重要检疫性杂草刺萼龙葵分子生物学检测的研究

刺萼龙葵属于茄属植物,是我国规定禁止进境的检疫性有害杂草。选取与刺萼龙葵种子形态非常近似的同科5种植物为研究对象,依据茄科植物GBSSI基因序列差异,设计刺萼龙葵的特异PCR引物SRSF1/SRSR1,扩增片段约为500bp,用于对刺萼龙葵的特异检测。本研究建立了刺萼龙葵快速简便、稳定可靠的分子生物学检测方法,一个工作日内完成检测。     

DNA条形码、形态学与地理分布相结合的植物果实(种子)鉴定方法——以口岸截获的外来入侵植物犬蔷薇(Rosa canina L.)为例

以单一方法鉴定物种具有参考文献(或比对数据)少、专业要求高的缺点。以新疆口岸进境货车截获的未知植物果实为例,从分子、形态、地理分布3个维度,对该未知果实逐级进行分类鉴定。根据ITS、atp F-H、psb K-I 3段植物DNA通用条形码序列,以及形态学和地理分布信息,最终确定该未知果实为原产于西亚的蔷薇科蔷薇属犬蔷薇(Rosa canina L.)。本方法简单、准确,弥补了现有单一检测鉴定技术的不足,适用于进境产品中植物果实或种子的检疫鉴定。     

番茄茎腐病菌的分子鉴定及致病力检测

本研究采用形态学观察、核糖体DNA转录间隔区(ITS)克隆、系统发育树和致病力检测等方法对河南周口地区番茄茎腐病菌进行分子鉴定和致病力检测,以期为茎腐病的抗病育种及病害防治提供一定的理论依据。形态学观察结果显示,从周口地区番茄上分离的茎腐病病原菌属于镰孢属,ITS序列分析及系统进化树分析进一步确定其为茄镰孢,分离物与茄镰孢福建分离物(JN232141.1)亲缘关系最近,聚在一个进化支上,致病力检测结果表明在测试的植物中该病菌对茄科植物龙葵的致病力最强,该病菌寄主范围广,可侵染茄科、十字花科多种植物。     

一品红亚属杂草DNA条形码的筛选研究

为筛选可用于一品红亚属植物鉴定的DNA条形码序列,通过比较ITS、ITS2、trn H-psb A、rbc L、ndh F的种内和种间变异,进行Barcoding gap检验和鉴定成功率分析。结果表明:ITS、ITS2和trn H-psb A的种内种间变异数据显示明显好于rbc L、ndh F。在Barcoding gap检验和鉴定效率分析方面,trn H-psb A明显好于其他4个区域,但该区域也存在一些明显的缺点。而ITS2与ndh F的组合序列,明显好于其单条序列,有望成为一品红亚属植物DNA条形码序列。     

美国苜蓿草中鳞球茎茎线虫的鉴定

2015年,宁波口岸从美国苜蓿草中分离到一种茎属线虫,通过形态学和分子生物学方法,鉴定为鳞球茎茎线虫,这是我国口岸首次从牧草中截获该线虫。部分茎属线虫的核糖体28S和ITS基因DNA序列分析表明,28S基因是茎属线虫合适的DNA条形码基因。     

刺萼龙葵的入侵机理与控制策略研究进展

刺萼龙葵(Solauum rostratum Dunal.)是一种世界性的恶性入侵杂草,扩散速度快,对环境为害严重。结合近年来的研究热点,从扩散机制、生物学特性、环境的可入侵性、植物与土壤的反馈作用以及防控与利用基础等多个方面解析其入侵扩张机制和控制策略。主要研究进展:1刺萼龙葵强大的繁殖和扩散能力、高度的抗逆性、入侵地植物种类的单一性以及入侵环境的脆弱性在其入侵过程中起着关键作用;2刺萼龙葵具有丰富的遗传多样性,在不同生境的长期作用下已产生一定程度的分化,形成了不同的地理种群;3刺萼龙葵不同生长阶段通过改变根际土壤微生物数量和种类来调节自身生长,并通过改变入侵地土壤微生物群落结构和功能,创造对自身生长有利的土壤环境,形成一个自我促进式的入侵机制;4采用人工物理清除和植物替代控制相结合的生态修复方法,可防止刺萼龙葵的再次入侵。刺萼龙葵的有效控制需要采取综合防治策略;5刺萼龙葵从药物开发和植物保护方面有很大的潜力可以挖掘。     

外来刺萼龙葵与本地龙葵的比较

外来入侵植物刺萼龙葵与我国本地龙葵在分类学上同属茄科（Solanaceae）茄属（Solanum）,但在形态特征、发生分布、生物学特性、化学成分及生物活性、重要性及利用价值方面却存在差异。本文主要从以上几个方面对刺萼龙葵和龙葵进行论述比较,以期为这2种茄属植物的深入研究提供参考。     

刺萼龙葵PDS基因VIGS载体的构建

病毒诱导的基因沉默(VIGS)是通过插入目的基因片段的重组病毒来抑制植物内源基因表达的遗传技术,主要用于基因的功能分析。茄科植物刺萼龙葵(Solanum rostratum)是一种外来恶性杂草,研究证实,在农杆菌GV3101介导下,刺萼龙葵的八氢番茄红素脱氢酶(PDS)基因能被部分沉默,导致叶片和花朵出现白化表型。半定量RT-PCR检测显示,被侵染叶片和花朵的mRNA显著降解。VIGS沉默体系的建立可适用于研究刺萼龙葵的部分功能基因,有助于深入了解其生长发育调控的分子机制。     

景天属多肉植物基于ITS条形码鉴定探析

在多个条形码基因研究的基础上,本研究以ITS序列为目的片段通过DNA条形码技术对口岸截获的景天属多肉植物进行快速鉴定。采用通用引物扩增景天属10种32个供试样本的ITS片段并双向测序拼接,利用MEGA7软件进行序列比对,分析种内种间K2P距离并作barcoding gap,构建系统发育树。结果表明供试材料ITS序列的种内变异明显小于种间差异,种内种间变异存在明显的间隔区,每种的所有样品在NJ树上严格聚成单系,种间区分明显。初步判定ITS具有作为景天属多肉植物DNA条形码的潜在可能。     

大穗看麦娘与日本看麦娘的DNA条形码鉴定

大穗看麦娘是我国麦田新发生的恶性杂草,与日本看麦娘苗期形态相近,导致难以识别和有效监测。本研究利用4个DNA 条形码候选序列(rbcL、matK、trnH-psbA和ITS2)对13份大穗看麦娘和10份日本看麦娘叶片材料进行分子鉴定,采用Vector NTI分析扩增的DNA序列峰图质量并比对碱基差异,通过MEGA 6.0软件中的K2P模型计算样本种内和种间的双参数遗传距离,采用邻接法构建系统发育树。结果表明,4个DNA 条形码序列中仅matK扩增测序结果不理想。日本看麦娘在4种DNA条形码序列中不存在种内差异,大穗看麦娘在rbcL、matK和ITS2序列中无种内差异,仅在trnH-psbA序列中存在7个差异位点。大穗看麦娘和日本看麦娘种间各DNA条形码序列均有差异,trnH-psbA、rbcL、matK和ITS2序列存在的差异位点数分别为6、3、14和28。ITS2的种间平均遗传距离大于rbcL,且具有特异性,适宜用于大穗看麦娘和日本看麦娘的准确鉴定。     

入侵植物黄花刺茄(Solanum rostratum Dunal.)在新疆的分布及其群落特点

黄花刺茄原产北美,2005年首次在新疆乌鲁木齐县萨尔达坂乡和昌吉市三工镇发现。采用样方法,对不同生境中黄花刺茄分布相对集中的14个样地所有物种的多度、频度和重要值进行观测和分析,进一步明确黄花刺茄在新疆的分布区及其群落特征,并揭示其最适生境。结果表明：黄花刺茄分布于新疆乌鲁木齐市(县)、昌吉市、石河子市、吐鲁番市和托克逊县,分布在海拔-12～1 325 m处。生境类型包括荒漠草原、荒漠和绿洲。在3种生境中,黄花刺茄的相对多度均处于首位,且在绿洲中达到最大值,是入侵区域最为重要的物种;在相对盖度上均为第一,且绿洲(54.27％)＞荒漠草原(35.07％)＞荒漠(26.40％)。基于重要值数据的分析表明,黄花刺茄在3种生境中的综合适应力均最大,且以绿洲中尤为突出,表明黄花刺茄在新疆荒漠草原和荒漠生境中尚处于局部危害阶段,但在绿洲中已处于蔓延期。     

Investigation and integrated molecular diagnosis of root-knot nematodes in Panax notoginseng root in the field

Root-knot nematodes (RKNs, Meloidogyne spp.) are damaging pests that can infect thousands of plant species and cause enormous crop losses worldwide. Panax notoginseng is a common host of root-knot nematodes. In this study, we surveyed notoginseng gardens and determined the incidence of RKNs. Among the gardens surveyed, 71 % were infected with RKNs, and the RKN incidence index ranged from 8 % to 47 % in three randomly infected gardens. Meloidogyne hapla was identified as the pathogenic nematode based on 18S ribosomal RNA analysis by DNA barcoding. The results were qualitatively and quantitatively confirmed using a real-time PCR assay according to variations in the ITS1 and ITS2 regions. These results indicated that the combination of DNA barcoding and real-time PCR is a reliable and precise method for identifying parasitic nematodes from mixed-infected plant roots in the field. In addition, the abundance of ITS1 and ITS2 displayed a similar trend to the numbers of RKNs in the three gardens, which suggests that the results of real-time PCR can be used to determine the damage caused by M. hapla in the field. Our studies show that RKNs are common and can cause serious damage to notoginseng. We present an integrated method of detecting mixed nematode species in the field and confirm M. hapla as the target for parasitic nematode control in notoginseng gardens. Our results contribute to the improvement of RKN control in notoginseng and further promote the sustainable development of medicinal plants.     

黄花刺茄浸提液对番茄种子萌发及幼苗生长的影响

为明确入侵植物黄花刺茄对当地主栽经济作物番茄种子萌发和幼苗生长的影响,本文采用培养皿滤纸法,在智能光照培养箱的变温控制条件下,分别研究了黄花刺茄根、茎、叶浸提液对番茄种子发芽率、发芽指数及幼苗根长和苗高的影响。结果表明:(1)黄花刺茄不同器官的浸提液对番茄种子发芽率、发芽指数、幼苗根长和苗高的影响不同,其茎、叶浸提液的化感作用较为显著(P<0.01)。(2)黄花刺茄同部位浸提液对番茄种子发芽率、发芽指数、幼苗根长和苗高不同指标的影响也不同。根浸提液对种子发芽率、幼苗根长影响不显著(P>0.05),但对种子发芽指数的影响极显著(P<0.01)。茎和叶浸提液各浓度对种子发芽指数的影响比种子发芽率、幼苗根长和苗高的影响显著(P<0.01)。     

Development of a PCR-based diagnostic test for Spongospora subterranea f.sp. nasturtii, the causal agent of crook root of watercress (Rorippa nasturtium-aquaticum)

The polymerase chain reaction (PCR) technique was utilized to obtain internal transcribed spacer ribosomal DNA (ITS rDNA) and small-subunit (18S) rDNA sequences from UK isolates of Spongospora subterranea f.sp. nasturtii , a plasmodiophorid pathogen of watercress ( Rorippa nasturtium-aquaticum ). ITS sequence data obtained from S. subterranea isolated from a range of UK sites were found to be identical. PCR primers were designed using these sequences and were shown to be capable of specific amplification of S. subterranea f.sp. nasturtii DNA from plant tissue and from water samples containing zoospores of the pathogen. As little as 5 ng total genomic DNA from infected plant material, or 1000 zoospores, was required for consistently successful amplification of DNA. A filtration-based method for obtaining pathogen DNA for PCR from watercress-bed water was developed.     

Molecular taxonomy and its application to ecological studies of Pythium species

More than 140 Pythium species have been described; 41 of these species have been described since 2000. Many Pythium species cause seed rot and damping off of seedlings. Recent changes in crop culture systems, such as the introduction of hydroponic cultures, have resulted in Pythium diseases of mature plants as well, with symptoms such as root rot, crown rot, and blight. In Japan, newly introduced ornamental and floral plants are vulnerable to high temperatures and humidity, resulting in severe Pythium infections. Once a pathogen invades a greenhouse, it quickly propagates and disperses, resulting in serious yield loss. It is very difficult to identify an isolate at the species level based on morphological characteristics because of overlaps in the size and shape of reproductive structures, heterothallism, and the continuing discovery of new species. New procedures based on DNA sequences have thus been introduced to identify species; in particular, the rDNA ITS region and the coxI gene have been recommended for DNA barcoding. BLAST searches and phylogenic analyses allow for easy and precise identification. PCR techniques are also being used to diagnose plant diseases, and real time PCR can be used for quantifying the pathogen to assess the risk of disease loss. Molecular techniques to analyze population structure help clarify transmission routes of pathogens and the ecology of Pythium flora in natural ecosystems. In this review we discuss recent advances in molecular methods for identifying, detecting, and classifying Pythium species.     

Review of the current situation for Solanum elaeagnifolium in the Mediterranean Basin

Silverleaf nightshade (Solanum elaeagnifolium, Cav.) is one of the important invasive plant species in Mediterranean Basin countries. Over the last 60 years, this plant has gone from a few accidental introductions to near monospecific populations in many areas of the Mediterranean Basin (in particular Greece and Morocco). Recent findings from Lebanon show that the invasion is ongoing. Solanum elaeagnifolium has a negative impact on crops, causing up to 75% yield loss, as well as an indirect impact by harbouring plant pests and diseases. Solanum elaeagnifolium is toxic to livestock and has a negative affect on the quality of life for humans. Losses of potential resource or revenue caused by its invasion include: decreased forage quality on grazing lands; decreased cropping land and amenity values of public space; increased water loss; increased water conveyancing costs; and increased forest restoration costs. Available control techniques need to be strengthened to reduce the impact of S. elaeagnifolium and prevent its spread. More attention needs to be devoted to biological control, which could provide regional management of this invasive alien plant. Sustainable management of S. elaeagnifolium will require coordination, education and support across the affected countries. Governments must have the means to detect, manage and control S. elaeagnifolium.     

烟草叶围细菌Tpb55菌株的鉴定及其抑菌活性

Tpb55是从烟草叶面生境中分离获得的一株拮抗细菌,本研究从形态、生理生化、16S-23S rDNA转录间隔区PCR（ITS-PCR）分析,初步明确了该菌株的分类地位。光学显微镜下观察到Tpb55菌体为杆状,革兰氏染色均匀,鞭毛周生,产芽孢,各项生理生化测试指标与枯草芽孢杆菌菌株表现一致;16S-23S rDNA ITS-PCR分析结果表明,Tpb55与枯草芽孢杆菌同源性达到99.75%。Tpb55代谢产物对7种病原真菌和3种病原细菌均有不同程度的抑菌作用,表明该菌株代谢产物具有较高的抑菌活性和较广的抑菌谱。进一步研究发现,该菌株代谢产物对有机溶剂、蛋白酶、热和pH值都有很高的稳定性,是具较好开发潜能的生防菌株。