柑橘溃疡病菌LAMP实时浊度检测方法的建立及应用

以柑橘溃疡病菌特异性高的假定蛋白基因（登录号：AE008923.1）为靶标，设计并筛选4条引物来特异性识别靶标序列中的6个独立区域，建立柑橘溃疡病菌LAMP实时浊度检测方法。该方法利用实时浊度仪检测反应过程中产生的焦磷酸镁白色沉淀，实现对LAMP整个反应过程的实时监控。从镁离子浓度、甜菜碱浓度、dNTPs浓度、引物浓度和反应温度5个变量参数对 LAMP检测体系进行优化，并对方法的特异性、灵敏度、稳定性及实际样品检测效果进行评价。结果显示，该体系经优化后稳定性良好，可在66 ℃ 恒温条件下，60 min内完成检测；DNA检测下限可达0.02 ng/μL，灵敏度与荧光定量PCR方法相同，比常规PCR方法高1个数量级；能快速、准确地对田间疑似样品进行检测，与荧光定量PCR方法的检出情况一致，没有漏检。结果说明柑橘溃疡病菌实时浊度LAMP检测方法操作简便、所需时间短，特异性与灵敏度高，为柑橘溃疡病菌的快速筛查提供较好的途径。     

进口大豆中菜豆晕疫病菌巢式PCR检测方法的建立

为实现对进口大豆样品中菜豆晕疫病菌的快速检测,本研究根据菜豆晕疫病菌的argK特异性序列设计引物52B/8F和24B/24F,建立了巢式PCR检测方法,并进行特异性和灵敏度验证。试验结果表明,利用此检测方法对多种参试菌株进行检测时,只有菜豆晕疫病菌呈阳性反应,而其他病菌不产生扩增反应;巢式PCR检测方法对菜豆晕疫病菌基因组DNA和菌悬液检测时,其灵敏度分别达到0.916×10-4ng/μL和2.4×103CFU/m L,比常规PCR灵敏度高1 000倍。在对100份进口大豆样品检测时,3份样品扩增到了特异性条带,测序分析结果证明3份大豆样品中携带的病菌确实为菜豆晕疫病菌。本研究所建立的巢式PCR检测方法具有特异性强、灵敏度高、检测时间短并且检测准确率高等特点,可用于口岸菜豆晕疫病菌的检测。     

巢式PCR检测琯溪蜜柚黑斑病病原菌

利用特异性引物从琯溪蜜柚黑斑病菌基因组DNA中扩增出一条分子量为487 bp的特异性条带,准确地与疮痂病、炭疽病和黄斑病菌等蜜柚常发性真菌病害区分开.采用巢式PCR对该引物的检测灵敏度进行测定,结果显示,对于黑斑病菌基因组DNA,巢式PCR的检测灵敏度为100 fg/μL,灵敏度比常规PCR至少提高100倍;对于发病组织,巢式PCR可从病斑组织含量为100 μg的DNA样品中检测到黑斑病菌.采用巢式PCR检测技术,可从蜜柚的黑斑病显症组织和未显症组织特异性地检测到病原菌,且检出率分别为96.67％和90％.     

柑桔黄龙病环介导等温扩增检测方法建立及应用

以柑桔黄龙病（Citrus Huanglongbing,HLB）病原菌16s rDNA保守区域为靶标设计LAMP引物,建立柑桔黄龙病的环介导等温核酸扩增（loop-mediated isothermal amplification assay,LAMP）检测技术。LAMP检测方法具有极高的检测特异性和灵敏性,其检测下限约为1 pg/μL质粒DNA,是PCR检测灵敏度的100倍,能快速、准确地对田间疑似样品进行检测。建立的柑桔黄龙病LAMP检测方法是对黄龙病检测方法的拓展和延伸,可以很好地应用于田间检测,可满足基层以及科研单位对该病害检测的需要。     

常规和巢式PCR对柑橘黄龙病菌的检测灵敏度比较

根据柑橘黄龙病(citrus Huanglongbing,HLB)病菌16SrDNA、16S/23S核糖体基因间隔区(ribosomal intergenic region,RIR)、核糖体蛋白β操纵子(β-operon)和外膜蛋白(out membrane protein,OMP)基因序列设计了8对引物,通过常规和巢式PCR方法对各引物的检测灵敏度进行了比较。结果表明,不同引物的检测灵敏度不同。对于上述任何基因,巢式PCR的灵敏度均比常规PCR至少高103倍;对于同一种基因,扩增短片段的引物比扩增长片段的引物灵敏度高或相当;在扩增产物大小相同时,用以扩增核糖体蛋白β操纵子基因的引物较其他三种稍高。对不同症状柑橘样品的检测进一步验证了该结果。因此,对于柑橘黄龙病的检测,可优先考虑使用巢式PCR方法,若使用常规PCR,则宜选用具有高灵敏度的扩增小片段引物。     

环状等温扩增技术快速检测转基因玉米MON863的研究

采用环状等温扩增技术(Loop-Mediated Isothermal Amplification,LAMP),针对转基因玉米品系MON863中玉米基因组与外源基因结合处设计4条特异性引物,建立MON863的快速检测技术体系,并对时间、温度等反应条件及体系灵敏度、特异性等进行探索。结果表明:该检测体系最适反应温度为63℃,反应时间60 min,灵敏度是常规定性PCR的10倍。该检测方法具有高度的特异性和稳定性,操作方便、简单、省时。     

基于环介导等温扩增技术快速检测茶白星病菌的方法研究

为建立检测茶白星病菌的快速实用分子生物学技术,以痂囊腔菌DNA为模板,根据这种茶白星病菌的基因序列,设计并筛选了4条针对目的基因6个位点的特异性引物,优化的体系条件外引物与内引物比例为1∶3,最佳反应温度与时间分别为63℃与1 h。灵敏度测试LAMP比PCR高10倍,特异性测试痂囊腔菌呈阳性,而炭疽病菌则呈阴性。结果表明,该方法快速、准确、灵敏、特异,具有良好的实用性。     

甘蔗黑穗病菌和赤腐病菌双重PCR检测体系的建立

目前,针对甘蔗黑穗病菌与赤腐病菌单个病菌检测体系已相继建立,但是仍然未见两个病原菌的双重PCR检测方法。在本研究中,根据黑穗病菌交配型b E基因、甘蔗赤腐病菌特异性SCAR片段序列的特异性引物,在二者单一PCR检测体系的基础上,建立并优化可同时检测黑穗病菌和赤腐病菌的双重PCR检测方法。建立的双重PCR检测体系可从黑穗病菌与赤腐病菌样品中分别扩增出320 bp和442 bp的特异条带。灵敏度分析结果表明,对混合模板黑穗病菌与赤腐病菌的最低检测水平量均为0.1ng。     

发酵豆乳主要乳酸菌实时荧光定量PCR检测方法的建立

为快速检测发酵豆乳中主要乳酸菌植物乳杆菌、副干酪乳杆菌含量,建立实时荧光定量聚合酶链式反应检测方法。根据植物乳杆菌、副干酪乳杆菌保守区域设计特异性引物和探针,验证建立的实时荧光定量PCR法的特异性、灵敏度和重复性,并与国标法进行比较。结果表明:引物特异性强,实时荧光定量PCR法特异性及重复性较好;植物乳杆菌和副干酪乳杆菌的检验灵敏度分别达到1.3×10~(-4)和1.0×10~(-5) ng·μL~(-1)。分别建立植物乳杆菌和副干酪乳杆菌标准菌株的实时荧光定量PCR检验法的标准曲线,得出R~2分别为0.994 3和0.999 6,表明线性关系较好,可进行发酵豆乳中2种菌株含量的检测,测得供试发酵豆乳中植物乳杆菌与副干酪乳杆菌比例为4∶1。实时荧光定量PCR法测得发酵豆乳中乳酸菌总量为(5.5±0.26)×10~9 CFU·mL~(-1),国标法检测为(5.3±0.43)×10~9 CFU·mL~(-1),2种方法检测结果无差异(P0.05),表明建立的实时荧光定量PCR方法可快速、准确地检测出发酵豆乳中植物乳杆菌和副干酪乳杆菌的含量。     

抗草甘膦转基因大豆及加工品LAMP检测研究

将环介导的等温核酸扩增技术应用于转基因大豆及加工品检测.针对抗草甘膦Roundup Ready转基因大豆及加工品外源基因EPSPS设计2对特异性引物进行扩增,成功建立起定性检测转基因大豆及加工品的LAMP检测方法.优化LAMP反应条件,反应温度为65℃,反应时间为1h.结果表明:该体系能快速、灵敏、有效地检测转基因大豆及加工品中整合的EPSPS基因,检测限为0.01%,低于国际现行最低检测量0.5%的要求.检测EPSPS基因操作简单,成本低、特异性强、灵敏度高.LAMP检测结果可信,稳定性好,可对目前批准的抗草甘膦RoundupReady转基因大豆及加工品进行定性检测.     

Comparative Assessment of the Efficacy of Various Nitrogen Fertilizers

Summary

The efficiency of N fertilizers is usually poor; often less than 50% of the applied N is taken up by the crop. This review focuses on various N fertilizers with respect to the significance of different N loss pathways, namely (i) ammonia volatilization, (ii) dinitrogen and nitrogen oxide emissions, and (iii) nitrate leaching. Further, the significance of biological N immobilization, ammonium fixation and, finally, the impact of nitrate vs. ammonium uptake on crop yield are also discussed. The reviewed literature shows that N fertilizers may differ markedly in their susceptibility to losses. There is, however, considerable scope to improve N efficiency of each N source by proper N management practices.     

Novel food and non-food uses for sorghum and millets

Sorghum and millets have considerable potential in foods and beverages. As they are gluten-free they are suitable for coeliacs. Sorghum is also a potentially important source of nutraceuticals such antioxidant phenolics and cholesterol-lowering waxes. Cakes, cookies, pasta, a parboiled rice-like product and snack foods have been successfully produced from sorghum and, in some cases, millets. Wheat-free sorghum or millet bread remains the main challenge. Additives such as native and pre-gelatinised starches, hydrocolloids, fat, egg and rye pentosans improve bread quality. However, specific volumes are lower than those for wheat bread or gluten-free breads based on pure starches, and in many cases, breads tend to stale faster. Lager and stout beers with sorghum are brewed commercially. Sorghum's high-starch gelatinisation temperature and low beta-amylase activity remain problems with regard to complete substitution of barley malt with sorghum malt . The role of the sorghum endosperm matrix protein and cell wall components in limiting extract is a research focus. Brewing with millets is still at an experimental stage. Sorghum could be important for bioethanol and other bio-industrial products. Bioethanol research has focused on improving the economics of the process through cultivar selection, method development for low-quality grain and pre-processing to recover valuable by-products. Potential by-products such as the kafirin prolamin proteins and the pericarp wax have potential as bioplastic films and coatings for foods, primarily due to their hydrophobicity.     

Evolutionary aspects of the trade-off between seed size and number in crops

Whereas the concept that availability of resources drives seed production is sound in principle, it is incomplete as there are many solutions to the allocation of resources that derive from the trade-off between number and size. This paper examines evolutionary aspects of this trade-off in annual grain crops. The analysis is centred on the working hypotheses that, for a given species and environment, allocation of resources to reproduction involves (H1) high plasticity in seed number, which allows for variable resource availability, and (H2) a relatively narrow range of seed size that results from evolutionary and agronomic selection. Comparisons between crops and fish are used to highlight common evolutionary elements in taxa where parents provide little or no care to their offspring, with the consequence that both number and early survival of offspring, hence fitness of parents, are partially related to embryo size and reserves.

The plasticity of seed number in relation to availability of resources is analysed against the established relationship between offspring number and parent growth rate during critical stages. The notion that seed size is under stabilising selection is analysed against three conditions: (1) mean seed size is conservative for a given species and environment, (2) seed size affects fitness, and (3) seed size is heritable. Databases from published papers were compiled to analyse the relative variability of seed size and number, and the heritability of seed size. Evidence for and against the link between seed size and parental fitness is revised using the Smith–Fretwell model as framework (Am. Nat., 108, 499–506).

The proposal of high plasticity of seed number and narrow variability of seed size resulting from stabilising natural selection is generally consistent with evolutionary and genetic considerations. Agronomic selection may have reinforced natural selection leading to relatively narrow seed size in species such as wheat and soybean, where cultivated types retained high plasticity for seed number. In contrast, selection for one or few inflorescences in crops like sunflower and maize may have morphologically reduced seed number plasticity and increased variability of seed size and its responsiveness to resource availability in relation to their wild ancestors.     

Citrus Flavonoids in Fruit and Traditional Chinese Medicinal Food Ingredients in China

Flavonoids-enriched tissues of citrus such as peel, immature fruit and flower are consumed as culinary seasonings, tea ingredients in China for centuries. This HPLC quantitative study on the five citrus flavonoids, naringin, hesperidin, neohesperidin, sinensetin and nobiletin on a wide range of Chinese citrus fruits and several Traditional Chinese Medicinal food ingredients in East China, revealed a great diversity in flavonoid composition. Huyou peel (C. paradisi cv. Changshanhuyou) was found to be the best naringin (3.25%) and neohesperidin (2.76%) source; C. aurantium, a major ingredient of several citrus-related TCM, is also a suitable source of naringin and neohesperidin, and a good juice source for flavanone glycosides; the peel of Wenzhoumiju (C. unshiu) is one of the richest local species in hesperidin (up to 6.25%); Zaoju (C. subcompressa) has the highest content of nobiletin (0.59%), a polymethoxylated flavone. LC-ES-MS analysis of Zanthoxylum genus for flavonoids revealed for the first time the presence of significant amounts (0.74%) of hesperidin in the root of Liangmianzhen (Z. nitidum (Roxb.) DC), a relative of Sichuan pepper, which is a spice widely used in China.     

Effect of a continuous hot water treatment of potato tubers on seed-borne fungal pathogens

Summary The viability of five pathogens was decreased by treatment with hot water when tested in vitro.Polyscytalum pustulans was most sensitive andRhizoctonia solani least sensitive. Potato tubers were exposed to 55°C for 5 min in a commercial continuous hot water treatment plant using naturally contaminated seed tubers and tubers which had been inoculated by dipping in comminuted cultures. The frequency of eyes colonised byP. pustulans, Helminthosporium solani, andR. solani was reduced to virtually zero and the effect persisted on tubers subsequently stored at 4°C and at 15°C for up to 16 wk. Results withColletotrichum coccodes were inconclusive. Treatment suppressedPenicillium spp. which, however, rapidly recolonised the eyes during storage, leading to higher contamination levels in the treated than in the untreated tubers. With tubers inoculated withPhoma foveata, good control was achieved when the incubation period before treatment was 10 d but not when the period was 42 d.     

Role of Benzoxazinones in Cereals

Summary

Heterocyclic 1,4-benzoxazin-3-ones and related benzox-azolinones are important natural products of the cereal crops maize, wheat and rye. Much research has focused on the role of these compounds as defensive agents against plant diseases and pests. Studies of a wide variety of biological effects on herbivores and nematodes, plant pathogenic fungi and bacteria are reviewed in this article. Phytotoxic activity of the compounds is also considered with respect to allelopathic interaction with higher plants. Recent investigations of molecular aspects of interactions of cyclic hydroxamic acids and related benzoxazo-linones with acceptor organisms have demonstrated different effective detoxification strategies and tolerance mechanisms. This research has greatly advanced our knowledge about the multiple roles that these allelochemicals play in ecological systems.     

Potential of dry fractionation of wheat bran for the development of food ingredients,part I: Influence of ultra-fine grinding

Wheat bran is an undervalued by-product of white flour and has great nutritional potential due to its high content in fibres and bioactive compounds. Micronized bran could be used as a food ingredient to improve the nutritional potential of cereal products, or be used as a starting material for other processes (bioactive compound extraction or bran fractionation). The aim of this work was to find a way to efficiently decrease the particle size of bran. The influence of the grinding temperature (ambient or cryogenic grinding) on the granulometric distribution of particles, their composition, and their microstructure was studied, at lab-scale and pilot-scale. It showed that the intrinsic characteristics of bran (glass transition within intermediate layers at −46 °C) had more influence on its grinding behaviour than the type of grinding device used: the particles size distributions obtained after grinding at lab-scale and pilot-scale were very similar. At both scales, the granulometric curves were narrow for cryogenic grinding, while for ambient grinding they were spread over the whole particle size range. Ultrafine particles were obtained in both ambient and cryogenic conditions. Negative temperatures, by increasing the material’s brittleness, favoured a fast fragmentation of bran: one step of cryogenic grinding allowed a median particle size of nearly 50 μm to be reached, whereas three successive steps of ambient grinding were needed for the same result. On the other hand, ambient temperature favoured the dissociation of the different constituent layers of wheat bran, and produced less composite particles than cryogenic grinding.     

Multiple use of plants: Studies on selenium incorporation in some agricultural species for the production of organic selenium compounds

The technique of spraying plants with inorganic forms of selenium can be employed for phytochemical production of organic selenium compounds. Fractionation of the plant material makes it possible to produce a highly concentrated and well defined selenium supplement with potential use in animal and human nutrition. The fractionation also gives opportunities to combine production of organic selenium compounds with other products, for example plant fibres. Multiple use of plants can contribute to a more efficient utilization of land area (in comparison to monocultures solely adapted to food production). It also gives the opportunity to develop systems suitable for long term fixation of carbon, as long as the plant material is not reoxidised to carbon dioxide. Plant fibres could provide raw material for the production of paper or building materials in combination with the production of organic selenium compounds preferentially accumulated in another fraction of the processed plant.     

Biological factors influencing the transmission of potato leafroll virus by different aphid species

Summary Macrosiphum euphorbiae, collected in the field from potato plants infected with potato leafroll virus (PLRV), transmitted the virus to fewer potato plants in a field trial than did laboratory-rearedMyzus persicae. In the laboratory,M. persicae was the only efficient vector of PLRV fromPhysalis floridana seedlings, potato sprouts or excised leaves toP. floridana. Two clones ofM. euphorbiae and one clone ofAulacorthum solani transmitted PLRV from infected potato plants toNicotiana clevelandii as effeciently asM. persicae but a clone ofAphis gossypii was an inefficient PLRV vector. An isolate of PLRV, whichM. persicae transmitted inefficiently from potato toN. clevelandii, was also transmitted inefficiently byM. euphorbiae andA. solani.