Mating disruption of Lasioderma serricorne (Coleoptera: Anobiidae) in stored product habitats using the synthetic pheromone serricornin

Cigarette beetles, Lasioderma serricorne (F.) (Coleoptera: Anobiidae), cause significant damage to the multibillion dollar food and tobacco industries worldwide each year. A non‐insecticidal alternative to manage L. serricorne is the application of mating disruption, in which high levels of synthetic sex pheromone are released to create an atmosphere that results in males failing to mate females, thereby causing population suppression or extinction. The reported work used synthetic serricornin, the predominant sex pheromone of L. serricorne, in mating disruption trials conducted in selected food‐ and feed‐processing facilities in South Carolina during 2010 and 2011. Mills subjected to mating disruption trials were monitored using oviposition cups filled with larval food and pheromone traps for males that contained monitoring lures. Immediately after deployment of mating disruption dispensers, trap captures declined significantly and indicated a reduction in population levels, that is, there was ‘trap shutdown’. A significant reduction was observed in numbers of adult beetles caught in the traps 8 weeks before and 8 weeks after treatment in both years. Beetle numbers from pheromone traps in untreated buildings remained at similar levels or increased after the time of mating disruption deployment in treated buildings. The numbers of adults that emerged from oviposition food cups were generally low and varied irregularly in treated and untreated buildings and were determined to be of little value for assessing treatment effects on reproduction. These initial field studies in the USA suggest that release of the synthetic sex pheromone of L. serricorne for mating disruption can significantly inhibit proper orientation behaviour of male L. serricorne to females and may lead to pest population decline from mating disruption.     

Orientation of the Cigarette Beetle, <Emphasis Type="Italic">Lasioderma serricorne</Emphasis> (F.) (Coleoptera: Anobiidae) to Plant-Derived Volatiles

Experiments were conducted to study the orientation of adult cigarette beetles,Lasioderma serricorne (F.), to plant volatiles in a walking bioassay. Seven out of sixteen test materials that displayed attractive responses were further studied for (1) responses to whole extracts from three types of solvents and (2) the effects of sex and mating status of L. serricorne on responses to plant volatiles. Bioassays with extracts revealed that responses of L. serricorne varied among the type of solvent extract. Volatiles from different Capsicum products attracted significantly more adult beetles than did volatiles from other materials. When virgin males, virgin females and mated females were bioassayed, mated females responded significantly more than the other groups to plant volatiles. Semiochemical-based pest management methods may be developed for L. serricorne with plant volatile-based attractants that may be identified and used alone or combined with a synthetic sex pheromone to manipulate beetle behavior. Mention of product or trade name in this paper does not constitute an endorsement or recommendation for its use by Oklahoma State University     

Enantiomeric composition of nicotine in tobacco leaf,cigarette, smokeless tobacco,and e‐liquid by normal phase high‐performance liquid chromatography

Evaluating the source of nicotine in e‐liquid is a problem. Tobacco‐derived nicotine contains predominantly (S)‐(?)‐nicotine, whereas tobacco‐free nicotine products may not. Thus, we developed a new normal phase high‐performance liquid chromatography method to determinate the enantiomeric composition of nicotine in 10 kinds of flue‐cured tobacco, 3 kinds of burley, 1 kind of cigar tobacco, 2 kinds of oriental tobacco, 5 kinds of Virginia cigarette, 5 kinds of blend cigarette, 10 kinds of e‐liquid, and 4 kinds of smokeless tobacco. The amount of (R)‐(+)‐nicotine ranged from ~0.02% to ~0.76% of total nicotine. An e‐liquid sample had the highest level of (R)‐(+)‐nicotine. The extraction and purification processes used to obtain commercial (S)‐(?)‐nicotine from the tobacco do not decrease the amount of (R)‐(+)‐nicotine in tobacco. So the amount of (R)‐(+)‐nicotine in samples in our work were the same as tobacco samples.     

烟草甲细胞色素P450还原酶基因的分子特征与表达模式分析

烟草甲Lasioderma serricorne是一种重要的仓储害虫,长期化学防治导致烟草甲已对多种传统熏蒸剂产生抗性,但其对新型熏蒸剂甲酸乙酯仍处于敏感水平。因此明确其体内细胞色素P450还原酶（cytochrome P450 reductase, CPR）对甲酸乙酯的代谢解毒功能,对开展该药剂的抗性监测及延缓抗性的发生发展具有重要意义。本研究旨在克隆烟草甲LsCPR基因,分析其分子特征和表达特性,为进一步明确其在烟草甲对甲酸乙酯解毒代谢过程中的作用奠定基础。利用RT-PCR技术克隆烟草甲LsCPR基因的开放阅读框（open reading frame, ORF）;利用生物信息学软件分析LsCPR编码蛋白的结构、特征和系统进化关系。采用实时定量PCR技术检测LsCPR在烟草甲不同发育阶段（低龄幼虫、高龄幼虫、蛹、低龄成虫、高龄成虫）、幼虫不同组织（表皮、肠道、脂肪体和马氏管）以及甲酸乙酯熏蒸胁迫后的表达模式。烟草甲LsCPR基因的ORF为2 046 bp（GenBank登录号：MZ423209）,编码681个氨基酸,具有典型的昆虫CPR基因FMN区域、NADPH区域和FAD等保守结构域。系统发育分析表明,烟草甲LsCPR与鞘翅目昆虫聚为一支,且与赤拟谷盗Tribolium castaneum CPR亲缘关系最近。LsCPR在烟草甲不同发育阶段均有表达,在高龄幼虫期的表达水平较高;在幼虫体内的表达部位主要在肠道,其次为脂肪体和马氏管,而在表皮的表达水平最低。LC10、LC30和LC50 3种浓度的甲酸乙酯处理24 h后,烟草甲LsCPR表达量随着胁迫浓度升高而上调且显著高于对照;甲酸乙酯LC50处理烟草甲不同时间（3、6、12、24和48 h）后,LsCPR基因上调表达,24 h时达到表达高峰。推测LsCPR是参与烟草甲代谢甲酸乙酯的候选基因。     

烟草TCP家族成员鉴定及表达分析

TCP家族作为植物特有的转录因子,在植物发育的不同方面发挥着重要作用。为筛选烟草中TCP家族成员,本研究通过全基因组同源比对,鉴定烟草与拟南芥TCP家族同源序列。利用生物信息学的方法分析其理化性质、系统进化关系、顺式作用元件等;筛选AtTCP3/AtTCP4的同源基因,并利用RT-qPCR检测在20% PEG6000处理下的基因表达量变化。结果表明烟草中含有TCP家族成员63个,其氨基酸序列长度范围为89−596 aa,蛋白亲水性(grand average of hydropathicity, GRAVY)范围为−1.147−0.125,等电点(isoelectric point, pI)范围为4.42−9.94,内含子个数为0−3,亚细胞定位均位于细胞核。保守结构域和系统进化关系分析结果表明,烟草TCP家族可分为PCF、CIN和CYC/TB1这3个亚家族且每个亚家族具有稳定序列。基因启动子区顺式作用元件结果表明,TCP家族基因含有低温顺式作用元件(LTR)及多种胁迫及代谢调控相关的元件(MYB、MYC)等顺式作用元件。基因表达模式分析表明,AtTCP3/AtTCP4同源基因(NtTCP6、NtTCP28、NtTCP30、NtTCP33、NtTCP42、NtTCP57和NtTCP63)在20% PEG6000处理下表达量显著上调表达/下调表达,并发现NtTCP30和NtTCP57基因对干旱胁迫响应较为明显。研究结果剖析了烟草基因组中的TCP家族,为烟草抗旱基因功能研究及品种培育提供了候选基因。     

The uptake and metabolism of 3H-benzylaminopurine in tobacco (Nicotiana tabacum L.) and cucumber (Cucumis sativus L.) explants

The uptake and metabolism of ³H-benzylaminopurine(³H-BAP) were studied in explanted stem pith andleaves of tobacco and in the hypocotyls and cotyledonsof cucumber. The explants were kept for 2, 5, 8 and 20h on MS medium with 0.8 mg.l^–1 2,4-D,0.5 mg.l^–1 BAP and 13.2 mg.l^–1 aspartic acid(induction medium) with or without ³H-BAP and¹⁴C-sucrose. The highest uptake of ³H-BAPwas observed in tobacco leaves and cucumbercotyledons. The major metabolite in both species was³H-benzylaminopurine riboside (³H-BAPR). Thehighest level was found in explanted cucumbercotyledons after 20 h in culture, the lowest inexplanted tobacco stem pith. Intensive 7-glucosylationof ³H-BAP was observed in explanted tobaccoleaves after 20 h in culture, where the levels of7-glucoside of ³H-BAP and of free ³H-BAPwere equal. To study the morphogenic effect of growth regulators(BAP and 2,4-D), the explants were subcultured aftershort-term induction (20 h) to MS medium without anygrowth regulators. In most cases, incubation of 20 hon induction medium was sufficient to induce therespective morphogenesis. Cucumber hypocotyl andtobacco stem pith explants formed a callus on theirbasal end. Root formation was observed on explantedcucumber cotyledons and shoot formation on tobaccoleaves. Long-term culture (3 weeks) of tobacco leaveson induction medium led to the formation of callus andglobules. The microscopic analysis of globulesindicated the presence of meristematic and tracheidalcells.     

微尺度下烟田铁的空间变异性及其与烟叶铁的相关分析

铁是作物必需的营养元素之一,是血红蛋白和细胞色素的组成成分,也是细胞色素氧化酶、过氧化氢酶、过氧化物酶等的组成成分,与烟叶香气前体物的形成密切相关.精准农业通常研究的是微尺度下(米级或亚米级)土壤养分的空间变异性.为此,在米级尺度下(11 m×10 m)对烟田全铁在种烟前后的空间变异性进行比较,并分析相应网格上烤后烟叶样品的铁含量,探讨土壤在种烟前后全铁含量与烟叶铁含量间的相互关系.结果表明:收完烟后试验地全铁含量平均值与种烟前相比有所下降,由30.3 g kg-1降到了24.79 g kg-1.种烟前土壤全铁具有中等的空间相关性(块金效应为48.8%),变异函数的最佳理论模型为线性模型,决定系数达到0.999,Block-Kriging内插图显示试验地全铁具有较强的空间异质性,方差分析的结果也表明取样点间的差异达极显著水平(p=0.00008).种烟后土壤全铁含量以球状模型为变异函数的最佳理论模型,决定系数达到0.956,Block-Kriging内插图显示种烟后土壤全铁含量的最高值比种烟前往南移动了一些,最低值也往北移了一些,整个试验地土壤全铁在种烟后的空间变异性比种烟前有所升高.相关分析表明,烟叶铁含量只与种烟前土壤全铁含量呈极显著相关,与种烟后土壤全铁含量、种烟前后土壤全铁的相对变化量都没有显著相关.根据相关分析的结果,建立了以种烟前土壤全铁含量为自变量,以烟叶全铁含量为因变量的回归方程,并对方程进行验证,结果表明在自变量X的分布区间内预测效果比较好.     

Cloning of Helicobacter pylori urease subunit B gene and its expression in tobacco (Nicotiana tabacum L.)

Vaccines produced by transgenic plants would have the potential to change the traditional means of production and inoculation of vaccines, and to reduce the cost of vaccine production. In the present study, an UreB antigen gene from a new Helicobacter pylori strain ZJC02 was cloned into the binary vector pBI121 which contains a CaMV35S promoter and a kanamycin resistance gene, and then transformed UreB into tobacco leaf-disc by Agrobacterium-mediated method. A total of 50 regenerated plants with kanamycin resistance were obtained in the selection media. The 35 putative transgenic individuals were tested and verified the presence and integration of the UreB into the nuclear genome of tobacco plants by PCR, PCR-southern, and Southern analyses. Expression of UreB gene in the tobacco plants was confirmed by RT-PCR and Western Blot analysis using polyclonal human antiserum. To our knowledge, this is the first report of the expression of Helicobacter pylori UreB antigen gene in a plant system, suggesting a major step in the production of plant-based vaccines for Helicobacter pylori.     

Effect of humidity and a superabsorbent polymer formulation on the efficacy of Heterorhabditis zealandica (Rhabditida: Heterorhabditidae) to control codling moth,Cydia pomonella (L.) (Lepidoptera: Tortricidae)

Adequate moisture levels are required for nematode survival and subsequent efficacy as entomopathogens. Formulation of nematodes aimed at aboveground applications may assist in maintaining such moisture levels. In this study, we report the effects of a superabsorbent polymer formulation, Zeba® on the performance of an entomopathogenic nematode, Heterorhabditis zealandica Poinar, for controlling diapausing codling moth, Cydia pomonella (L.) larvae in cryptic habitats on trees. Water activity (a_w-value) on bark was considered to be an indication of moisture levels on trees in cryptic habitats where codling moth larvae are known to occur, thereby influencing nematode efficacy. H. zealandica was only able to infect codling moth larvae at a_w≥0.92, with a_w50=0.94 and a_w90=0.96. Laboratory experiments in which nematode concentration was investigated indicated a positive linear relationship between the concentration of nematodes applied and the level of control obtained, with the highest level of mortality recorded at 80 IJs/larva, requiring at least 4 h of conditions conducive to nematode activity to ensure infectivity and subsequent efficacy. Further experimentation showed that the use of the Zeba formulation, together with the nematodes, improved the level of control obtained at 60% and 80% RH in the laboratory and that it also enhanced the survival and infection-ability of the nematodes in the field. The study conclusively illustrates that the tested formulation assisted in maintaining adequate moisture levels on the application substratum, as required for nematode survival and subsequent efficacy.     

普通烟草CESA基因家族成员的鉴定、亚细胞定位及表达分析

纤维素合成酶蛋白(cellulose-synthase proteins, CESA)是一类质膜定位蛋白,以蛋白复合体的形式存在于质膜上合成纤维素,在细胞壁建成和植物生长发育过程中起着非常重要的作用。本研究利用CESA蛋白保守域序列PF03552检索普通烟草(Nicotiana tabacum L.)蛋白序列,并通过拟南芥(Arabidopsis thaliana)10个CESA蛋白序列在普通烟草基因组数据库中利用TBLASTN程序进行比对,共获得21条NtCESA基因候选序列,对这些序列进行蛋白序列理化性质分析、系统进化树构建、基因结构分析、保守结构域及跨膜区分析和组织表达模式分析,并对NtCESA9和NtCESA14两个蛋白进行了亚细胞定位实验。结果表明：获得的21条NtCESA蛋白序列的理化性质相似;系统进化分析将21个NtCESA基因和10个AtCESA基因分成5个分支,每一个分支各成员之间的进化相对保守,基因结构类似,不同分支之间的基因结构差异也较小;NtCESA蛋白结构域相对保守,都含有CESA蛋白典型的N端锌指结构、C端跨膜区和DDD-QXXRW保守功能域;组织表达分析结果表明,大部分NtCESA基因在幼苗和成熟期烟草的根、叶、胚芽和愈伤组织中都有表达,同一个分支中的基因表达模式基本一致,并且NtCESA基因参与初/次生细胞壁纤维素的合成与该基因编码蛋白的跨膜区数目存在关联,表明NtCESA基因家族成员功能上的复杂性;亚细胞定位结果证实NtCESA9和NtCESA14为质膜定位蛋白。本研究为烟草CESA基因家族功能的深入研究奠定了基础。     

Antisense Expression of a NBS-LRR Sequence in Sunflower (Helianthus annuus L.) and Tobacco (Nicotiana tabacum L.): Evidence for a Dual Role in Plant Development and Fungal Resistance

A partial sunflower cDNA clone, PLFOR48, segregating with a resistance marker to Plasmopara halstedii, the causal agent of downy mildew, has been cloned from the mildew resistant sunflower line, RHA 266. PLFOR48 encodes a putative protein with a nucleotide-binding site and a leucine-rich repeat domain, showing significant homology with previously cloned resistance genes belonging to the TIR-NBS-LRR family. Southern blot analysis of non-transgenic sunflower suggests that PLFOR48 is part of a multigenic family. The potential role of PLFOR48 sequence in sunflower resistance to mildew was studied, by assessing loss of function, using expression of the antisense cDNA in RHA 266 sunflower line. Quite unexpectedly, transgenic sunflower lines displayed severe developmental abnormalities, and in particular, on the main meristems of homozygote T2 progeny, thus hampering any further challenge inoculation with Plasmopara halstedii. The presence of homologous sequences to PLFOR48 in Nicotiana tabacum var Samsun NN, as demonstrated by Southern blotting, drove us to consider tobacco as an additional model to investigate the potential role of this sequence in fungal resistance. Expression of the same antisense cDNA in transgenic tobacco lines gave rise to higher degree of susceptibility to Phytophthora parasitica, as well as to severe alterations in seed development. These results suggest that PLFOR48 and homologous sequences could be involved in both regulating developmental pathways and controlling resistance to fungal pathogens.     

Multiple paternity in a natural population of a wild tobacco fly, Bactrocera cacuminata (Diptera: Tephritidae), assessed by microsatellite DNA markers

Mating frequency has important implications for patterns of sexual selection and sexual conflict and hence for issues such as speciation and the maintenance of genetic diversity. Knowledge of natural mating patterns can also lead to more effective control of pest tephritid species, in which suppression programmes, such as the sterile insect technique (SIT) are employed. Multiple mating by females may compromise the success of SIT. We investigated the level of polyandry and sperm utilization in a Brisbane field population of the tropical fruit fly, Bactrocera cacuminata (Hering), using seven polymorphic microsatellite loci. The offspring of 22 wild-caught gravid females were genotyped to determine the number of males siring each brood and paternity skew, using the programs gerud and scare. Our data showed that 22.7% of females produced offspring sired by at least two males. The mean number of mates per female was 1.72. Paternal contributions of double-sired broods were skewed with the most successful male having sired between 76.9% and 87.5% of the offspring. These results have implications for SIT, because the level of remating we have identified would indicate that wild females could mate with one or more resident fertile males.     

Biodegradation and detoxification of nicotine in tobacco solid waste by a Pseudomonas sp.

A Pseudomonas sp. grew with nicotine optimally 3 g l^–1 and at 30 °C and pH 7. Nicotine was fully degraded within 10 h. The resting cells degraded nicotine in tobacco solid waste completely within 6 h in 0.02 m sodium phosphate buffer (pH 7) at maximally 56 mg nicotine h^–1 g dry cell^–1.     

生物质发酵产热-小型温室棚联用对春冬季烟苗生长温度调控的数值研究

小型温室棚作为农业生产的重要设施,可为农作物创造一个温度适宜的、受保护的生长空间,保证作物正常生长、促进作物早熟。以确保重庆地区春冬季烟苗正常发育、维持温室内适宜的土壤温度为导向,对温室棚内温度进行了试验和数值模拟,探究了温室棚内温度分布规律,并在此基础上分析生物质发酵产热对温室棚内土壤温度场的影响。结果表明：试验中埋设稻壳的温室棚内土壤温度平均提高3.5℃且温室棚中平均温度低于10℃的天数明显减少。埋设稻壳的温室棚中烟苗茎高和叶片数增长值最大,平均茎高从移栽时的7.8 cm增长到了12 cm,叶片数从平均5片增长到了11片,均大于未埋设稻壳试验组,这表明土壤中埋设稻壳有助于烟草的生长发育。数值研究表明温室棚内不同深度的土壤层温度变化状况一致,均呈现先降低后升高再降低的趋势。地温随土壤深度的增加而降低,地表下10 cm、20 cm的土壤层区域温度高于10.6℃、8℃,有利于烟苗在低温环境下正常生长。且土壤温度存在一定的昼夜温差,更利于烟苗发育。埋设稻壳的温室土壤层温度变化趋势与未埋设稻壳的温室一致,但其土壤层平均温度、最低温度至最高温度以及昼夜温差分别升高了2.3℃、1.1—5.5℃、...     

应用^45Ca示踪技术研究烤烟对钙肥的吸收及分布规律

应用放射性核素^45Ca示踪方法,以烤烟品种“K326”为材料进行盆栽试验,研究在不同施钙水平下,烤烟对钙素营养的吸收分配规律。结果表明：烟株在旺长期体内积存Ca量最高。烤烟吸收的Ca肥量随施Ca水平的提高或生育期的进程而增加,有显著的正相关性。将烟吸收的钙肥在烟株的地上部分主要分配在叶中,其次是茎,花与侧芽较少。烟叶中吸收Ca肥量占地上部分的百分数随生育期进程下降,而烟茎的却增加。烟株地上部吸收的Ca肥量占施入量的百分数随施Ca水平的提高而减少,但随生育期进程而增加。     

旺长期遮光及光照转换对不同肥料条件下烟草叶片光合特性的影响

采用盆栽方法研究了旺长期遮光及光照转换对不同肥料条件下烟草叶片光合速率(Pn)与荧光特性的影响。结果表明,遮光促进两种肥料条件下烟草叶片叶绿素(Chl)和类胡萝卜素(Car)含量的积累,却降低它们的Pn。其中施无机肥的烟草叶片Chl含量增加较多,而50%无机肥+50%饼肥配施烟草叶片的Car增幅显著,从而,施无机肥烟草叶片Chl/Car上升,而无机肥+饼肥配施烟草叶片Chl/Car却下降。无论从自然光转至遮荫条件下还是从遮荫条件转至自然光下,两种肥料条件下生长的烟草叶片的Pn、实际光化学效率(ΦPSⅡ)、最大光化学效率(Fv/Fm)及光化学猝灭系数(qP)均急剧下降,但与施无机肥相比,无机肥+饼肥配施烟草叶片保持较高的Pn、ΦPSⅡ、Fv/Fm、及qP,可能是饼肥促进了栽培条件下烟草植株的光生态适应性。