棉花黄萎菌拮抗菌BDT-25的鉴定及抗菌蛋白产生条件研究

拮抗细菌BDT_25是从根际土壤中分离到的一株对棉花黄萎病菌具有强烈抑制作用的芽孢杆菌。经生理生化特征的鉴定,证明BDT_25菌株为枯草芽孢杆菌(Bacillus subtilis)。此外,该菌株还对棉花枯萎病菌、棉花立枯病菌等10种其他病原真菌的生长具有显著的抑制作用,为一株广谱拮抗细菌菌株。通过对BDT_25菌株培养条件的研究发现,采用NB液体培养基,28℃,170 r/min培养48 h所得发酵液对棉花黄萎病菌的抑制作用最强。其发酵液经硫酸铵沉淀粗提到对棉花黄萎病菌具有抑制作用的提取物,进一步实验表明,该拮抗物质对热和多种蛋白酶敏感,证实其为抗菌蛋白。     

棉花黄萎病菌VdSge1基因的敲除与功能分析

为了明确棉花黄萎病菌大丽轮枝菌(Verticillium dahliae Kleb.)的VdSge1基因功能,本研究利用基因同源重组原理,通过构建敲除载体,对棉花黄萎病菌落叶型强致病力菌株V592的VdSge1基因进行了敲除,获得了4个目标基因敲除的突变体;以野生型菌株V592为对照,通过对敲除突变体生物学性状和致病性测定,结果表明,突变体的菌落生长速度、产孢量明显高于野生型,并且丧失对棉花的致病性。利用q RT-PCR对其他基因在突变体中的表达情况进行分析,结果表明,VdSge1基因敲除后,VMK1、VGB和VdGARP1的表达量随之下降,而VDH1和PevD1的表达却明显上升。由此说明,VdSge1基因与大丽轮枝菌的菌落生长速度、产孢量及致病性密切相关,并且可以影响其他一些基因的表达。     

Creation of a New Resistant Germplasm to Verticillium Wilt by Distant Hybridization in Upland Cotton

To create new germplasm lines resistant to Verticillium wilt in upland cotton, 65 distant hybridization germplasm lines (DHGLs) in upland cotton genetic background were cultivated by interspecific hybridization between Gossypium hirsutum and wild species including G. anomalum, G. armourianum, G. aridum, G. raimondii, G. mustelinum, interspecific F₁ backcrossing with G. hirsutum for four generations, and selfing for four generations, followed by a conventional breeding program. The results of agronomic trait identification during 2011-2012 indicated that average plant heights of DHGLs were closely similar to commercial cultivars of upland cotton, while average fruit branches, fruit nodes, bolls of DHGLs individual plant were lower than those in commercial cultivars of upland cotton. Average single boll weight and lint percentage of DHGLs were lower than commercial cultivars of upland cotton. Fiber length, strength, fineness and maturity of DHGLs were reasonably collocated. Fiber of most lines was suitable for spinning extra high count yarn, but the main fiber quality indices of commercial cultivars of upland cotton were not well coordinated. Identification of resistance to Verticillium wilt in defoliation disease nursery during 2012-2013 indicated that five DHGLs resistant to Verticillium wilt . Suyuan 040, Suyuan 045 and Suyuan 061 were highly resistant to Verticillium wilt with a disease index of 8.33, 4.35 and 7.79, respectively. Suyuan 030 and Suyuan 034 were resistant to Verticillium wilt with a disease index of 12.35 and 13.70, respectively. The genetic relationship of new germplasm lines resistant to Verticillium wilt were traced and showed that Suyuan 040 and Suyuan 045 were DHGLs of G. raimondii, Suyuan 061 was DHGL of G.mustelinum, Suyuan 030 and Suyuan 034 were DHGLs of G. aridum.     

不同强度温度刺激对棉花黄萎病的影响

采用三个不同抗性的棉花品种、三个不同的黄萎病菌系和三个温度梯度,完全随机区组设计,在人工气候箱中研究刺激不同时间对棉花黄萎病发病的影响。结果表明,刺激温度的高低和品种的抗性对棉花黄萎病的发生与扩展有显著的影响,而菌系致病力以及刺激时间长短对棉花黄萎病的发生也存在显著差异,但影响较小;品种的抗病性对棉花黄萎病的发生起着至关重要的制约作用;温度对黄萎病的发生与扩展起着关键作用。初步明确了影响棉花黄萎病发生与扩展的几个因子间的互作效应及其影响程度。     

棉花黄萎病菌毒素ELISA检测方法的建立及其应用

以纯化的棉花黄萎病菌毒素(PLPC)免疫新西兰白兔制备了PLPC特异性抗血清,建立了可特异性检测棉花黄萎病菌毒素的A蛋白双抗体夹心ELISA方法。应用建立的ELISA方法测定了病菌培养滤液、人工接种幼苗和大田病株不同组织中的毒素,结果表明:不同产毒能力菌株(VD 8和VD-5)在25℃下振荡培养3d后就能在培养滤液中检测到毒素,这比生物测定要早2～3d。将VD-8菌株的孢子以灌根方式接种泗棉3号幼苗5d后,就能从接种幼苗的茎杆和叶柄中检测到毒素,这比幼苗自然发病显症要早3～5d。在测定的30份大田病株茎杆、叶柄、叶脉样品中,阳性样品率为100%。这些结果表明建立的ELISA方法可用于菌株产毒能力的测定和大田棉花黄萎病的早期诊断。     

连作对棉田土壤枯、黄萎病菌数量及细菌群落的影响

采用选择培养法测定新疆库尔勒不同连作年限棉田土壤枯、黄萎病病菌数量的变化,运用PCR-DGGE(Polymerase chain reaction-denatured gradient gel electrophoresis)技术分析棉花连作对土壤细菌群落结构和多样性的影响。结果表明,不同连作年限棉田的枯萎病菌和黄萎病菌量具有相似的变化特征,即在5~20年连作年限内,土壤中病原菌量随着连作年限的延长而增加,20年连作土壤病原菌数量达到最高峰;连作超过20年,土壤菌源数量开始下降。不同连作年限棉田土壤中枯、黄萎病病原菌的数量与发病株率呈显著正相关关系。连作棉田土壤细菌群落的DGGE图谱分析显示,连作棉田土壤细菌种群的组成、结构、丰富度均发生变化。连作5~40年的棉田土壤细菌种群Shannon-Wiener多样性指数(H')、均匀度(EH′)和丰富度(S)指数均降低,且连作年限越长降低越突出。     

棉酚及色素腺体对棉花抗枯萎病性的作用

本文对低酚棉与有酚棉的抗病和感病品种及其杂交后代进行了研究。结果表明，在种子中棉酚含量及色素腺体密度与品种的抗枯萎病性没有相关性。在幼苗中，低酚棉幼苗棉酚含量均显著高于有酚棉品种，但它们之间的抗枯萎病性也没有显著差异。在低酚棉与有酚棉品种杂交的Ｆ２代群体中，无色素腺体和稀色素腺体类型苗的抗病性明显高于腺体密度较高的类型苗。对７个杂交组合Ｆ２代中有色素腺体和无色素腺体幼苗的抗病性分析表明，无色素腺体类型苗的病指显著低于有色素腺体类型苗的病指。幼苗人工接种枯萎菌后，棉酚含量表现升高，但抗病品种低于感病品种。     

棉花抗黄萎病基因的QTL定位

以高感黄萎病的陆地棉品种"邯郸208"与高抗黄萎病海岛棉品种"Pima90"的136个F2单株为作图群体,构建了一个包括17个连锁群、标记间平均间距18.61cM、全长1842.8cM的陆海种间分子标记遗传连锁图,该图约覆盖棉花基因组的36.8%。单因子方差分析和复合区间作图检测到与黄萎病抗性相关的3个QTL,分别位于第四连锁群和第七连锁群上,分别解释表型变异方差的15.39%、54.11%和57.18%。初步认为海岛棉"Pima90"对陆地棉"邯郸208"的黄萎病抗性由两个主效QTL和一个微效QTL共同控制。     

Use of culture-derived Fusarium oxysporum f. sp. cubense, race 1 filtrates for rapid and non-destructive in vitro differentiation between resistant and susceptible clones of field-grown banana

Banana and plantain are among the most important food crops in developing countries but production is threatened by increasing virulent forms of Fusarium oxysporum f. sp. cubense. Chemical control is not economically effective and,therefore, breeding programs are necessary. Traditional field studies of new genotype resistance to this disease are time-consuming and destructive. Therefore,we developed a rapid and non-destructive procedure to differentiate field-grown banana resistant from susceptible clones. This procedure implicates application of culture filtrates of Fusarium oxysporum f. sp. cubense race 1 onto banana leaves. The relationship between duration of the fungal in vitro incubation, and the fungal culture fresh mass, the culture filtrate absorbency, and the Gross Michel (susceptible cultivar)leaf lesion area (after application of the culture filtrate) were similar and at 24day-incubation the highest values of the recorded indicators were observed. A comparison between Gross Michel and FHIA-01(resistant) was also performed. The most relevant differences between cultivars were observed at 48 hours after application of the culture filtrate, and in the middle-aged leaves. The position of the culture filtrate application in the leaf limb (distal, middle, proximal) was not determinant. A wider comparison among banana cultivars confirmed previous results informed by other researchers using different systems to study this plant-fungus interaction. Such a confirmation validates the effectiveness of the procedure described here to select rapid and non-destructively banana resistance to this disease at field level. This revised version was published online in August 2006 with corrections to the Cover Date.     

不溶性胞壁结合酚类物质在棉花对枯萎病抗性中的作用

分析了枯萎病菌侵染后棉花叶片和根茎部组织中不溶性胞壁结合酚类物质 (胞壁结合的简单酚、复杂酚聚合物及黄酮醇 )含量的变化。结果表明 ,抗病品种棉苗中不溶性胞壁结合酚类物质含量高于感病品种 ,同一品种内根茎部组织中含量较高 ;氟乐灵播前土壤处理可诱发棉苗产生对枯萎病的诱导抗性 ,同时也促进了棉苗组织中不溶性胞壁结合酚类物质的积累 ;枯萎病菌侵染后棉苗组织中不溶性胞壁结合酚类物质含量明显增加 ,抗病品种棉苗和经氟乐灵处理的棉苗受侵后不溶性胞壁结合酚类物质含量的增加分别大于感病品种棉苗和未经氟乐灵处理棉苗中的增加水平。这些结果说明 ,不溶性胞壁结合酚类物质在棉花对枯萎病的抗性及由氟乐灵诱发的诱导抗性中起作用