大豆猝死综合症病菌检测技术研究

 大豆猝死综合症是美国等几个大豆主产国的一种较新的、危害性很大的大豆真菌病害,它分别由两种镰刀菌Fusarium virguliforme和Fusanum tucumaniae引致。本研究确立了从土壤中筛选、分离大豆猝死综合症病菌的选择性培养基PCNB;明确了中国具有对该病菌敏感的大豆品种;利用Southern杂交技术,将致病菌和近似菌的基因组DNA分别用EcoRI酶切,与mtDNA探2u18和4u40杂交的RFLP图谱可成功的鉴定F.virguliforme,F.tucumaniae和近似菌F.phaseoli。由此建立了,通过选择性培养基分离进口大豆中的土壤,进而以Southern杂交技术鉴定大豆猝死综合症病菌的检测方法。     

大豆猝死综合症

大豆猝死综合症（Ｓｕｄｄｅｎｄｅａｔｈｓｙｎ－ｄｒｏｍｅ,以下简称ＳＤＳ）是近年来大豆上发生的一种相对新的病害。Ｈ．Ｊ．Ｗａｌｔｅｒｓ于１９７１年在阿肯色州首次观察到该病害,直到１９８２年才由Ｍ．Ｃ．Ｈｉｒｅｌ［１］给此病害命名。在适宜的环境条件下,...     

大豆猝死综合症病原菌北美种的PCR鉴定

 应用等位基因特异性PCR(allele-specific PCR,AS-PCR)技术,建立了大豆猝死综合症(sudden death syndrome of soybean,SDS)病原菌北美种Fusarium virguliforme与其近似种Fusarium phaseoli(菜豆根腐病菌)的鉴别方法。针对翻译延长因子(EF-1α)基因上的3个SNP(单核苷酸多态性)位点,参照引物设计原则和等位基因特异引物(allele-specific primer,ASP)的要求设计了1对引物Fsg-α-1/2,能特异地扩增F.virguliforme,产生327 bp的电泳条带。另外,根据ITS区序列设计了1对通用引物Fu1/2,能扩增F.virguliforme和F.phaseoli,产生452 bp的电泳条带。本实验在传统的AS-PCR基础上进行改进,引入Fu1/2作为阳性质控引物,将ASP的特异性与双重PCR的严谨性相结合,建立了简便可靠的SDS病原菌鉴定方法。     

橡胶树白粉病菌分子检测技术的建立

根据橡胶树白粉菌(Oidium heveae Steinm.)基因组中的特有保守序列OHS,设计两对特异性引物OHF1/OHR1和OHF2/OHR2。以不同地区收集的6份O.heveae(OH1~OH6)和橡胶树胶孢炭疽病菌(Colletotrichum gloeosporioides Penz.)等5种非靶标病原菌及健康橡胶树叶片基因组DNA为模板,建立了橡胶树白粉菌PCR及nested-PCR分子检测技术,并验证了检测体系的特异性和灵敏度。结果表明,引物OHF1/OHR1和OHF2/OHR2对橡胶树白粉菌均具有较高的特异性和灵敏度。其中引物OHF2/OHR2能检测到10pg/μL的橡胶树白粉菌DNA,而以OHF1/OHR1和OHF2/OHR2组合进行nested-PCR,其最低DNA检测浓度达到0.01fg/μL。人工接种试验中,当孢子接种量为2×10~3个/叶时,PCR和nested-PCR检测体系可分别在接种4d和24h后检测到目的条带。表明nested-PCR对在叶片组织中处于潜育期的橡胶树白粉菌的检测更有效,可为橡胶树白粉菌的检测提供一种简便而准确的检测方法。     

进境美国大豆中大豆茎褐腐病菌的截获

 从进境的美国大豆豆秆样品中分离到2株疑似大豆茎褐腐病菌Phialophora gregata的分离物247-8和8300-5,2株分离物在PGM培养基上菌落圆形,边缘规则,白色至暗褐色,表面隆起,粗糙,轮纹状。分生孢子卵形至椭圆形,无色,平滑,单胞,平均大小4.3 μm×1.9 μm。分生孢子梗具有瓶梗状结构,无色,无隔膜或有隔膜,大小(5~16)μm×(2~3) μm,呈桶型或长颈瓶型。特异性引物BSRIGS1/2、BSR1/2和Pgl/4分别扩增分离物247-8的DNA得到预期1 022 bp、483 bp和499 bp的产物;分离物8300-5的DNA经PCR扩增分别得到834 bp、483 bp和499 bp的预期条带。2株分离物的ITS区序列完全一致,与GenBank中大豆茎褐腐病菌(登录号AB190396、DQ459387、DQ459386和AF132804)的序列相似性为100%。人工接种大豆幼嫩植株茎基部均产生大豆茎褐腐病菌的典型症状。根据分离物形态特征、PCR检测、序列分析以及致病性测试结果,将进境美国大豆样品中的分离物247-8和8300-5鉴定为大豆茎褐腐病菌P. gregata。     

水稻白叶枯病菌和细菌性条斑病菌的分子标记筛选及检测

 水稻白叶枯病菌（Xanthomonas oryzae pv. oryzae）和细菌性条斑病菌(Xanthomonas oryzae pv. oryzicola)是水稻种子产地检疫中最重要的两种检疫对象,且同属于水稻黄单胞杆菌。本研究基于生物信息学技术构建比较基因组学算法对两种病原的全基因组序列比对分析,得到一系列能够区分两种病原的特异性PCR引物。结合简单的PCR技术及全自动DNA分析系统,我们选取了12对引物分别对23株水稻白叶枯病菌和5株水稻细菌性条斑病菌及其它相关菌株进行验证。结果获得了2对显性标记(Xoo-Hpa1和Xoc-ORF2)以及3对共显性分子标记(M568、M897和M1575)可以达到理想的区分检测两种病原的效果。分子标记的检测灵敏度从5×10⁴到5 × 10⁷cfu·mL^-1不等,且从水稻种子浸提液中也能成功地检测水稻白叶枯病菌和细菌性条斑病菌。本研究丰富了检测标记的靶位点,并有效的结合了高通量检测的手段对多位点联合分析,增强了检测的可靠性,有望在今后的植物检疫及病原鉴定中发挥着重要的作用。     

瓜黑星病菌、枯萎病菌和蔓枯病菌的三重PCR检测

通过测定黄瓜黑星病菌(Cladosporium cucumerinum)rDNA的ITS序列,比对近缘种及瓜类几种重要病原菌的ITS序列,设计出特异性引物HX-1/HX-2,经过对引物HX-1/HX-2PCR条件的优化,可以扩增出1条190bp的黄瓜黑星病菌特异性DNA条带,灵敏度达到1pg/μL。进一步将引物HX-1/HX-2和瓜类枯萎病菌、瓜类蔓枯病菌特异检测引物Fn-1/Fn-2、Mn-1/Mn-2组合,建立三重PCR体系,可一次检测出瓜类黑星病菌、瓜类枯萎病菌、瓜类蔓枯病菌3种瓜类植物重要的病原菌。建立了可以应用于田间瓜类黑星病菌PCR检测技术和瓜类主要病害三重PCR检测技术,对瓜类病害的诊断和防治具有重要的指导作用。     

三重PCR检测黄瓜炭疽病菌、菌核病菌和细菌性萎蔫病菌

 本试验建立一种可同时检测黄瓜炭疽病(Colletotrichum orbiculare)、黄瓜菌核病(Sclerotinia sclerotiorum(Lib.)de Bary)和黄瓜细菌性萎蔫病(Erwinia tracheiphila)等黄瓜主要病害病原菌的三重PCR检测体系。采用正交试验设计方法, 对三重PCR的影响因素分析研究, 进行退火温度优化, 并以3个引物组、Taq DNA聚合酶、dNTP和Mg²⁺ 共6因素3水平进行多重PCR体系优化, 成功建立了适合黄瓜主要病害的三重PCR最佳检测体系, 即25 μL的反应体系中含有0.24 μmol·L^-1 CY1/CY2;0.72 μmol·L^-1 SSFWD/SSREV;0.336 μmol·L^-1 ET-P1/ ET-P2;1 U Taq聚合酶;0.15 mmol·L^-1 dNTP;1 mmol·L^-1 MgCl₂, 最适退火温度为63℃。该方法能够快速从田间黄瓜发病植株和根围土壤中将黄瓜炭疽病菌、黄瓜菌核病菌和黄瓜细菌性萎蔫病菌检测出来, 灵敏度可以达到10 pg·μL^-1。     

烟草根黑腐病菌的PCR分子检测

 根据烟草根黑腐病菌(Thielaviopsis basicola)与其它烟草病原真菌核糖体基因转录间隔区(internal transcribed spa-cer,ITS)序列间的差异,设计了一对特异性引物TB-5/TB-3,用于T. basicola的分子检测。利用该对引物对包括T. basicola在内的13个烟草病原菌菌株的基因组DNA进行PCR扩增,结果表明:只有T. basicola能扩增到一条400bp左右的特异性条带,其它菌株及阴性对照均无扩增产物。对烟草组织和土壤的检测结果也表明,该对引物能特异性的检测到T. basicola基因组DNA的存在。该引物对T. basicola基因组DNA检测的灵敏度为100fg/μL。     

大豆疫霉根腐病菌检测鉴定方法及病害传播途径研究

 研究了导致黑龙江省大豆苗期死亡和成株期茎部褐色坏死的病原菌的形态、生理及致病性,在此基础上着重研究了该菌的血清学、同工酶标记、RAPD、致病性分化及病害传播途径,结果如下。     

3种瓜类枯萎病菌Fusarium oxysporum的分子检测

 Fusarium oxysporum is one of the most important phytopathogens and cause Fusarium wilt disease in cucumber, watermelon and melon, etc.In this study, a pair of species-specific primers Fc-1 and Fc-2 was synthesized based on differences in internal transcribed spacer sequences of Fusarium genus.With the primers, a specific 315 bp PCR product was amplified from five F.oxysporum isolates isolated from cucumber, watermelon and melon, infected cucumber and watermelon tissues, while no product was obtained from other fourteen fungi, healthy cucumber and watermelon tissues.The detection sensitivity is 100 fg for genomic DNA of F.oxysporum and 1 000 spores/g soil for the soil pathogens.In contrast, the nested PCR with two pairs of primers(ITS1/ITS4 and Fc-1/Fc-2) increased the sensitivity by 100-fold.In addition, one-step PCR could also detect F.oxysporum in symptomless cucumber root of 7 dpi(days post inoculation) and in infected cucumber and watermelon tissues at the early stage of disease development.Therefore, the developed PCR-based method enabled rapid, sensitive and reliable detection of F.oxysporum.It also provides the detection method for early monitoring and diagnosis of the pathogen as well as the plant disease management guidance.     

Molecular detection of Fusarium solani f. sp. glycines in soybean roots and soil

A polymerase chain reaction (PCR)-based method was developed to detect DNA of Fusarium solani f. sp. glycines , the cause of soybean sudden death syndrome. Two pairs of primers, Fsg1/Fsg2 designed from the mitochondrial small subunit ribosomal RNA gene, and FsgEF1/FsgEF2 designed from the translation elongation factor 1-α gene, produced PCR products of 438 and 237 bp, respectively. Primer specificity was tested with DNA from 82 F. solani f. sp. glycines , 55 F. solani non-SDS isolates, 43 isolates of 17 soybean fungal pathogens and the oomycete Phytophthora sojae , and soybean. The sensitivity of primer Fsg1/Fsg2 was 10 pg while that of FsgEF1/FsgEF2 was 1 ng when using F. solani f. sp. glycines total genomic DNA or down to 10³ macroconidia g⁻¹ soil. Nested PCR increased the sensitivity of the PCR assay 1000-fold to 10 fg using primers Fsg1/Fsg2, and 1 pg using primers FsgEF1/FsgEF2. F. solani f. sp. glycines DNA was detected in field-grown soybean roots and soil by PCR using either single pairs of primers or the combination of two pairs of primers. The occurrence of F. solani f. sp. glycines was determined using nested PCR for 47 soil samples collected from soybean fields in 20 counties of Illinois in 1999. F. solani f. sp. glycines was detected in soil samples from all five Illinois Agricultural Statistic Districts including 100, 89, 50, 92 and 50% of the samples from East, Central, North-east and West Districts, respectively.     

利用伏马毒素合成基因对玉米种子寄藏镰刀菌的分子检测

我国玉米种子普遍受到拟轮生镰刀菌(Fusarium verticillioides)的侵染,该菌产生的伏马毒素对国家种质库中玉米种质的安全保存有着潜在的影响。fum1基因是F.verticillioides等镰刀菌中伏马毒素生物合成的必需基因。选用3对已知引物Fum5F/Fum5R、P1/P2和P3/P4分别对从玉米种子中分离出的9株镰刀菌和1株阴性对照菌链格孢菌进行PCR扩增检测,在F001和F003菌株中扩增出相应的特异性表达片段。测序表明,这些片段的长度分别为846bp、888bp和703bp,与已知fum1基因(AF155773)的同源性达99%以上。3对引物的检测灵敏度达到88pg/mL。结果表明,这3对引物都能够有效地检测产生伏马毒素的镰刀菌菌株,但引物Fum5F/Fum5R所扩增片段完全来自fum1基因内部,具有更强的特异性。在此定性检测基础上,需要进一步研究定量检测方法并进一步研究伏马毒素对农作物种质保存的影响。     

尖镰孢菌(Fusarium oxysporum)的快速分子检测

 由尖镰孢菌(Fusarium oxysporum Schlecht.)引起的大豆枯萎病是危害大豆生产的主要土传病害^［1］。该菌在土壤和病残体上均可长期生存造成危害。快速准确地在发病初期植株和带病土壤中进行鉴定和检测对防治该病害至关重要。     

马铃薯干腐病优势病原菌Fusarium sambucinum 分子检测技术的建立

马铃薯干腐病是马铃薯最重要的贮藏期病害之一,现已成为马铃薯贮藏期烂窖的重要原因。实现病原菌的快速检测对病害诊断和科学防控具有重要的实践意义。本研究基于镰刀菌翻译延伸因子序列设计了一对检测接骨木镰刀菌Fusarium sambucinum的特异引物Fs-F/Fs-R。该特异引物可从接骨木镰刀菌中获得309bp的特异性扩增片段,而其他种类镰刀菌及马铃薯重要病害病原菌中均无此片段,说明该引物具有专一性。该体系的灵敏度检测结果表明,最低能检测到的接骨木镰刀菌基因组DNA浓度为70pg/μL。该引物也适用于发病马铃薯块茎中接骨木镰刀菌的快速检测。     

Soybean sudden death syndrome: Fungal pathogenesis and plant response

Soybean sudden death syndrome (SDS) is a fungal disease caused by members of clade 2 of the Fusarium solani species complex (FSSC). These fungi are soilborne pathogens that infect soybean plants through the roots and produce toxins that translocate to aerial parts of the plant, inducing foliar chlorosis and necrosis followed by premature defoliation. Here, we first give the current state of knowledge of early pathogen detection and infection establishment for the SDS pathosystem. Subsequently, we discuss the nature and activity of secreted toxins, followed by an overview of changes in plant metabolism and factors that influence fungus–soybean interaction. Finally, we summarize the advances in plant disease resistance, symptom evaluation, and treatment.     

Pathogenicity and toxicity of Fusarium tucumaniae and Fusarium crassistipitatum to soybean and Arabidopsis thaliana

Sudden death syndrome (SDS) of soybean is a fungal disease caused by at least four distinct Fusarium species: F. tucumaniae, F. virguliforme, F. brasiliense, and F. crassistipitatum. All four species are present in Argentina. These fungi are soilborne pathogens that only colonize roots and cause root necrosis. However, damage also reaches the aboveground part of the plant, and foliar chlorosis and necrosis, followed by premature defoliation, can be observed. Although the pathogenicity and phytotoxicity of F. virguliforme has been well characterized, knowledge regarding disease development by other fungal species is scarce. In this study, two plant species, soybean (Glycine max) and Arabidopsis thaliana, and isolates from two fungal species, F. tucumaniae and F. crassistipitatum, were used to comparatively analyse the fungal pathogenicity and the phytotoxicity of volatile organic compounds (VOCs) and cell-free culture filtrates. Fungal inoculation had a significant effect on plant growth, regardless of the plant species. In addition, infected soybean plants showed disease incidence and foliar and root symptoms. Inhibition of A. thaliana growth was not due to VOCs emitted by fungi. Instead, both pathogens were shown to produce toxins that caused typical SDS foliar symptoms in soybean and root length reduction in A. thaliana. As far as we know, this is the first report that demonstrates that F. tucumaniae and F. crassistipitatum affect A. thaliana growth and emit VOCs, and that F. crassistipitatum produces toxins.     

棉花枯萎病菌新生理型菌株的分子鉴定

 Fusarium oxysporum f.sp. vasinfectum(Fov)已报道有8个生理小种和一个澳大利亚生理型,在我国只报道有3、7、8号小种。前期研究发现,我国还有一个与3、7、8号小种具有较大遗传差异的Fov新生理型。本研究通过对3、7、8号小种和新生理型菌株的翻译延伸因子(EF-1α)基因序列比对分析,发现该新生理型菌株具有特异性碱基序列。根据这些特异性碱基序列,设计了一对针对新生理型菌株的特异性引物,并证明该引物可以特异性检测Fov新生理型菌株。利用该引物对采自河北省主要植棉区的77株Fov菌株进行筛选,鉴定出2株潜在的新生理型菌株。从这2个Fov菌株中克隆出EF-1α和β-tubulin基因序列,通过构建系统发育树,证明这2个Fov菌株为新生理型菌株,而且与澳大利亚生理型菌株具有较近的亲缘关系。本研究建立的分子检测方法可用于棉花枯萎病菌新生理型菌株的鉴定。     

南方镰孢Fusarium meridionale特异性PCR检测方法的建立与应用

为建立快速、稳定的南方镰孢Fusarium meridionale特异性检测方法,对已报道的镰孢属reductase-like基因部分序列进行比对分析,寻找特异性SNP位点,设计出特异性检测引物F-Fm/R-Fm3。利用该引物对包括南方镰孢在内的30株镰孢的基因组DNA进行PCR扩增。结果显示仅在7株南方镰孢中均扩增出400 bp左右的特异性条带。PCR灵敏度试验结果表明该方法的检测灵敏度达到500 pg基因组DNA。