麦根腐平脐蠕孢病菌的菌落变异研究

小麦根腐平脐蠕孢病菌在ＰＳＡ培养基上，极易发生菌落变异，变异后菌落呈半月形，扇形，放射形，不规则形。变异在培养中不稳定，可以恢复原菌落状态。变异后的菌落生长速度下降，菌体干重和产孢量降低，致病力明显低于不产生变异的菌株。     

接菌时期与保湿时间对麦根腐平脐蠕孢小麦黑胚病的影响

麦根腐平脐蠕孢(Bipolaris sorokiniana)是小麦黑胚病的重要致病真菌。建立小麦对B.sorokiniana黑胚病的抗性评价方法是培育抗病品种的基础。就不同接菌时期与保湿天数对B.sorokiniana引发的黑胚病严重度进行了研究。结果表明,接菌时间越早、保湿时间越长B.sorokiniana引发的小麦黑胚病越严重。适宜接菌鉴定条件为:花后5~10d接菌,套袋保湿5~7d。     

稻平脐蠕孢的分离、鉴定及对作物的安全性

为了筛选获得可用于防治李氏禾的病原菌菌株,采用孢子形态观察和ITS1-5.8S-ITS2序列分析法对分离自云南西双版纳稻田感病李氏禾植株上的两株病原真菌菌株进行了鉴定,采用离体叶片生测法评价其除草活性,采用盆栽生测法评价其对主要作物的安全性.结果表明,两株病原真菌分别被鉴定为稻平脐蠕孢(Bipolaris oryzae)和稻平脐蠕孢有性型[Bipolaris oryzae (teleomorph:Cochliobolus miyabeanus)].稻平脐蠕孢发酵液对李氏禾的致病性最高为88.9;稻平脐蠕孢有性型发酵液配制的菌悬液对李氏禾的致病性最高为100.两个菌株对水稻、玉米、黄瓜及番茄等主要农作物安全.     

棘孢木霉JM-1菌株对麦根腐离蠕孢的拮抗机制

为了研究木霉菌JM-1菌株对麦根腐离蠕孢CY6菌株的拮抗机制,本研究结合形态学和分子生物学鉴定方法,明确JM-1菌株的分类地位,并采用平板对峙培养法和显微观察,分析JM-1菌株对CY6菌株的拮抗作用。结果表明,木霉菌JM-1菌株为棘孢木霉(Trichoderma asperellum),可通过空间和营养竞争抑制CY6菌株的生长,抑菌率为77.22%;显微观察发现,棘孢木霉JM-1对CY6菌株有明显的重寄生作用,同时,JM-1菌株可产生某些抗生物质,使CY6菌株的菌丝、分生孢子发生变形和消解。因此,棘孢木霉JM-1菌株可通过竞争作用、重寄生作用和抗生作用等多重生防机制抑制麦根腐离蠕孢的生长。     

内脐蠕孢属、平脐蠕孢属和凸脐蠕孢属的分类鉴定

本文是继前一个研究之后对来自东北的24种禾本科植物和海南省的一种双子叶植物(橡胶树)上的14种“长蠕孢菌”的分类鉴定的继续,共鉴定出内脐蠕孢属(Drechslera)1种,平脐蠕孢属(Bipolaris)9种和凸脐蠕孢属(Exserohilum)4种。其中双色平脐蠕孢(B.bicolor)和虉草内脐蠕孢(D.tetrarrhenae) 为国内新记录种;狗牙根平脐蠕孢(B.cynodontis)、长孢平脐蠕孢(B.urochloae) 和环形凸脐蠕孢(E.holmii)3个种首次在东北地区报道;此外,还报道了橡胶树平脐蠕孢(B.heveae) 的一个新寄生植物——燕麦(Avena sativaL.)。     

内脐蠕孢属、平脐蠕孢属和凸脐蠕孢属的研究

本文报道了平脐蠕孢属(Bipolaris)一个新种,一个新组合,5个新记录以及凸脐蠕孢属(Exserohilum)一个新记录。讨论了内脐蠕孢属(Drechslera)、平脐蠕孢属、凸脐蠕孢属与长蠕孢属(Helminthosporium)的关系,并就我国已报道的长蠕孢属真菌按Alcorn等~([1,3,4,5,24])人的意见,逐个进行了订正。     

4种杀菌剂对玉米大斑凸脐孺孢病菌的室内毒力测定

采用室内生长速率法测定了4种杀菌剂对玉米大斑凸脐孺孢病菌(Exserohilum turcicum)的毒力。筛选出的2种有明显抑菌作用的药剂70%甲基硫菌灵WP和70%噁霉福美双WP,在5.0μg/mL和10.0μg/mL时抑菌菌率均达100%,在1.25μg/mL和2.5μg/mL时抑菌菌率分别达90.48%~99.05%和90.95%~99.29%,抑菌效果显著,其EC_(50)分别为0.5322μg/mL和0.4892μg/mL。     

农杆菌介导稻瘟病菌绿色荧光蛋白（GFP）遗传转化研究

稻瘟病是水稻生产中的主要病害,抗病品种选育和利用是控制稻瘟病最有效措施。研究稻瘟病菌与水稻品种互作机制是培育抗病品种的基础。GFP（Green fluorescent protein）基因因其具有片段小、易与多种不同蛋白质N端或C端融合等特点,广泛应用于病菌与寄主植物互作研究中。研究以pBGgHg作为转化载体,根癌农杆菌C58C1作为转化介体,转化稻瘟病菌的强致病菌株py1022。研究发现,250μg·mL-1潮霉素B能完全抑制稻瘟病菌生长,利用根癌农杆菌介导稻瘟病菌遗传转化的最佳条件为：稻瘟病菌分生孢子浓度为1×105个·mL-1,共培养时间为4 d,共培养AS浓度为200μmol·mL-1,共培养温度为28℃。随机挑取8个转化子分别进行PCR扩增后测序、荧光显微镜下观察、致病力及稳定性测定,结果表明GFP成功转化到稻瘟病菌中,可为稻瘟病菌与水稻品种的互作机制研究提供技术支撑。     

Jellyfish Green Fluorescent Protein （GFP） as a Reporter for Fusarium gramminearum Development on Wheat

The plasmid pGPDGFP under the control ofpgpdA promotor was used together with vector pAN7-1 containing the hygromycin resistance cassette to co-transform protoplasts of HG1, Fusarium graminearum from Hubei Province, China. Twelve out of 14 hygromycin-resistant transformants showed green signal under the UV light and contained one or several copies ofgfp, as indicated by Southern analysis of genomic DNA digested with different restriction enzymes and hybridized to the gfp probe. A single gfp copy transformant （HG1C5） was selected for further evaluation of 80 Chinese wheat cultivars or advanced lines. The results showed different resistance type to F. graminearum were observed. GFP signals observed in the rachis and adjacent spikes of 70 Chinese wheat lines such as Chuanchongzu 104 indicated both type I （host resistance to the initial infection by the fungus） and type II （resistance to the spread of FHB symptoms within an infected spike） were not observed. While other 10 lines showed type II resistance to F. graminearum with GFP signals only in inoculated spikelets. Development of the mycelium can be intuitively observed and the resistance of wheat to F. graminearum can be identified at 7 days post inoculation （dpi） in this way. The results showed no differences were evaluated between the transformed HG1C5 and the non-transgene artificial inoculation by SAS paired chi-square test and McNernar＇s test （P=-0.0625）.     

浙恢7954农杆菌介导法遗传转化培养基的优化

以综合性状优良的籼稻品种浙恢7954的成熟胚为材料,比较了在遗传转化不同阶段培养基成分对愈伤组织生长及植株再生的影响,进行遗传转化体系的优化。结果表明,最优的愈伤组织诱导培养基为Y1、愈伤组织抗性选择培养基为J3、愈伤组织分化培养基为D6,在此条件下,GFP转化率达26.8%。     

利用绿色荧光蛋白优化辣椒遗传转化体系

【目的】农杆菌介导的辣椒遗传转化较为困难,至今仍未建立高效转化体系。绿色荧光蛋白(GFP)基因是植物遗传转化中常用的报告基因,以GFP为报告基因,优化农杆菌介导的辣椒遗传转化体系。【方法】利用GFP表达系统,统计3个辣椒品种(‘HP’‘8214’和‘L55’)中3种不同外植体(子叶、下胚轴和Flamingo-bill外植体)的不定芽分化率、不定根分化率与荧光阳性率,探究农杆菌侵染浓度、侵染时间、预培养时间和共培养时间等因素对不定芽、不定根分化率及荧光阳性率的影响。【结果】3个辣椒品种的Flamingo-bill外植体不定芽分化率均显著高于下胚轴与子叶外植体,其中‘L55’ Flamingo-bill外植体的不定芽分化率最高、达77.59%,故选用‘L55’ Flamingo-bill外植体进行后续研究。4种农杆菌不同侵染浓度和时间组合下,‘L55’Flamingo-bill外植体均可产生不定芽、不定根及表达GFP的愈伤组织,当农杆菌侵染浓度为OD₆₀₀=0.05,侵染时间为30 min时,不定芽分化率和荧光阳性率最高,分别达48.39%、4.84%。在6种不同预培...     

Early Identification of Stable Transformation Events by Combined Use of Antibiotic Selection and Vital Detection of Green Fluorescent Protein (GFP) in Carrot (Daucus carota L.) Callus

Genetic transformation is a useful technique to complement conventional breeding in crop improvement. Although carrot has been a model organism for in vitro embryogenesis study, genetic transformation of carrot is still lengthy and labor intensive. An efficient transformation and detection system is desirable. Direct infection of Agrobacterium to carrot calli has provided an easy way for carrot genetic transformation. To improve the efficiency of antibiotic selection in this method, we report the combined use of an improved green-fluorescent protein, referred to as smGFP, to establish a versatile selection method for carrot callus transformation system. By combining antibiotic selection with the bright fluorescence observed in the callus tissue, we were able to easily identify stable transformants in early stage of the transformation process. In addition to the GFP expression of the callus cells, the transgenic nature of callus cells was confirmed with Southern and Western analysis. We found we can link the simplicity of carrot-callus-cell transformation, early detection of stable transformants with antibiotic selection, visualization of GFP fluorescence, and molecular analysis (Southern and Western) of callus tissue (non-photosynthetic tissue) to provide a more efficient way in identifying stable transformants at early stage of carrot transformation.     

Early Identification of Stable Transformation Events by Combined Use of Antibiotic Selection and Vital Detection of Green Fluorescent Protein （GFP） in Carrot （Daucus carota L.） Callus

Genetic transformation is a useful technique to complement conventional breeding in crop improvement. Although carrot has been a model organism for in vitro embryogenesis study, genetic transformation of carrot is still lengthy and labor intensive. An efficient transformation and detection system is desirable. Direct infection of Agrobacterium to carrot calli has provided an easy way for carrot genetic transformation. To improve the efficiency of antibiotic selection in this method, we report the combined use of an improved green-fluorescent protein, referred to as smGFP, to establish a versatile selection method for carrot callus transformation system. By combining antibiotic selection with the bright fluorescence observed in the callus tissue, we were able to easily identify stable transformants in early stage of the transformation process. In addition to the GFP expression of the callus cells, the transgenic nature of callus cells was confirmed with Southern and Western analysis. We found we can link the simplicity of carrot-callus-cell transformation, early detection of stable transformants with antibiotic selection, visualization of GFP fluorescence, and molecular analysis （Southern and Western） of callus tissue （non-photosynthetic tissue） to provide a more efficient way in identifying stable transformants at early stage of carrot transformation.