杀菌剂对玉米穗腐病菌的毒力及毒素产生的影响

为筛选防治玉米穗腐病同时减少病原菌毒素积累的化学药剂,采用菌丝生长速率法、涂布平板法分别测定了4种药剂多菌灵、百菌清、吡唑醚菌酯和氰烯菌酯对玉米穗腐病的3种主要致病镰孢菌拟轮生镰孢菌、层出镰孢菌和禾谷镰孢菌菌丝生长和孢子萌发的影响,并采用高效液相色谱法测定了4种药剂对其毒素积累的影响。结果表明:层出镰孢菌菌株127-1和禾谷镰孢菌菌株125-3的最佳产毒时间为第10天。供试4种药剂对3种镰孢菌菌丝生长的EC50值范围为0.18~5.71μg/m L,对孢子萌发的EC50值范围为0.02~8.14μg/m L,均表现出一定的抑制作用。但对3种镰孢菌产伏马毒素、玉米赤霉烯酮和脱氧雪腐镰孢菌烯醇的影响不尽相同,吡唑醚菌酯浓度在1~500μg/m L时能够抑制拟轮生镰孢菌和层出镰孢菌产生伏马毒素,氰烯菌酯能够在0.05~0.8μg/m L浓度范围内显著抑制禾谷镰孢菌产生玉米赤霉烯酮,当多菌灵浓度为0.05~0.5μg/m L时,几乎可以完全抑制禾谷镰孢菌产生脱氧雪腐镰孢菌烯醇。     

大豆根腐病致病镰孢菌的多重PCR检测技术

为建立大豆根腐病镰孢菌的多重PCR检测方法,以四川大豆根腐病致病菌包括尖孢镰孢菌、腐皮镰孢菌、禾谷镰刀菌和木贼镰孢菌为对象,设计镰孢菌翻译延伸因子基因EF-1α的种特异引物,建立多重PCR扩增体系,并进行优化与验证。结果表明:25μL体系为最优镰孢菌多重PCR扩增体系,4种镰孢菌等体积混合DNA 4.0μL,各镰孢菌特异正向引物1.0μL,共用反向引物4.0μL,最佳退火温度为54℃,当循环30次时,能清晰地扩增出各镰孢菌EF-1α条带,对4种镰孢菌混合DNA的检测灵敏度可达0.1 ng/μL。室内环境样本验证结果表明,依据EF-1α扩增片段大小,该体系能够特异地检测出大豆黄化苗与致病镰孢菌混合样本中的镰孢菌,但无法从其它真菌的DNA中扩增获得目的片段。表明基于EF-1α基因特异引物建立的镰孢菌多重PCR检测技术可快速、特异地检测大豆根腐病镰孢菌。     

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。     

利用Tri8基因区分中国小麦赤霉病菌的毒素化学型

由镰孢菌(Fusarium spp.)引起的小麦赤霉病在全世界均有发生,我国小麦赤霉病主要由禾谷镰孢菌复合种(Fusarium graminearum species complex,FGSC)所引起,根据产生单族毒素种类分为三种化学型,分别为3-乙酰脱氧雪腐镰孢菌烯醇(3-AcDON)、15-乙酰脱氧雪腐镰孢菌烯醇(15-AcDON)和雪腐镰孢菌烯醇(NIV)化学型。为了更方便地对禾谷镰孢菌复合种进行化学型的区分,本研究利用三对引物分别扩增了三种化学型菌株的Tri8基因并进行了序列测定,根据不同化学型菌株的基因序列差异,进行了特异性引物设计和筛选。利用设计的引物同时扩增三种化学型的Tri8基因并用AvaI内切酶进行酶切,根据酶切片段的大小区分三种化学型。研究表明该方法可准确有效地区分我国小麦赤霉病菌的化学型。     

玉米圆斑病病原的快速检测

玉米生平脐蠕孢菌(Bipolaris zeicola)是引起玉米圆斑病的病原菌。本研究通过对玉米生平脐蠕孢菌及其近似种的EF-1α基因(elongation factor 1α)部分序列进行比对,设计出玉米生平脐蠕孢菌的特异性引物Y-EF-F和Y-EF-R,利用该引物可以从B.zeicola中扩增出137 bp的特异片段,而其余的17个参试菌株扩增结果为阴性。灵敏度实验表明该对引物可以检测到目标DNA的浓度为1 pg·μL~(-1)。用B.zeicola接种玉米叶片、苞叶以及玉米粒,然后以接种发病的病组织DNA为模板,利用引物Y-EF-F和Y-EF-R进行PCR扩增,可以扩增出137 bp的特异性条带,而健康玉米组织DNA中未能扩增出任何条带。用B.zeicola孢子悬浮液接种大田玉米叶片,接种第3 d可以检测到未发病组织中有B.zeicola病原菌,第5 d可以看到明显的病斑。研究结果表明该方法可用于快速、准确和灵敏地检测玉米组织中的潜伏期玉米生平脐蠕孢菌,为玉米圆斑病的快速检测,进而及早采取防治措施提供积极的指导。     

禾谷镰孢荧光定量检测体系的建立及其在玉米苗枯病上的应用

为实现对田间土壤中禾谷镰孢Fusarium graminearum的定量检测,本研究构建了土壤含菌量与玉米苗枯病病情指数的回归模型。基于禾谷镰孢甾醇14α-去甲基化酶基因CYP51C序列,设计特异性引物HQ1-F/HQ1-R,利用引物建立实时荧光定量PCR(RT-q PCR)体系,选取4个玉米自交系品种进行室内苗枯病接种试验,调查其病情指数,利用RT-qPCR体系检测土壤禾谷镰孢含菌量,并对病情指数和土壤禾谷镰孢含菌量进行回归。结果表明,仅禾谷镰孢扩增出目的条带并且可从多种病原菌土壤中检测出。RT-qPCR的熔解曲线具有单一吸收峰,扩增曲线的循环阈值与模板浓度呈良好的线性关系,扩增效率为104.7%,标准曲线为y=-3.2137x+34.9560(R~2=0.9968),最低可检测到1 pg/μL的DNA。随着土壤禾谷镰孢接菌量的增加,单位土壤禾谷镰孢含菌量呈线性增加,即y=13.603x-85.370(R~2=0.9998)。4个玉米品种的病情指数与土壤禾谷镰孢含菌量的回归曲线分别为y=0.0789x+22.0590(R~2=0.7949)、y=0.0304x+7.8686(R~2=0.9579)、y=0.0458x+23.7540(R~2=0.5420)、y=0.0471x+32.0760(R~2=0.6753)。     

小麦赤霉病菌拮抗菌筛选及最适培养条件初步研究

<正>小麦赤霉病(Fusarium head blight)是我国小麦生产上最重要的真菌病害之一。已有的研究表明,在我国其致病菌的优势种主要为Fusarium graminearum和F. asiaticum。此病害具有爆发速度快、流行范围广等特点[1,2],而且其致病菌还可产生脱氧雪腐镰刀菌烯醇(deoxynivalenol, DON)和玉米赤霉烯酮(zearalenone, ZEN)等毒素,人畜吃过会产生不同程度的中毒反应[3]。目前小麦     

RNA-Seq解析cAMP促进禾谷镰孢菌DON毒素产生的分子基础

 禾谷镰孢菌(Fusarium graminearum)引起的小麦赤霉病(Fusarium head blight)不仅造成小麦产量损失,而且病菌在侵染过程中会释放大量真菌毒素,导致小麦籽粒污染,严重威胁人畜健康。实验室前期研究发现外源添加环磷酸腺苷(cAMP)可以促进禾谷镰孢菌脱氧雪腐镰孢菌烯醇毒素(Deoxynivalenol, DON)产生,但其分子机制尚不清楚。本研究利用转录组分析了cAMP处理后禾谷镰孢菌基因的表达情况,探究了cAMP促进DON毒素合成的潜在机制。研究发现响应cAMP处理的差异表达基因有4 470个,其中1 818个基因上调,2 652个基因下调。负责DON毒素合成TRI基因簇的所有基因在cAMP处理下均上调表达,表明cAMP通过诱导TRI基因簇表达促进DON毒素合成。进一步分析了cAMP下游依赖性蛋白激酶A(PKA)的敲除突变体Δpka的转录组数据,发现几乎所有TRI基因簇基因均下调表达,并且cAMP处理上调表达而Δpka突变体中下调表达的基因中显著富集真菌毒素代谢相关的基因,该结果进一步表明cAMP通过PKA通路调控DON毒素合成。此外,cAMP处理后,可诱导DON毒素产生的γ-氨基丁酸(gamma-aminobutyric acid, GABA)合成相关基因上调表达,而GABA分解相关基因下调表达。这表明禾谷镰孢菌可通过调节细胞内的GABA水平促进DON毒素合成。     

香蕉黑腐病菌(Botryodiplodia theobromae)的PCR检测

 根据香蕉黑腐病菌可可球二孢菌(Botryodiplodia theobromae)与其它香蕉病原真菌核糖体基因转录间隔区(rDNA-ITS)ITS1和ITS2间序列差异,设计了特异引物Bth-S(5'-TCTCCCACCCTTTGTGAAC-3')和Bth-A(5'-AAAAGT-TCAGAAGGTTCGTC-3'),利用此引物对包括可可球二孢菌在内的21个菌株基因组DNA进行PCR扩增,结果只有4个可可球二孢菌菌株扩增到422bp特异带,其它17个菌株无扩增产物。灵敏度测试结果表明此特异引物能对1pg的可可球二孢菌基因组DNA进行扩增。对自然感染黑腐病的香蕉果实组织和接种可可球二孢菌或多种香蕉病原真菌混合接种的果实组织进行检测,Bth-S和Bth-A引物对不仅能够在自然感染黑腐病果实组织中特异检测到可可球二孢菌,而且能在未显症和发病的接菌香蕉果实组织中特异检测得到可可球二孢菌。这为香蕉可可球二孢菌潜伏侵染检测提供了技术支持。     

双重PCR检测马铃薯晚疫病菌和青枯病菌方法的建立及应用

 利用真菌通用引物ITS1和ITS4扩增马铃薯晚疫病菌转录间隔区并进行序列测定,通过序列比较,设计了1对马铃薯晚疫病菌的特异引物INF1/INF2,并对15种不同真菌、细菌和7种疫霉属和腐霉属卵菌基因组DNA进行PCR扩增,结果只有不同来源的马铃薯晚疫病菌株可获得324 bp的特异带。将引物INF1/INF2与卵菌通用引物进行巢式PCR扩增后,其检测灵敏度在DNA水平上可达30 fg。运用设计的引物与马铃薯青枯病菌特异引物结合建立了双重PCR体系,能从马铃薯晚疫病菌和马铃薯青枯病菌总基因组DNA以及人工接种和自然发病的马铃薯植株中分别或同时扩增到324 bp和281 bp的特异片段。实现了同时对马铃薯晚疫病菌和马铃薯青枯病菌的快速可靠检测。     

Rapid preparation of Fusarium DNA from cereals for diagnostic PCR u sing sonification and an extraction kit

Several PCR methods have recently been developed for Fusarium analysis in pure cultures. To use these new techniques in mycological studies and in industrial quality control, a protocol was set up for the rapid preparation of fungal DNA from cereals. An ultrasonification probe (sonotrode) and a lysis buffer were used for mechanical lysis of mycelia from infected grains. Following ultrasonification, DNA was isolated using a commercially available kit. DNA preparation was completed within 5 min per sample. The method resulted in DNA of sufficient quality and quantity for diagnostic PCR. Group- and species-specific primers were used to detect DNA of Fusarium graminearum and F. culmorum in species-specific assays as well as trichothecene-producing Fusarium spp. in a group-specific system. A minimum of one F. graminearum -infected grain added to an uninfected 40 g wheat sample was detectable with a species-specific PCR. The PCR signals produced with primers specific for the tri5 gene of trichothecene-producing Fusarium spp. and with primers for the detection of F. graminearum (gaoA) were in accordance with corresponding concentrations of deoxynivalenol (DON) found in samples by HPLC analysis. The speed of the protocol developed may promote the use of PCR in routine applications in an agro-industrial context.     

Molecular characterization of the Fusarium graminearum species complex in Japan

Members of the Fusarium graminearum species complex are important cereal pathogens worldwide and belong to one of at least nine phylogenetically distinct species. We examined 298 strains of the F. graminearum species complex collected from wheat or barley in Japan to determine the species and trichothecene chemotype. Phylogenetic analyses and species-diagnostic polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLPs) revealed the presence and differential distribution of F. graminearum sensu stricto (s. str.) and F. asiaticum in Japan. F. graminearum s. str. is predominant in the north, especially in the Hokkaido area, while F. asiaticum is predominant in southern regions. In the Tohoku area, these species co-occurred. Trichothecene chemotyping of all strains by multiplex PCR revealed significantly different chemotype compositions of these species. All 50 strains of F. graminearum s. str. were of a 15- or 3-acetyl deoxynivalenol type, while 173 (70%) out of 246 strains of F. asiaticum were of a nivalenol type. The possibility of gene flow between the two species was investigated by use of 15 PCR-RFLP markers developed in this study. However, no obvious hybrids were detected from 98 strains examined, including strains collected from regions where both species co-occur.     

Real-time PCR Quantification and Mycotoxin Production of Fusarium graminearum in Wheat Inoculated with Isolates Collected from Potato, Sugar Beet, and Wheat

ABSTRACT Fusarium graminearum causes Fusarium head blight (FHB) in small grains worldwide. Although primarily a pathogen of cereals, it also can infect noncereal crops such as potato and sugar beet in the United States. We used a real-time polymerase chain reaction (PCR) method based on intergenic sequences specific to the trichodiene synthase gene (Tri5) from F. graminearum. TaqMan probe and primers were designed and used to estimate DNA content of the pathogen (FgDNA) in the susceptible wheat cv. Grandin after inoculation with the 21 isolates of F. graminearum collected from potato, sugar beet, and wheat. The presence of nine mycotoxins was analyzed in the inoculated wheat heads by gas chromatography and mass spectrometry. All isolates contained the Tri5 gene and were virulent to cv. Grandin. Isolates of F. graminearum differed significantly in virulence (expressed as disease severity), FgDNA content, and mycotoxin accumulation. Potato isolates showed greater variability in producing different mycotoxins than sugar beet and wheat isolates. Correlation analysis showed a significant (P < 0.001) positive relationship between FgDNA content and FHB severity or deoxynivalenol (DON) production. Moreover, a significant (P < 0.001) positive correlation between FHB severity and DON content was observed. Our findings revealed that F. graminearum causing potato dry rot and sugar beet decay could be potential sources of inoculum for FHB epidemics in wheat. Real-time PCR assay provides sensitive and accurate quantification of F. graminearum in wheat and can be useful for monitoring the colonization of wheat grains by F. graminearum in controlled environments, and evaluating wheat germplasms for resistance to FHB.     

玉米细菌性枯萎病菌PCR检测

 根据GenBank中玉米细菌性枯萎病菌及其近似种的16S序列差异,设计了一对玉米细菌性枯萎病菌特异性引物Ps2r/Ps3r,该引物能从供试的7株玉米细菌性枯萎病菌中特异性扩增出一条268 bp的预期条带,供试的32株近似种菌株都没有扩增产物;与国内外文献报道的其它5对特异性引物相比,除引物PSA/PSB外,引物DEP1/DEP2、ES16/ESIG2c、HRP1d/HRP3c和CPSL1/CPSR2c在不同程度上对部分近似种菌株出现了扩增。试验结果表明,引物Ps2r/Ps3r和PSA/PSB能特异性扩增玉米细菌性枯萎病菌,得到预期的扩增产物。对不同系列稀释度的DNA和玉米样品中病菌的检测结果表明,由引物Ps1/Ps4和Ps2r/Ps3r组合的巢式PCR方法的检测灵敏度高于引物ITSA/ITSB和PSA/PSB组合的巢式PCR方法,也高于Bio-PCR检测方法;前者可以检测到玉米种子中300 cfu/sample的目的细菌,该检测方法在进境玉米种子样品玉米细菌性枯萎病菌的检疫中具有比较理想的应有潜力和推广价值。     

Fusarium populations on Chinese barley show a dramatic gradient in mycotoxin profiles

We report on a large gene bank of Fusarium isolates established by a broad survey conducted in 2005 in which infected barley ears were collected in 23 counties of seven provinces and two municipalities along the Yangtze River in China. In total, 1,894 single spore isolates were obtained. The isolates were characterized at the species level by a newly developed and robust set of diagnostic primers based on single nucleotide polymorphisms (SNPs) among members of the F. graminearum clade. In addition, we determined their chemotype using previously described polymerase chain reaction (PCR) primers. The results showed that in all regions F. asiaticum was the predominant species causing Fusarium head blight (FHB) on barley in China (N = 1,706), while in the upper valleys of the Yangtze River also F. graminearum sensu stricto, F. meridionale, and F. proliferatum were found. Major differences in the chemotypes were found in the F. asiaticum populations, from very high to exclusive nivalenol (NIV) chemotypes in the mountainous upper valleys of the Yangtze River to predominantly deoxynivalenol (DON) chemotypes in the middle and lower valleys. In contrast to the F. asiaticum isolates from three counties in Sichuan province, which were largely NIV producers (278 of 291), F. graminearum isolates from these sampling sites were for the vast majority (27 of 28) DON producers, indicating that despite sharing the same habitat, these sympatric species apparently have unique mycotoxin chemotypes.     

长江中下游地区亚洲镰刀菌NX-2毒素群体的检测

由禾谷镰刀菌复合种(Fusarium graminearum species complex,FGSC)引起的麦类赤霉病,是农业生产上的重要病害。为明确中国长江中下游冬小麦主产区小麦赤霉病菌种的构成及其地理分布,对2008年从江苏、浙江和湖北3省采集的656株小麦赤霉病菌株进行了分类鉴定。结果显示,其中558个菌株为亚洲镰刀菌(Fusarium asiaticum),98个菌株为禾谷镰刀菌(Fusarium graminearum sensu stricto),表明中国长江中下游冬小麦主产区小麦赤霉病的主要致病菌是亚洲镰刀菌。选择亚洲镰刀菌(F.asiaticum)为研究对象,通过PCR-RFLP的方法对其进行产NX-2毒素菌株的检测。结果没有检测到产NX-2毒素菌株,表明中国长江中下游冬小麦主产区并未出现NX-2毒素群体。本研究旨在了解NX-2毒素群体在中国长江中下游地区的地理分布,为进一步研究麦类赤霉病菌群体遗传多样性和演化趋势奠定基础,为麦类赤霉病的防治和毒素污染的控制提供理论依据。     

PIRA-PCR for detection of Fusarium graminearum genotypes with moderate resistance to carbendazim

PIRA-PCR ( p rimer- i ntroduced r estriction a nalysis PCR) was developed to detect isolates of Fusarium graminearum with moderate resistance to carbendazim, a methyl benzimidazole carbamate (MBC)-group fungicide. Two primer pairs were designed and synthesized according to the nucleotide sequence of the β ₂-tubulin gene from F. graminearum. Fragments of 164 bp were amplified by nested PCR from isolates differing in carbendazim sensitivity. A Hin dIII restriction enzyme recognition site was introduced artificially by inner primers to detect a mutation at codon 167, and Taa I ( Tsp 4CI) restriction enzyme was used to detect a mutation at codon 200. The sensitivity of isolates to carbendazim was determined by analyzing electrophoresis patterns of the resulting PCR products after simultaneous digestion with both Hin dIII and Taa I. Results from PIRA-PCR and a conventional method (mycelial growth on agar) were identical but PIRA-PCR required only 7–8 h while the conventional method required 5–7 days. This study demonstrates that PIRA-PCR not only monitors the appearance of moderately resistant isolates, but can be useful for detecting genotypes of F. graminearum with moderate resistance to carbendazim.     

苇状羊茅与多年生黑麦草内生真菌菌丝基因组DNA提取的优化及PCR初步检测

感染内生真菌的禾草在牧草和草坪业上具有重要的生态和经济意义，家畜采食感染Neotyphodium coenophialum和N.lolii的苇状羊茅和多年生黑麦草会发生中毒。本研究收集天津口岸1998年以来进境的部分苇状羊茅和多年生黑麦草种子，对经镜检确认带有内生真菌的种子进行分离培养，对疑似菌株的菌丝用改进的Moiler等方法进行基因组DNA抽提，测定浓度及纯度，对照原方法，DNA的纯度有较大提高，浓度略有上升。Tubulin2基因的引物IS1-IS3扩增结果显示为单一的条带，结合形态学和序列比对，分离培养得到的菌株可以基本确定为N．coenophialum和N。lolii。根据Genbank中N.coenophialum和N.lolii的NC25基因序列设计出引物F1-R1，扩增得到能区分开N.coenophialum和N．lolii的单一条带（相差160bp），建立了N．coenophialum和N．lolii的PCR检测方法，结果准确可靠。     

应用PCR技术检测玉米中的禾谷镰刀菌

本实验通过用PCR方法来实现对禾谷镰刀菌的快速检测,经对霉变玉米样品、玉米茎腐病组织及玉米穗腐病标本的检测,证明该方法是一种高效、灵敏的方法,具有重要的实际应用价值.