水稻细菌性谷枯病菌和水稻白叶枯病菌荧光RPA快速检测方法的建立

本研究分别针对我国进境植物检疫性有害生物水稻细菌性谷枯病菌及水稻白叶枯病菌建立了光RPA检测方法，并对其灵敏度、特异性以及对实际种子样本的检测能力进行了测试评价。结果表明，本研究建立的两种方法均能够在20 min内特异性检测到目标菌株，对水稻细菌性谷枯病菌(Burkholderia glumae)菌液的检测下限达到了11.4 CFU/反应，DNA检测下限达到了4.83×10^-5ng/反应；对水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae)菌液的检测下限达到了4.42 CFU/反应，DNA检测下限达到了3.83×10^-4ng/反应。两种方法均能够成功应用于带菌种子样品的快速检测。     

向日葵黑茎病菌RPA/CRISPR-Cas12a快速检测方法的建立

向日葵黑茎病菌是向日葵上的一种毁灭性真菌,是我国的植物检疫性有害生物。为了准确快速检测向日葵黑茎病菌,本研究根据向日葵黑茎病菌及其近似种的ITS序列差异,设计特异性RPA引物和CRISPR-Cas12a crRNA,建立了RPA等温扩增技术结合CRISPR-Cas12a检测的快速检测方法。通过条件优化,RPA/CRISPR-Cas12a检测体系在37℃恒温条件下,RPA反应30 min,CRISPR/Cas12a反应20 min,即可特异性检测向日葵黑茎病菌。荧光法检测灵敏度与实时荧光PCR的灵敏度相当,最低检测量为0.1 pg,试纸条法检测最低检测量为1 pg。由于试纸条检测结果可用肉眼观察,快速便携,操作简单,更适合用于田间和口岸的向日葵黑茎病菌快速早期检测;而荧光检测灵敏度高,对环境要求高,更适合用于实验室检测。     

两种向日葵检疫性真菌病害的多重DPO-PCR检测方法

本研究根据向日葵白锈病菌大亚基核糖体RNA基因序列,向日葵黑茎病菌的ITS-5.8S r RNA基因序列,分别设计特异性DPO(dual priming oligonucleotide)引物,建立同时检测这两种检疫性病菌的多重DPO-PCR检测方法,并对其特异性和灵敏度进行评价。结果表明,所设计的DPO引物特异性强,仅向日葵白锈病菌和向日葵黑茎病菌可分别扩增出307 bp与388 bp的特异性条带,其他参照菌株及阴性对照均无条带;检测体系对混合模板中向日葵白锈病菌和向日葵黑茎病菌的DNA灵敏度均达0.05 ng/μL;且该检测方法对退火温度不敏感,适用范围广。该方法能够准确、快速的检测向日葵白锈病菌和向日葵黑茎病菌,适合于口岸实验室的快速检测。     

向日葵黄萎病病原菌轮枝菌的多重DPO-PCR检测方法

为建立可同时快速检测引起向日葵黄萎病害的2种检疫性病原菌大丽轮枝菌Verticillium dahliae Kleb.和黑白轮枝菌V.albo-atrum Reinke et Berthold的方法,根据2种病原菌的β-tubulin基因分别设计特异性DPO引物,建立多重DPO-PCR检测方法,并对其特异性和灵敏度进行评价。结果表明,所设计的DPO引物特异性强,仅大丽轮枝菌和黑白轮枝菌可分别扩增出225 bp与151 bp的特异性条带,其它向日葵病害的7种病原菌及阴性对照均无目的条带;反应体系中引物终浓度为0.2μmol/L、退火温度为60℃时,30个扩增循环的检测灵敏度均可达0.05 ng菌丝DNA量;在45~65℃退火温度范围内均可高效扩增靶基因片段,表明该方法退火温度范围宽。所建立的检测方法能够准确、高效地检测引起向日葵黄萎病的大丽轮枝菌和黑白轮枝菌,可用于向日葵种子带菌筛查及田间病害诊断检测。     

番茄细菌性叶斑病菌RPA检测技术

番茄细菌性叶斑病菌(Pseudomonas syringae pv.tomato,Pst)是我国进境植物检疫性有害生物,可随种子进行远距离传播。快速简便的检测对于防止该病害的扩散传播具有重要意义。依据番茄细菌性叶斑病菌的hrpZPst基因序列,设计并筛选出特异性扩增引物,建立了番茄细菌性叶斑病菌的重组酶聚合酶等温扩增(RPA)检测方法。该方法可从10种不同的植物病原细菌中特异性地检测到3个参试番茄细菌性叶斑病菌株。对病菌DNA的检测灵敏度为75fg/μL,与PCR凝胶电泳相当。该方法扩增核酸时间短、效率高、对设备的要求低,适合于基层简易实验室的快速检测。本文为该病原菌的检测提供了新方法。     

小麦全蚀病菌重组酶聚合酶检测方法的建立及应用

由禾顶囊壳小麦变种Gaeumannomyces graminis var.tritici引起的小麦全蚀病是危害极大的小麦土传真菌病害。本研究以小麦全蚀病菌β-tubulin为靶标基因设计重组酶聚合酶扩增技术(recombinase polymerase amplification,RPA)引物Gg-RPA-F/Gg-RPA-R和RPA探针Gg-LF-Probe,结合侧流层析试纸条,建立了小麦全蚀病菌RPA快速检测方法,该方法在38℃恒温条件下30 min内完成可视化检测,摆脱PCR仪等仪器设备的限制。小麦全蚀病菌RPA快速检测方法具有较高的检测特异性,其检测极限为10 pg/μL,与普通PCR一致。此外,小麦全蚀病菌RPA快速检测方法可从土壤中快速检测到小麦全蚀病菌,具有较强的实用性。因此,本研究建立的小麦全蚀病菌RPA快速检测方法具备简便高效、实用性强的特点,为小麦全蚀病菌的快速检测和病害早期诊断提供新技术。     

不同引物PCR检测向日葵黑茎病菌的灵敏度比较及应用

为准确快速筛查进境油葵种子中携带的向日葵黑茎病菌Plenodomus lindquistii,采用常规PCR方法比较了3对向日葵黑茎病菌PCR检测引物320FOR/320RVE、LEPB/LEPF和LLF/LLR的灵敏度,对哈萨克斯坦进境120批油葵种子样品进行PCR检测,并对检测为阳性的样品进行产物序列测定及病原菌分离。结果显示,引物320FOR/320RVE的检测灵敏度最高,可达10-5ng/μL,引物LEPB/LEPF和LLF/LLR分别为10-4ng/μL和10-3ng/μL。利用引物320FOR/320RVE、LEPB/LEPF和LLF/LLR对120批进境油葵种子样品进行检测,阳性检出率分别为65%、50%和45%,阳性检出率高低与引物的检测灵敏度相一致。阳性样品扩增的产物序列与Gen Bank中向日葵黑茎病菌的序列同源性最高;分离的病原菌菌落为乳白色至灰白色,分生孢子器黑褐色、球形并产生透明状分生孢子液,分生孢子单胞、无色、肾型、两端有油球,与已报道的向日葵黑茎病菌的病原特征描述一致,初步鉴定哈萨克斯坦进境油葵种子中存在向日葵黑茎病菌。     

木尔坦棉花曲叶病毒RPA快速检测方法的建立

木尔坦棉花曲叶病毒Cotton leaf curl Multan virus(CLCuMuV)引起的病害是世界棉花生产上的毁灭性灾害,也是我国进境植物检疫性有害生物之一。因此,建立快速检测技术对CLCuMuV的检疫和防控具有重要意义。本研究根据CLCuMuV外壳蛋白(CP)基因序列设计引物,建立了该病毒的重组酶聚合酶等温扩增(recombinase polymerase amplification, RPA)检测方法,并评价了该方法的灵敏度和特异性,进一步测定了对田间疑似病样的检测准确性。结果表明,建立的RPA检测方法仅能从感染CLCuMuV的样品中扩增出目的条带,而感染同属的其他5种病毒的样品中未扩增出目的条带。该方法检测灵敏度是常规PCR的10倍,且对田间疑似病样的检出结果与PCR试验结果一致。因此,本研究所建立的CLCuMuV RPA快速检测方法具有特异、灵敏、准确、操作简便、无需特殊设备等优点,这些为CLCuMuV的快速检测提供了一种新技术。     

进境苹果中3种检疫性真菌三重PCR同步检测方法的建立

苹果壳色单隔孢溃疡病菌(Botryosphaeria stevensii)、美澳型核果褐腐病菌(Monilinia fructicola)和丁香疫霉病菌(Phytophthora syringae)是为害多种水果和其他植物的重要检疫性真菌,均能侵染苹果果实导致烂果,并造成严重的经济损失。本研究设计和筛选了3对特异性引物,建立了同步检测3种检疫性真菌的普通PCR和三重PCR方法。设计的引物Bs-368 F/R、M.cola 986F/R和Psy1/Psy2能分别特异性地扩增出B.stevensii、M.fructicola和P.syringae所有供试菌株的DNA,而对相应多个近似种的DNA则无扩增。灵敏度试验结果表明,上述3对引物分别检测B.stevensii、M.fructicola和P.syringae DNA的灵敏度是41.7 pg、47.4 pg和0.375 pg,三重PCR同时检测到3种病菌DNA的灵敏度是4.44 ng。该方法可以满足对进境苹果果实携带的苹果壳色单隔孢溃疡病菌、美澳型核果褐腐病菌和丁香疫霉病菌的快速初筛,加速进境新鲜水果的快检快放。     

向日葵黑斑病研究进展及其综合防治

向日葵黑斑病是一种重要的真菌病害,我国于20世纪60年代首次报道,目前对向日葵生产仍存在巨大威胁。本文综述了向日葵黑斑病的病原和寄主范围、向日葵黑斑病的症状和危害、国外种传黑斑病菌的检测、种传Alternaria helianthi对种子萌发和种苗活力的影响以及国内外有关黑斑病菌A. helianthi和A. alternata毒素的相关研究和向日葵黑斑病的综合防治。     

Molecular Variability Within Diaporthe/Phomopsis helianthi from France

ABSTRACT Diaporthe/Phomopsis helianthi causes brown stem canker of sunflower (Helianthus annuus) and is responsible for considerable yield loss. This species shows considerable variation for morphological characters, growth, and pathogenicity. Molecular variability of two sample groups was assessed with amplified fragment length polymorphism (AFLP) markers. Isolates of the first sample were collected from infected sunflower tissues from the main regions in France where the crop is grown, whereas isolates from the second sample came from stems within a single field of sunflower. A soybean strain was taken as an outgroup for AFLP analyses. Within sample one, the greatest genetic distance among isolates was 0.97, whereas it was 0.44 within sample two isolates. For the whole of France, the average genetic distance was 0.68, whereas in the one field it was 0.12. Nei's genetic diversity indices were 0.20 and 0.06 for France and for one field, respectively. The greatest genetic distance was found between isolates from the most northern crops. The greatest genetic distance between D. helianthi isolates and the strain isolated from soybean was similar to that observed for D. helianthi isolates from different geographical areas. The problems in defining the genus Phomopsis are discussed. It is shown that internal transcribed spacer sequencing could be a useful criteria for Diaporthe/Phomopsis species determination. The considerable genetic variability of the pathogen could lead to the occurrence of new strains that could be more aggressive or more resistant to chemical control.     

Interactions Between French Isolates of Phomopsis/Diaporthe Helianthi Munt.-Cvet. et al. and Sunflower (Helianthus annuus L.) Genotypes

Three artificial infection tests measuring the rate of mycelial growth of 7 Phomopsis/Diaporthe helianthi isolates were used on leaves, stems and capitula of 6 sunflower hybrids. Isolates and hybrids were chosen to cover the range of variability and resistance levels known at the present time. Significant genotype and isolate effects and isolate×genotype interactions were shown in all the tests, with some changes in order of hybrids according to the isolate used for infection. Consequences of interactions in breeding for stable resistance to P./D. helianthi are discussed.     

Molecular Detection of Diaporthe phaseolorum and Phomopsis longicolla from Soybean Seeds

ABSTRACT Species-specific detection of Diaporthe phaseolorum and Phomopsis longicolla from soybean seeds was accomplished using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and TaqMan chemistry. To use these detection systems, fungal DNA was released from soybean seed coats using an ultrasonic processor to break the cells. DNA fragment lengths ranged from 200 to 1,200 base pairs (bp), with the majority of fragments <500 bp. Based on DNA sequences of the internal transcribed spacer (ITS) regions of ribosomal DNA, three TaqMan primer/probe sets were designed. Primer/probe set PL-5 amplified a 96-bp fragment within the ITS1 region of P. longicolla, D. phaseolorum var. caulivora, D. phaseolorum var. meridionalis, and D. phaseolorum var. sojae. Set PL-3 amplified a 86-bp DNA fragment within the ITS2 region of P. longicolla. Set DPC-3 amplified a 151-bp DNA fragment within the ITS2 region of D. phaseolorum var. caulivora. TaqMan primer/probe sets were able to detect as little as 0.15 fg (four copies) of plasmid DNA. When using PCR-RFLP for Diaporthe and Phomopsis detection, the sensitivity was as low as 100 pg of pure DNA. Among 13 soybean seed lots from Italy and the United States, the total Diaporthe and Phomopsis detected using a traditional seed-plating technique ranged from 0 to 32%. P. longicolla was most prevalent, followed by D. phaseolorum var. sojae. D. phaseolorum var. caulivora, which only occurred in 0.5% of the Italian seed lots, was not detected in the U.S. seed lots. D. phaseolorum var. meridionalis was not detected in either the U.S. or Italian seed lots. Using TaqMan primer/probe set PL-3, the frequency of P. longicolla was 18% in seed lot I3, similar to the frequency obtained from PCR-RFLP and potato dextrose agar plating detection. The frequencies of D. phaseolorum and P. longicolla in each seed lot obtained by the different detection methods were comparable with respect to total infection and individual species detection. However, TaqMan detection provided the fastest results of all the methods tested.     

The geographical distribution of sunflower diseases in China

In various areas of China, the fourth largest sunflower-producing country in the world, 25 sunflower diseases have been reported: 19 caused by fungi, one virus disease and five caused by different species of broomrape. Their geographical distribution and economic significance are discussed. Fungi are the most prevalent disease causing agents in every region. More species of broomrape have been recorded in China than in any other country in the world. Orobanche coerulescens has not previously been reported to attack sunflower but is an important cause of disease in China. The number of diseases recorded differs among the regions, with 14 in North-east China, 11 each in North, North-west and South-west China, 10 in East China and six in Central and South China. This distribution broadly corresponds to the intensity of sunflower production. Only Puccinia helianthi and Septoria helianthi are reported in all the regions; 64% of the diseases are restricted to one or two regions. Alternaria helianthi, Septoria helianthi, Sclerotinia sclerotiorum and Orobanche coerulescens are considered to be the most important, causing 10-50% losses in yield, and even crop failure in some areas in some years. Much work remains to be done on disease control to promote the development of sunflower production in China.     

向日葵链格孢的分类研究

引起油葵黑斑病的病原菌在我国一直采用向日葵链格孢Alternaria helianthi这个学名。然而国际上已更名为向日葵拟链格孢Alternariaster helianthi。2017年从陕西省华阴市采集到油葵黑斑病样品。通过单孢分离获得6株病原菌菌株,致病性检测后选取2个代表菌株进行形态学研究,并基于ITS和LSU基因进行系统发育分析。结果表明,2个病原菌菌株为小球腔菌科Leptosphaeriaceae拟链格孢属Alternariaster向日葵拟链格孢Alternariaster helianthi。     

向日葵品种叶片组织结构与抗锈病的关系

向日葵锈病是向日葵的重要病害之一,在世界向日葵生产地区普遍发生,给向日葵产业带来巨大的经济损失。本研究通过比较向日葵抗、感锈病品种叶片组织结构的差异,以揭示此类病菌与寄主间的相互作用及其寄主抗病机理,研究结果表明：抗病品种叶片蜡质含量高于感病品种,且栅栏组织双层,排列整齐、紧密,可以抵抗病菌的侵入和扩展,而气孔密度与抗病性没有相关性。     

甘蔗白叶病植原体巢式PCR检测方法的建立及初步应用

甘蔗白叶病(sugarcane white leaf,SCWL)是由植原体引起的重要甘蔗病害[1],广泛分布在印度、泰国等许多国家[1,2].我国甘蔗产区的栽培品种也有SCWL的发生[3].甘蔗是无性繁殖作物,植原体可通过繁殖种苗进行传播,台湾斑纹叶蝉(Matsumuratetlix hiroglyhious)通过咬食感染甘蔗植株的韧皮部可引起该病害[4].     

3种甘薯病毒多重RT-PCR检测方法的建立及应用

正病毒病是引起甘薯品质降低和减产的重要原因之一,现已报道30多种能侵染甘薯的病毒~([1,2])。山东省是甘薯种植大省,病毒种类近10种~([3,4])。甘薯羽状斑驳病毒(Sweet potato feathery mottle virus,SPFM V)、甘薯潜隐病毒(Sweet potato latent virus,SPLV)是为害甘薯的主要病毒,在全国甘薯种植区广泛分布~([5,6])。甘薯病毒2(Sweet potato virus 2,SPV2)为Potyvirus的一个暂定种,多与同属的其他病毒混合侵染~([7])。多重PCR技术由Chamberian等~([8])1988年首次提出,可实现多基因的同时扩增,具有节省时间、提高效率的优点,已初