protocol, plant genome DNA could be extracted easily from dormant buds, mature leaves, and other parts of plant. Our results showed that the purified DNA was of high purity and could be analyzed by PCR. Furthermore, this CTAB-silica method took much less time for a successful DNA purification process compared to the traditional methods (CTAB and SDS). By our method,the suitable DNA can be extracted and purified from over 10 plant samples by one person in an hour.     

Methodological comparison of DNA extraction from Holcocerrus hippophaecolus （Lepidoptera： Cossidae） for AFLP analysis

Amplified fragment length polymorphism （AFLP） is a powerful DNA fingerprinting technique for studying genetic relationships and genetic diversity in insects. However, the crucial prerequisite for AFLP analysis is to extract DNA of high quality. In this study, we evaluate four different protocols （SDS method, improved SDS method, CTAB method and a complex method with SDS and CTAB） for isolating DNA from the seabuckthorn carpenter moth （Holcocerrus hippophaecolus （Lepidoptera： Cossidae））. The results indicate that the CTAB method does not produce DNA suitable for AFLP analysis. The SDS method and the complex method with SDS and CTAB are comparatively time-consuming and resulted in low yields of DNA and were therefore not used for AFLP assay. The improved SDS method is recommended for preparing DNA templates from H. hippophaecolus for AFLP analysis.     

Comparisons of extraction and purification methods of soil microorganism DNA from rhizosphere soil

Microorganism DNA of rhizosphere soil from Pinus koraiensis and Pinus sylvestriformis were extracted by proteinase K based on SDS method, CTAB method, PVP （polyvinylpolypyrrolidone） method, and freezing and thawing method and the crude DNA from rhizosphere soil were purified by dialysis method, silver beads absorption method, and squeezing DNA gel method. The results of different extracting and purifying methods were compared and evaluated. Results indicated that the best method of extraction for microorganism DNA in rhizosphere soil was proteinse K based on SDS method with high salt concentration of 1.0% （w/v） NaCl, which could effectively eliminate humic acids and other impurities. The dialysis method was suitable to purify DNA from rhizosphere soil because of effectively removing brown matters and humic acids and the purified products were suited to PCR amplification. Squeezing DNA gel method was also a good purification method with the advantage of inexpensive in cost and efficient in use.     

Biological characters and rDNA ITS sequences of pathogen of poplar leaf blight

DNA was extracted from the strain of pathogen of poplar leaf blight using a modified CTAB method. ITS sequence （601bp） was initially amplified from the pathogen by using the universal primers ITSl and ITS4 （registered No, DQ011257）. Comparing to the nucleotide sequences acquired from GenBank database, the strain is clustered into the homogeneity with Alternaria alternate （AY787684） and Alternaria alternate （AY354228）, with a homology of 98%, thus the strain was checked as Alternaria alternata （Fr.） Keissler. The optimal conditions for conidia germination and mycelium growth of the pathogen were tested, The optimal temperature for conidia germinating and mycelium growth is 25℃, and the optimal pH value is 6. Mycelium grows rather slowly at 10℃ and 30℃ and growth stops at above 35 ℃. Among the six culture mediums tested, PDA ＋ poplar leaf juice medium is most favorable for mycelium growth.     

Comparison of methods for extracting high-throughput sequencing RNA from Korean pine seeds

Korean pine(Pinus koraiensis Sieb. et Zucc.) is an ecologically and economically important tree species in East Asia. Molecular studies of seed development of this species are limited due to the lack of effective RNA extraction protocols. This study aimed to obtain an effective method to extract high-quality RNA from Korean pine seeds. The TRIzol kit and CTAB methods were used to extract the total RNA from Korean pine seeds at different developmental stages. The bands of RNA extracted by CTAB were not clear, whereas the bands of RNA extracted by the TRIzol kit were brighter and clearer, indicating higher quality and integrity of the RNA products extracted by the TRIzol kit. The 28 S r RNA band was approximately1.5- to 2-fold brighter than the 18 S r RNA band on the agarose gel electrophoresis. The absorbance value A260/280 was 1.8–2.0, and the absorbance value A 260/230 was [1.9. The Bioanalyzer RNA integrity results showed that the RNA integrity number of the RNA extracted using the TRIzol kit was acceptable for high-throughputsequencing. Therefore, the total RNA extracted using the TRIzol kit method can be used for high-throughput sequencing and other molecular biology experiments.     

Tissue Culture Technology for Populus × euramericana Hybrids: Effect of Genotype on in Vitro Regeneration

In order to select a suitable genotype for the studies on genetic transformation, the difference among three genotypes (DN04-18, DN04-25 and DN04-26) of Populus × euramericana (Populus deltoides × P. nigra) in terms of shoot regeneration was investigated in this paper. An efficient in vitro micropropagation method was successfully established for genotype DN04-18. The high frequency shoot regeneration (90.0%) from leaf discs was obtained in genotype DN04-18 on the modified Murashige and Skoog (mMS) medium w...     

Callus induction and plant regeneration from mature bluegrass (Poa pratensis L.) seeds

A protocol was discussed for high efficient plant regeneration from seven bluegrass (Poa pratensis L.) cultivars via an indirect callus induction and somatic embryogenesis method. Mature seeds were used as explants for callus initiation. Callus induction and proliferation efficiencies were investigated on NB, modified MS (MMS) and MS media, supplemented with 2.0 mg·L-1 2,4-dichlorophenoxyacetic acid (2,4-D). The MMS medium performed best. Based on the MMS medium, direct and indirect callus induction effects of bluegrass from mature seeds were compared at the range of 1-5 mg·L-1 2,4-D contained in the medium. Under the direct callus induction method, the most suitable 2,4-D concentrations varied among cultivars. Under the indirect callus induction method, a significantly high callus induction frequency (93.33%-98.33%) was obtained and there were barely any statistically significant differences among the tested genetically diverse cultivars. Somatic embryos were promoted on the MMS medium supplemented with 3 mg·L-1 2,4-D, 0.1 mg·L-1 kinetin and 0.8 mg·L-1 CuSO4. Embryogenetic calli developed into plantlets on the MMS medium containing different concentrations of thidiazuron (TDZ), and the differentiation frequencies varied in the range from 20.15% to 77.65%. The 0.25 mg·L-1 TDZ was generally the most suitable concentration for the tested cultivars.     

Agrobacterium-mediated genetic transformation of Camptotheca acuminata

UGPase gene related with wood cellulose synthesis was transferred into C. acuminata using the method of Agrobacte- rium-mediated genetic transformation, and an efficient transformation system was developed for C. acuminata on the basis of evaluations of several factors affecting Agrobacterium-mediated DNA transfer rate. The highest transformation rate was achieved when pre-cultttred leaf explants were infected with an Agrobacterium culture corresponding to OD600 （0.5） for 10 min, and cultured on explant regeneration medium for three days. The results of Southern hybridization showed that genomic DNA of the kanamycin-resistant shoots to an UGPase gene probe substantiated the integration of the transgene. Transformation efficiency （6%） was achieved under the optimized transformation procedure, This system should facilitate the introduction of important useful genes into C, acuminata.     

Antimicrobial activity of some endemic plants growing in the Eastern Black Sea Region, Turkey

The Eastern Black Sea Region has an extensive flora because of ample rainfall lasting all year. In this study, antimicrobial effects of 74 crude extracts of 22 endemic plants were investigated. Among the 30 active crude extracts, water-insoluble crude extracts from Betula medwediewii, Heracleum platytaenium, Primula longipes, Anthemis cretica ssp. argaea and Centaurea helenioides were the prominent ones with their MIC values.     

Photosynthetic characteristics of Fagus sylvatica and Quercus robur established for stand conversion from Picea abies

Efforts in Europe to convert Norway spruce (Picea abies) plantations to broadleaf or mixed broadleaf-conifer forests could be bolstered by an increased understanding of how artificial regeneration acclimates and functions under a range of Norway spruce stand conditions. We studied foliage characteristics and leaf-level photosynthesis on 7-year-old European beech (Fagus sylvatica) and pedunculate oak (Quercus robur) regeneration established in open patches and shelterwoods of a partially harvested Norway spruce plantation in southwestern Sweden. Both species exhibited morphological plasticity at the leaf level by developing leaf blades in patches with an average mass per unit area (LMA) 54% greater than of those in shelterwoods, and at the plant level by maintaining a leaf area ratio (LAR) in shelterwoods that was 78% greater than in patches. However, we observed interspecific differences in photosynthetic capacity relative to spruce canopy openness. Photosynthetic capacity (A₁₆₀₀, net photosynthesis at a photosynthetic photon flux density of 1600 μmol photons m⁻² s⁻¹) of beech in respect to the canopy gradient was best related to leaf mass, and declined substantially with increasing canopy openness primarily because leaf nitrogen (N) in this species decreased about 0.9 mg g⁻¹ with each 10% rise in canopy openness. In contrast, A₁₆₀₀ of oak showed a weak response to mass-based N, and furthermore the percentage of N remained constant in oak leaf tissues across the canopy gradient. Therefore, oak photosynthetic capacity along the canopy gradient was best related to leaf area, and increased as the spruce canopy thinned primarily because LMA rose 8.6 g m⁻² for each 10% increase in canopy openness. These findings support the premise that spruce stand structure regulates photosynthetic capacity of beech through processes that determine N status of this species; leaf N (mass basis) was greatest under relatively closed spruce canopies where leaves apparently acclimate by enhancing light harvesting mechanisms. Spruce stand structure regulates photosynthetic capacity of oak through processes that control LMA; LMA was greatest under open spruce canopies of high light availability where leaves apparently acclimate by enhancing CO₂ fixation mechanisms.     

Aromatic plant-derived essential oil: an alternative larvicide for mosquito control

Five aromatic plants, Carum carvi (caraway), Apium graveolens (celery), Foeniculum vulgare (fennel), Zanthoxylum limonella (mullilam) and Curcuma zedoaria (zedoary) were selected for investigating larvicidal potential against mosquito vectors. Two laboratory-reared mosquito species, Anopheles dirus, the major malaria vector in Thailand, and Aedes aegypti, the main vector of dengue and dengue hemorrhagic fever in urban areas, were used. All of the volatile oils exerted significant larvicidal activity against the two mosquito species after 24-h exposure. Essential oil from mullilam was the most effective against the larvae of A. aegypti, while A. dirus larvae showed the highest susceptibility to zedoary oil.     

Antibacterial activity of plant extracts from Brazilian southeast region

A screening was conducted with 26 plants collected in the Brazilian southeast region, to identify plant extracts with antibacterial properties against Aeromonas hydrophila, Bacillus subtilis, Pseudomonas aeruginosa and Staphylococcus aureus. Initially, the agar diffusion method was employed. Then, those extracts presenting activity were submitted to a broth microdilution assay to determine the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC). It was observed that 13 of the tested extracts showed antibacterial activity. The best results were obtained with those from Lantana lilacina and Phyllanthus tenellus.     

利用三种梢栖真菌生物防治落叶松枯梢病的研究

从落叶松梢栖真菌筛选出对落叶松枯梢病病原菌有抑制作用的菌株,对落叶松枯梢病进行生物防治。将自落叶松梢部分离到的11种梢栖真菌与落叶松枯梢病病原菌进行对峙培养,根据被覆盖程度、抑菌率、拮抗系数和防治预试验的结果,综合评价后发现:粪生粪壳、深绿木霉和球毛壳3种梢栖真菌对落叶松枯梢病有良好的拮抗作用。利用这3个菌株在黑龙江省勃利县通天一林场国富沟进行林间防治试验,结果表明:防治效果为粪生粪壳>代森锰锌400倍液>深绿木霉>球毛壳;深绿木霉和粪生粪壳喷洒浓度50%和球毛壳喷洒浓度75%时,真菌多样性最多,梢栖真菌系统较其他浓度更为稳定,与化学防治相比,既达到了防治目的又具有不污染环境的优势。     

Growth and physiological response of six Australian rainforest tree species to a light gradient

An understanding of growth and photosynthetic potential of subtropical rainforest species to variations in light environment can be useful for determining the sequence of species introductions in rainforest restoration projects and mixed species plantations. We examined the growth and physiology of six Australian subtropical rainforest tree species in a greenhouse consisting of three artificial light environments (10%, 30%, and 60% full sunlight). Morphological responses followed the typical sun-shade dichotomy, with early and late secondary species (Elaeocarpus grandis, Flindersia brayleyana, Flindersia schottiana, and Gmelina leichhardtii) displaying higher relative growth rate (RGR) compared to mature stage species (Cryptocarya erythroxylon and Heritiera trifoliolatum). Growth and photosynthetic performance of most species reached a maximum in 30–60% full sunlight. Physiological responses provided limited evidence of a distinct dichotomy between early and late successional species. E. grandis and F. brayleyana, provided a clear representation of early successional species, with marked increase in A_max in high light and an ability to down regulate photosynthetic machinery in low light conditions. The remaining species (F. schottiana, G. leichhardtii, and H. trifoliolatum) were better represented as falling along a shade-tolerant continuum, with limited ability to adjust physiologically to an increase or decrease in light, maintaining similar A_max across all light environments. Results show that most species belong to a shade-tolerant constituency, with an ability to grow and persist across a wide range of light environments. The species offer a wide range of potential planting scenarios and silvicultural options, with ample potential to achieve rapid canopy closure and rainforest restoration goals.     

Predisposition to bark beetle attack by root herbivores and associated pathogens: Roles in forest decline,gap formation,and persistence of endemic bark beetle populations

Bark beetles are largely known for their ability to undergo intermittent population eruptions that transform entire landscapes and pose significant economic hardships. However, most species do not undergo outbreaks, and eruptive species usually exert only minor disturbances. Understanding the dynamics of tree-killing noneruptive species can provide insights into how beetles persist at low densities, and how some spatiotemporal patterns of host predisposition may more likely favor breaching eruptive thresholds than others. Elucidating mechanisms behind low-density populations is challenging, however, due to the requirement of long-term monitoring and high degrees of spatial and temporal covariance. We censused more than 2700 trees annually over 7 years, and at the end of 17 years, in a mature red pine plantation. Trees were measured for the presence of bark beetles and wood borers that breed within the primary stem, root weevils that breed in root collars, and bark beetles that breed in basal stems. We quantify the sequence of events that drive this decline syndrome, with the primary emergent pattern being an interaction between below- and above-ground herbivores and their fungal symbionts. This interaction results in an expanding forest gap, with subsequent colonization by early-successional vegetation. Spatial position strongly affects the likelihood of tree mortality. A red pine is initially very likely to avoid attack by tree-killing Ips beetles, but attack becomes increasingly likely as the belowground complex spreads to neighboring trees and eventually make trees susceptible. This system is largely internally driven, as there are strong gap edge, but not stand-edge, effects. Additional stressors, such as drought, can provide an intermittent source of susceptible trees to Ips beetles, and elevated temperature slightly accentuates this effect. New gaps can arise from such trees as they subsequently become epicenters for the full complex of organisms associated with this decline, but this is not common. As Ips populations rise, there is some element of positive feedback, in that the proportion of killed trees that were not first colonized by root organisms increases. This positive feedback is very weak, however, and we propose the slope between beetle population density and reliance on host stress as a quantitative distinction along a gradient from noneruptive through eruptive species. Almost all trees colonized by Ips were subsequently colonized by wood borers, likely a source of negative feedback. We discuss implications to our overall understanding of cross-scale interactions, between-guild interactions, forest declines, and eruptive thresholds.     

Growth and production varies between pair-wise mixtures and monoculture plantations in North Viet Nam

The potential benefits of species mixture were investigated using pair-wise comparisons of four timber tree species in northern Viet Nam. Chukrasia tabularis, Canarium album, Michelia mediocris and Eucalyptus urophylla were grown in monocultures and in pair-wise mixtures. The trial was established as a randomized block design with each treatment replicated four times. Volume production gain or loss in mixtures was assessed by calculating a mixture index, which is defined as Relative Yield Total (RYT). At age 48 months, the trial indicated mixed performance with both positive and negative impacts of growing some species in mixtures compared to monocultures. The largest gain was shown in the mixture of a shade-intolerant species, Chukrasia with a more shade-tolerant species, Michelia (47% gain in relative yield), and Eucalyptus with Michelia (45% gain in relative yield) at the first 38 months. The other three mixtures tested, and which were not successful (i.e. had lower relative yields) at 38 months, was the mixture of all shade-intolerant species, including Chukrasia with Canarium, Canarium with Eucalyptus and Chukrasia with Eucalyptus. This suggests that species with different shade tolerances can form complementary pair-wise mixtures, but this changed significantly (P < 0.05) over the following 10 months. At age 48 months the RYT of Chukrasia with Michelia increased by 12%, those of Michelia with Eucalyptus decreased by 20% at 48 months compared to 38 months, suggesting that Eucalyptus should be thinned at around year 5 years. Likewise, the RYT of Michelia with Canarium declined significantly by 23% at 48 months. The RYT of other tested mixtures remained almost unchanged over time.     

Ungulate herbivory of regenerating conifers in relation to foliar nutrition and terpenoid production

Ungulate browsing greatly influences regeneration dynamics of some forest ecosystems, yet the relationship between browse susceptibility and foliar chemistry of forest tree seedlings is not well understood. We applied field fertilization (15N-9P-10K controlled-release fertilizer at 0, 20, 40, and 60 g per seedling) and investigated how subsequent changes in terpenoid production and foliar nutrition influence ungulate browse preference for Douglas-fir (Pseudotsugamenziesii (Mirb.) Franco), western hemlock (Tsuga heterophylla Raf. Sarg.), and western redcedar (Thujaplicata Donn ex D. Don) seedlings across four sites in northwestern Oregon, USA. Fertilization increased foliar N concentration of all three species, but above-ground growth of only Douglas-fir and western hemlock. Foliar monoterpene concentrations for western hemlock and western redcedar also increased at higher fertilization rates, while Douglas-fir monoterpene production was not affected by fertilization. Regardless of monoterpene levels, ungulate browse preference was greater for fertilized western hemlock seedlings. The opposite response, however, was observed for western redcedar at two of the four study sites where the likelihood of browse was greater for non-fertilized than fertilized seedlings. Differences in browse preference among species may depend on the type and amount of individual monoterpenes manufactured in response to fertilization. Western redcedar produce α- and β-thujone, oxygenated monoterpenes known to promote gastroenteritis and possibly inhibit microbial rumen activity, which were absent from Douglas-fir or western hemlock. Higher concentrations of α- and β-thujone associated with increasing fertilizer rate provide a plausible explanation as to why ungulates preferred non-fertilized western redcedar. Our results illustrate species-specific adaptation in browse avoidance and selective ungulate browsing behavior of individual trees as linked to foliar chemistry.