基于TaqMan实时荧光PCR的拟松材线虫分子鉴定技术

拟松材线虫与松材线虫的亲缘关系极为接近,是松材线虫病病原鉴定的主要干扰物种。为了排除松材线虫病病原鉴定过程中拟松材线虫的干扰,采用TaqMan实时荧光PCR技术,以核糖体DNA内转录间隔区(ITS)为模板设计拟松材线虫特异性引物和TaqMan探针,开展拟松材线虫分子检测和鉴定技术研究。结果表明:拟松材线虫ITS2区域存在较多的碱基差异,以此为模板设计的拟松材线虫引物和TaqMan探针组合具有较强的特异性;该组合灵敏性高,能够实现对拟松材线虫单条线虫甚至单个卵的分子检测和鉴定。结合现有的松材线虫分子鉴定技术,可以为进一步构建松材线虫双重检测技术体系奠定基础,为林业和海关检疫提供可靠的技术支撑。     

“松材线虫SCAR标记与系列分子检测技术及试剂盒研制”项目通过鉴定

2006年5月，由南京林业大学森林资源与环境学院叶建仁教授主持完成的“松材线虫SCAR标记与系列分子检测技术及试剂盒研制”通过了江苏省科技厅组织的科技成果鉴定。该项研究对松材线虫与拟松材线虫特异片段进行了系统筛选，共获得了7个松材线虫DNA特异片段与5个拟松材线虫DNA特异片段，为松材线虫与拟松材线虫的分子鉴定奠定了基础。首次将2个松材线虫与2个拟松材线虫的DNA特异片段成功转化为SCAR标记，丰富了松材线虫与拟松材线虫分子标记方法。首次采用SCAR标记方法成功构建了松材线虫检测试剂盒，PCR检测过程仅需2．2小时。首次成功标记了一个可用于检测松材线虫的非放射性探针DIG-F2／R1。用该探针对线虫基因组DNA点杂交，松材线虫均表现有较强的杂交信号，而拟松材线虫、霍夫曼尼伞滑刃线虫、大核滑刃线虫、长尾属线虫等均无杂交信号。     

松材线虫分子生物学检测技术研究进展

概述松材线虫分子生物学检测技术的基础，介绍了DNA探针技术、基于PCR的检测技术和卫星DNA技术等分子生物学检测方法，并对PCR-RAPD、PCR-RFLP和PCR-SSCP技术进行了比较。     

松材线虫cul-1基因的表达特性与功能

【目的】研究松材线虫Bxy-cul-1基因的表达特性和生物学功能,明确该基因在松材线虫生长发育中的作用,为从生长发育角度探索特异性的线虫种群增长控制措施提供理论基础。【方法】根据松材线虫基因组数据设计引物、克隆Bxy-cul-1基因,对Bxy-cul-1进行序列、系统发育和蛋白结构预测等生物信息学分析。利用实时荧光定量PCR技术和原位杂交技术探究Bxy-cul-1基因在松材线虫各龄期的表达水平和表达部位,明确其时空动态表达特性,采用RNA干扰技术探究该基因在松材线虫生长发育中的作用。【结果】生物信息学分析结果显示,Bxy-cul-1基因CDS全长2 292 bp,编码763个氨基酸,属于Cullin蛋白家族。原位杂交结果表明,Bxy-cul-1基因在松材线虫各发育阶段均有表达,胚胎期全胚胎表达,2龄期广泛表达,3龄期和4龄期主要在肠道、体壁肌肉和尾部表达;成虫期,Bxy-cul-1基因在雌虫的卵母细胞和阴户、雄虫的腹部、交合刺和尾部表达。实时荧光定量PCR结果显示,Bxy-cul-1基因在松材线虫2龄期表达量最高,胚胎期次之,3龄期、4龄期、成虫期表达量依次递减。对松材线虫胚胎干扰后发...     

松材线虫分子检测技术研究进展

由于松材线虫病引起松树的毁灭性危害,松材线虫的准确、快速鉴定工作被认为是松材线虫病防控的最重要的环节之一。针对松材线虫形态鉴定存在的不足,一系列松材线虫分子检测技术如限制性片段长度多态性、随机扩增多态性、特异引物PCR、实时定量PCR、核酸杂交检测等技术相继被开发利用。其中实时定量PCR检测技术与SCAR标记检测技术具有检测时间短、检测准确性高。该两项技术已广泛应用于我国生产实践中。     

一种适于PCR和LAMP检测的松木中松材线虫DNA快速提取方法

【目的】本研究旨在探索快速、简便地直接从病木中提取松材线虫 DNA 的方法。【方法】运用 Chelex-100结合异硫氰酸胍蛋白变性缓冲液建立新的高效快速提取微量木块中线虫 DNA 的方法,并通过普通 PCR 与环介导等温扩增( LAMP)对提取的 DNA进行检测验证。【结果】建立了提取线虫 DNA 的新方法 Chelex-100法,并对影响提取效率的Chelex-100浓度、冻融时间和煮沸时间进行了优化。Chelex-100法最适Chelex-100终浓度为1.5%( W/V),最适冻融时间为5 min,最适煮沸时间为8 min。与传统的 CTAB法和蛋白酶 K 法比较,Chelex-100法提取的 DNA得率高,相同条件下,该方法提取的 DNA浓度远远大于常规方法。3种方法的 OD260/280比值从大到小依次为CTAB法＞蛋白酶K法≥Chelex-100法,Chelex-100法的OD260/280值显著低于CTAB法,而略低于或等同于蛋白酶K法,但这并不影响对提取的 DNA 进行 PCR 扩增。采用 Chelex-100法提取松木中松材线虫的 DNA,结合常规的PCR和 LAMP检测,检测灵敏度高,特异性强,稳定性好;提取的松材线虫 DNA 的75倍稀释液再稀释32倍后进行PCR扩增,扩增产物电泳依然有清晰的条带。用新方法提取病木中线虫的 DNA 后进行检测,所有带病松木样品的检测结果均呈阳性,而健康黑松、马尾松、油松木屑及盘多毛孢样品的检测结果均呈阴性。此外,该提取方法简便快速,20 min内即可完成整个 DNA的提取;经济方便,试剂保藏无特殊要求,单个样品提取耗费低于3.5元。【结论】Chelex-100法是一种快速有效的提取松木中松材线虫DNA的方法,此方法结合PCR和LAMP检测技术将进一步提高松材线虫的检测效率、灵敏度和准确性,可为松材线虫的野外检测提供可靠的技术依据。     

松墨天牛携带的松材线虫PCR检测技术

利用本研究筛选出的一对松材线虫特异性PCR引物(上游引物:5'-CTACGTGCTGTTGTTGAGTTGGC-3',下游引物:5'-TGGTGCCTAACATTGCGCGA-3'),从松材线虫DNA中,扩增出一条长度403 bp(基因库登陆号:DQ855275)的特异性DNA片段.该引物可以将松材线虫DNA与松墨天牛组织以及拟松材线虫、畸刺伞滑刃线虫、变异伞滑刃线虫、小角伞滑刃线虫、利昂伞滑刃线虫、湖南伞滑刃线虫、红松滑刃线虫、滑刃属线虫、斯坦纳长尾线虫、小茎线虫、剑尾齿杆双胃线虫和寄生小杆属线虫等12种线虫的DNA区分开来.在此基础上,建立一套利用PCR技术直接从受感染的松墨天牛组织中检测出松材线虫的技术,并在实际运用中得到检验.     

松材线虫实时PCR检测方法的建立

对南京林业大学开发的松材线虫实时PCR检测方法的操作过程进行了规范化.介绍了检测松材线虫所需要的试剂及使用方法,对仪器的稳定性进行了验证,对检测结果的判读方法进行了规定.规范化的检测方法灵敏度高,特异性强,应用方便.     

松材线虫病双基因检测方法的建立

采用松材线虫的核糖体DNA的内转录区序列及cathepsin L-like cysteine proteinase为目的片段设计2对引物,建立了可特异性检测松材线虫的双基因PCR检测法。利用这2对引物,松材线虫可扩增出大小分别为490 bp及264 bp的产物,而其他对照组均无扩增。此检测方法经过实际应用检验,灵敏度与常规PCR一致,而特异性更高,可以应用于实验室的常规检测需求。     

落叶松自然条件下感染松材线虫初报

对2018年辽宁抚顺市出现枯死现象的长白落叶松Larix olgensis、日本落叶松L. kaempferi和华北落叶松L. principis-rupprechtii取样调查,通过显微镜形态学鉴定和实时荧光定量PCR方法确定3种落叶松体内均含松材线虫Bursaphelenchus xylophilus。由此确定松材线虫在我国自然条件下侵染落叶松。这是我国关于落叶松自然感染松材线虫的首次报道。     

Decomposition and nutrient loss from prunings of different contour hedgerow species in tea plantations in the sloping highlands of Sri Lanka

Contour hedgerows of multipurpose tree species in the sloping tea lands of Sri Lanka are expected to reduce soil erosion and also add significant amounts of plant nutrients to the soil via periodic prunings. The objective of this experiment was to characterize the biomass decomposition pattern and quantify the amount of nutrients added through prunings of six tree species (Calliandra calothyrsus, Senna spectabilis, Euphatorium innulifolium, Flemingia congesta, Gliricidia sepium and Tithonia diversifolia) currently being used in hedgerows associated with tea. Withered leaf and stem prunings (50 g) were enclosed in 2-mm litter bags, placed at 5-cm depth and retrieved after one, three, six, nine and 12 weeks. Loss of initial dry weight, N, P and K was measured. Single exponential decay function adequately described both dry weight and nutrient loss. Tree species differed significantly in their rate of breakdown with decomposition constants (k) varying from 0.0299 to 0.2006 week⁻¹ for leaves and from 0.0225 to 0.0633 week⁻¹ for stems. Gliricidia showed the highest k for leaves with the rest in the following descending order: Senna > Tithonia ≥ Euphatorium > Calliandra > Flemingia. A similar pattern was observed for loss of all nutrients with Calliandra and Flemingia always having lower k values than the rest. Although N immobilization was not observed, immobilization of P and K was observed during the first week of incubation in some species, particularly in stem prunings. Annual biomass of prunings differed significantly between tree species in the following descending order: Calliandra > Senna > Flemingia > Tithonia > Gliricidia > Euphatorium. Calliandra added the greatest amount of nutrients annually to the soil with Euphatorium adding the least. Calliandra prunings provided the annual total K requirement and 49% of the N requirement of mature tea. However, none of the species provided more than 5% of the P requirement. It is concluded that among the tree species tested, Calliandra and Flemingia are the most suitable for contour hedgerows in tea plantations of this agroclimatic region because of their higher soil nutrient enrichment capacity and slower decomposition rates which would minimize leaching losses. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of Seed Weight and Seed Type on Early Seedling Growth of Pinus sylvestris under Harsh and Optimal Conditions

The effects of seed weight and seed type on seedling growth of Pinus sylvestris (L.) were studied by seeding individually weighed orchard and stand seed in different mixtures under harsh (direct seeding in field) and optimal (seeding in nursery) conditions. In the nursery experiment an increase in the seed weight from 3 to 7 mg increased the seedling height by 10-27% and total weight by 27-113%, and decreased the height/diameter ratio by 5-6% after 2 yrs. With elimination of seed weight effects, orchard seedlings were 2% taller than stand seedlings in year 2. Without elimination of seed weight effects, orchard seedlings were 7-13% taller. In the field experiment an increase in the seed weight from 3 to 7 mg increased seedling height by 18-65%, stem volume by 81-274% and the number of top-buds by 23-34% in year 5. After elimination of seed weight effects, orchard seedlings were 7-13% taller than stand seedlings and without elimination of seed weight effects 20-21% taller after 5 yrs. Even after elimination of both seed weight and genetic effects orchard seedlings were 3-9% larger than stand seedlings in the field experiment. In conclusion, the influence of seed weight and seed type on growth traits and slenderness is highly significant and the influence seems to be greater in harsh conditions.     

Modelling of adequate pretwist for obtaining straight timber

Abstract

Wood in general and wooden studs in particular are often distorted owing to uneven shrinkage during the drying process in the sawmill. Twist is often the most detrimental of all types of distortion, and it is caused by spiral grain in combination with variations in moisture content. For sawmills, the objective is to produce dried, straight boards, and one method of dealing with boards with excessive spiral grain is to sort them out and then dry them in a pretwisted position to obtain straight boards after drying. A model using the finite element (FE) method for the simulation of drying twist distortions was first calibrated against laboratory experiments in which boards were dried with and without restraints and pretwists. After the calibration, the FE results were compared with industrial test results for boards that were dried without restraints or with restraints with zero pretwist, i.e. straight restraints. The FE model used an elastic–ideally plastic material model to obtain permanent deformations. The calibration was to set the yield stresses so that there was a good match between FE results and results from the laboratory experiments. The comparison between the industrial test results and the FE results showed that the FE model is capable of realistic simulations of drying boards with and without restraints and presumably also pretwists.     

Potential to harness superior nutritional qualities of exotic baobabs if local adaptation can be conferred through grafting

Baobab leaves form an important part of the local diet in Sahel countries and elsewhere in Africa. Existing leaf nutritional data and agroforestry performance information are based solely on Adansonia digitata L., the baobab of continental Africa. The introduction potential of Adansonia species from the center of diversity in Madagascar and from Australia remains untapped. To assess this potential, the mineral contents and B₁ and B₂ vitamin levels of dried baobab leaves were determined for five-year old trees of A. digitata, A. gibbosa (A. Cunn.) Guymer ex D. Baum, A. rubrostipa Jum. & H. Perrier (syn. A. fony Baill.), A. perrieri Capuron and A. za Baill. grown in an introduction trial in Mali. Nutritional data were evaluated against survival and vigor to identify promising germplasm. Leaf vitamin and crude protein contents were highest in the Madagascar species, especially A. rubrostipa (B₁ 88 mg 100 g⁻¹, B₂ 187 mg 100 g⁻¹, protein 20.7% dry weight). However, the local species far outperformed the introductions in survival, tree height, basal diameter and resistance to termites. We suggest grafting as a way of harnessing the vigor of well-adapted local baobab varieties to the superior nutritional profiles of A. rubrostipa and others. Cross-species grafting tests in Adansonia were successful, thus creating new agroforestry possibilities with different scion/rootstock combinations.     

Growth of Phlebiopsis gigantea in wood of seven conifer species

Heterobasidion parviporum and Heterobasidion annosum are widely distributed root‐rot fungi that infect conifers throughout Europe. Infection of conifer stumps by spores of these pathogens can be controlled by treating fresh stumps with a competing non‐pathogenic fungus, Phlebiopsis gigantea. In this study, growth of three Latvian strains of P. gigantea and the biological control agent ‘Rotstop’ strain was evaluated in stem pieces of Norway spruce, Scots pine, lodgepole pine, Douglas‐fir, Weymouth pine, Siberian larch and Sitka spruce. The growth rates of one H. parviporum and one H. annosum isolate were also measured in the same stem pieces. The growth rate of P. gigantea varied greatly in wood of different conifer species. It was higher in the three pine species, lower in Norway spruce and lowest in Sitka spruce and Siberian larch, and in Douglas‐fir, this fungus did not grow. The largest area of wood occupied by P. gigantea was in lodgepole pine. Growth of Latvian isolates of P. gigantea in the wood of Pinus and Picea species was comparable to that of the Rotstop isolate. Consequently, stump treatment with local P. gigantea isolates should be recommended. However, our results suggest that Douglas‐fir stump treatment against Heterobasidion by P. gigantea may be ineffective and other stump treatment methods should be considered.     

Growth of Mixed Birch-Coniferous Stands in Relation to Pure Coniferous Stands at Similar Sites in South-eastern Norway

Mean age, mean and top heights and yield were studied in 20 mixed stands of birch ( Betula pubescens Ehrh. and B. pendula Roth) and Picea abies (L.) Karst. and nine mixed stands of birch and Pinus sylvestris L. in south-eastern Norway. Each mixed stand and the adjacent pure coniferous stand (control) were growing under the same site conditions and had not been commercially thinned. There were no significant differences in mean age at breast height or in top heights between birch and conifers in the mixed stands, while mean height was significantly higher for birch than for spruce. A growth index was calculated based on total volume and age at breast height. For the spruce sites the growth in young mixed stands (birch < 17 m) was superior to that of pure spruce, while the difference was insignificant in older stands. The growth index correlated positively with the ratio between generatively and vegetatively regenerated birch trees, and negatively with the age of the oldest species in the mixture and with site quality. For the pine sites there was no significant difference in the growth index between mixed birch-pine and pure pine stands. A mixture effect of birch on the volume yield of spruce or pine could not be demonstrated.     

Defoliation by the Needle-shortening Pine Gall Midge,Thecodiplosis brachyntera,on Pines in Central Sweden

An outbreak of the needle-shortening pine gall midge, Thecodiplosis brachyntera , occurred on Scots pine, Pinus sylvestris (L.) and lodgepole pine, P. contorta (Dougl.) in central Sweden during 1995-1998. The larval feeding of T. brachyntera kills current-year needles and heavy infestation causes severe defoliation. A pattern of fluctuating defoliation among years and sites was found on both pine species. Defoliation was evenly distributed in the crown of P. sylvestris during years of high as well as low infestation; apical whorls had as much defoliation as basal whorls. In P. contorta , however, there was a tendency for defoliation to be concentrated on branches in the most apical whorls. Growth ring increments on P. sylvestris with high levels of defoliation (71%) did not differ from trees with moderate levels (26%) of defoliation. Shoot lengths were significantly shorter, however, on heavily infested trees the year after defoliation.     

Biogeochemical impacts of clearfelling and reforestation on blanket-peatland streams: II. major ions and dissolved organic carbon

Streams and drains in blanket-peatland forest in western Ireland were sampled weekly over 5 years, 1996–2000, using continuous, depth-proportional passive sampling. Analysis was for pH, alkalinity by Gran titration, anions by IC, metals by ICP, aluminium speciation by loaded-resin exchange, and dissolved organic carbon (DOC) by absorbance at 320 nm.

Effects of felling are identified graphically from two forest drains whose catchments (both about 1 ha) were clearfelled, and partial clearfelling of one larger catchment (somewhat over 1 km²), in summer 1999. Calcium concentrations and alkalinity were increased in the drain- and streamwater from the felled areas. The pH was increased in drains, while in the larger stream, pH range decreased while the mean increased. Phosphorus increased markedly with clearfell, in the absence of recent fertilising, and also increased with fertilising; these results are reported in an accompanying paper. Ammonium-nitrogen concentrations were increased in the two drains with felling, and temporarily decreased in the larger stream. Nitrate increased in some cases. Potassium and manganese concentrations also increased. Concentrations of DOC and organic monomeric aluminium increased gradually, subject to a continuing strong annual cycle. There were no clearfelling effects on concentrations of solphate, suspended solids or inorganic monomeric aluminium. In the two drains (fully clearfelled), concentrations of sodium, chloride and magnesium, and conductivity, were all reduced after felling. Fertilising effects other than for phosphorus were indistinct.

Effects of the combined clearfell–reforestation treatment were testable statistically, using randomised intervention analysis, between two larger streams (1 km²) as a treatment–control comparison. There were statistically significant positive responses in streamwater concentrations of nitrate, potassium, calcium, DOC and aluminium fractions. The implications for management practice depend on whether the loss of nutrients is sustainable for future crop productivity, and whether critical limits and loads of receiving ecosystems are exceeded.     

Effect of Drought on Growth and Mortality of Actively Growing Norway Spruce Container Seedlings Planted in Summer

Survival, root egress and height growth of 13-month-old actively growing Norway spruce ( Picea abies (L.) Karst.) container seedlings exposed to preplanting drying treatments (0, 4, and 8 days) and postplanting drought periods (1, 2, 3, and 4 weeks) were studied in the field between 5 July and 18 August 1999. The mortality of seedlings increased and the height growth and root egress decreased throughout the postplanting drought period. Postplanting performance was also affected by preplanting drying treatments. The results indicate that no risk of excessive mortality and growth restrictions occurs when actively growing seedlings are planted in summer, provided that seedlings are well watered before planting and the drought period does not exceed 3 weeks.     

Rehabilitation of diseased cacao fields in Peru through shade regulation and timing of biocontrol measures

The objective of this case study was to test the effectiveness of biocontrol for the rehabilitation of abandoned, diseased (moniliasis, witches' broom and black pod) cocoa and to develop testable hypotheses regarding the effect of shade and biocontrol timing. Three trial fields differed in their shade regimes. Biocontrol was administered starting either at flowering or pod filling. Disease incidence and yield were compared with cultural control alone. Biocontrol reduced moniliasis from 49% to 25%, independent of the shading regime and application time. Witches' broom was most severe without shade and unaffected by biocontrol. No significant effects on black pod were observed. Although biocontrol increased percentage healthy fruit independent of shading and timing, only the shaded plot with biocontrol from flowering onwards responded with increased yield. Our hypothesis is: 'In the rehabilitation of abandoned cacao, only fields under sustainable shade management and early onset of biocontrol can realise their production potential.' This revised version was published online in June 2006 with corrections to the Cover Date.