棉花与番茄抗棉花黄萎病不依赖于Ve1

黄萎病是我国棉花的主要病害之一,发掘抗病基因和阐明抗病机制是开展棉花抗病分子育种的基础.本研究将目前唯一的植物抗黄萎病主效基因Ve1分别在本氏烟和陆地棉中超量表达,以探讨其在防控棉花黄萎病中的价值.研究发现,Ve1基因在本氏烟中超量表达后并未对番茄大丽轮枝菌2个生理小种和棉花黄萎病菌产生明显抗性.RT-PCR分析表明,Ve1并不能激活烟草抗病相关基因的表达,推测本氏烟中可能不存在完整的Ve1介导的抗黄萎病信号路径.Ve1超量表达的转基因棉花接种棉花黄萎病菌"V991"后表现出与本氏烟类似的结果,同时发现,陆地棉对番茄大丽轮枝菌1号生理小种表现出高抗性.利用番茄抗/感黄萎病近等基因系"Craigella GCR218"/"Craigella GCR26"进一步研究发现,2个番茄材料均对棉花落叶型强致病力黄萎病菌"V991"免疫,这暗示番茄对棉花黄萎病菌的抗性不依赖于Ve1.分子鉴定表明,棉花黄萎病菌与番茄大丽轮枝菌1号和2号生理小种存在明显区别,番茄大丽轮枝菌1号和2号生理小种均属于非落叶型黄萎病菌.本研究中鉴定的棉花黄萎病菌均不含有ave1基因,这可能是在棉花中超量表达Ve1并不能增强其棉花黄萎病菌抗性的直接原因.通过病毒介导的基因沉默,在抑制GbSERK1的表达后能显著削弱海岛棉对黄萎病菌的抗性,证实"海7124"中存在类似于Ve1的下游抗病信号路径.本研究还对棉花类受体蛋白的进化以及Ve1抗病信号路径在棉花抗黄萎病机制研究中的价值进行了探讨.     

我国首获转基因抗黄萎病棉花新株系

我国科学家在世界上首次获得转基因抗黄萎病棉花新株系。中国农业大学植物病理系齐俊生博士课题组,在导师李怀方教授的指导下,经过6年的努力,从“海岛棉”中分离、克隆出有自主知识产权的抗黄萎病基因“At7”,并导入到“陆地棉”,从而培育出高抗黄萎病的新体系。     

大丽轮枝孢微菌核的形成条件

以棉花黄萎病菌Verticillium dahliae XJ2008菌株为试材,研究了培养基类型、pH值、温度等因素对棉花黄萎病菌微菌核形成的影响,确立了微菌核形成的最佳培养条件,并对采自新疆、江苏、河南、陕西、山东等地的15个棉花黄萎病菌菌株进行了测试。结果表明,适合于棉花黄萎病菌微菌核大量产生的培养基为基础改良培养基（BMM）、pH值为9.5－11.5、温度为20℃。在该条件下,15个大丽轮枝孢菌株在接种后第12天即可产生大量的微菌核,不同菌株产生微菌核的数量及大小间存在着显著差异,但均与病菌的致病性     

4种木霉菌对棉花黄萎病菌抑制作用的测定

以绿色木霉(Trichoderma viride)、康氏木霉(Trichoderma koningii)和二株未知木霉菌株(Trichoderma spp.)为供试木霉菌株,采用对峙培养法测定了不同温度处理下对棉花黄萎病菌(Verticilliam dahliae)的拮抗作用。结果表明,木霉在不同温度下对棉花黄萎病菌的抑制作用不一,其中以30℃和25℃黑暗条件下木霉对棉花黄萎病菌菌丝生长的抑制作用最强,对峙培养5d后,绿色木霉、康氏木霉和二株未知木霉菌在30℃条件下对棉花黄萎病菌的抑制率分别达到67.3%、65.9%、56.8%、65.9%,25℃条件下的抑制率分别为65.9%、72.9%、65.9%、78.8%;20℃下木霉菌株对棉花黄萎病菌的抑制作用次之,10℃、15℃和35℃条件下木霉菌丝扩散速度较慢,且孢子产生量少,不能有效地抑制棉花黄萎病菌菌丝的扩展,表明环境温度过高或过低对木霉菌丝的生长及分生孢子的产生均有较大影响。该研究为筛选棉花黄萎病菌更为有效的生防木霉菌株提供理论依据。     

棉花黄萎病拮抗菌KF-43-1的鉴定与3种处理田间防治效果对比

【背景】棉花黄萎病具有棉花“癌症”之称,对棉花的产量和纤维质量造成较重危害,生物防治以绿色安全等优点成为防治棉花黄萎病应用领域的重点研究内容,因此开发新的生物菌剂防治棉花黄萎病对棉花生产工作具有重要意义。【目的】通过对具有拮抗棉花黄萎病菌作用的放线菌KF-43-1进行初步鉴定及生理生化测定,明确最佳使用方式,为开发新的防治棉花黄萎病生物菌剂提供支持。【方法】对菌株KF-43-1进行分子鉴定,观察其形态特征及生长特性,测定菌株KF-43-1对病原真菌的抑菌效果,明确菌株KF-43-1的生理生化特性,验证放线菌KF-43-1不同使用方式对棉花黄萎病的田间防治效果。【结果】培养特征和显微特征及分子鉴定结果将菌株KF-43-1鉴定为白浅灰链霉菌(Streptomyces albogriseolus);菌株KF-43-1对病原菌V991抑制效果达到82.05%,防效高于放线菌5406,对尖孢镰刀菌萎蔫专化型ST89抑制效果达到25.81%,低于放线菌5406的防效;菌株KF-43-1无荧光反应,能够使明胶液化、淀粉水解、甲基红试验阳性、可以产生黑色素,不具备分解纤维素的能力,菌株KF-43-1在pH 7.0-8.0时生长情况良好,在盐浓度为2%的高氏一号培养基上生长受到抑制并表现出一定的耐盐性,使用拮抗菌KF-43-1菌液制作种衣剂对棉花黄萎病的防效高于离心液包衣和离心液叶面喷施,说明拮抗菌KF-43-1在土壤中定殖后对棉花黄萎病暴发具有更好的抑制效果。【结论】棉花黄萎病拮抗放线菌KF-43-1作为一种新型生防菌,可为开发防治棉花黄萎病的生物菌剂提供生防菌资源,具有良好的开发价值和应用价值。     

棉花枯黄萎病及其抗性鉴定技术

棉花枯黄萎病是我国最主要的棉花病害,种植抗病品种是有效的防治方法。抗病鉴定是棉花抗病育种的关键技术之一。人工病圃鉴定是棉花抗病鉴定的主要方法,相对抗性指数(IR)或相对抗病效果(ER)最适合作为抗病鉴定指标,根据棉花的抗病程度可将棉花的抗病水平分为免疫、高抗、抗病、耐病和感病5级。     

天麻抗真菌蛋白基因(gafp)转化彩色棉的研究

天麻抗真菌蛋白(gastrodia antifungal protein简称GAFP)是从我国传统中药天麻(Gastrodia elata B1．)中分离到的一种具有广谱抗真菌活性的蛋白质,它对许多植物真菌病包括棉花枯萎病、黄萎病等的致病菌离体具有很强的抑制作用,因此,在植物抗真菌病基因工程上有很重要的应用价值。本研究通过花粉管通道法,将GAFP的基因．gafp转入3个新疆彩色棉品种中,通过田间抗病筛选和分子检测,得到了高抗黄萎病的转基因植株,两株Southem杂交阳性植株LB-5-8和ZB-1—49对黄萎病表现整株免疫。RT-PCR的结果显示,LB-5-8和ZB-1—49中均有gafp的正确转录;离体的抑菌实验也表明,它们的蛋白粗提物对棉花黄萎病致病菌离体有明显的抑制,表明了gafp在转基因植株中的正确表达,翻译的产物具有活性。经过进一步选育和扩繁,发现转基因彩色棉后代具有稳定的、较强的抗黄萎病能力,本研究为通过植物抗病基因工程的方法防治棉花黄萎病提供了一条新的途径。     

棉花黄萎病菌的侵染过程

深入揭示黄萎病菌的致病机理,为培育高抗新品种提供理论依据和技术途径,是有效控制黄萎病的根本.本文系统探讨了土壤中微菌核或孢子受寄主根系分泌物的刺激,开始萌发、产生的菌丝在寄主根表面定殖、穿过表皮、在皮层中发生及在寄主体内扩展和症状形成等黄萎病菌侵染棉花的过程,阐述了寄主植物形成多级防御反应阻击病原菌的入侵.对黄萎病菌侵染寄主植物过程及其机理下一步研究的问题和内容进行了讨论.     

棉花黄萎病病原菌诱导侵染对转基因抗病棉花生理性状的影响

通过研究棉花黄萎病病原菌诱导侵染对转基因抗病棉花生理性状的影响,旨在为提高转基因抗病棉花抗病性提供理论依据。Byd、28p两品种棉花为供试材料,应用花粉管通道法将几丁质酶与β-1,3-葡聚糖双价抗病基因导入棉花株系中,多年筛选获得不同抗性水平的棉花株系;棉种经棉花黄萎病病菌侵染后,苗期测定棉株内的生理生化变化。结果显示,转基因抗病棉株体内的酶活性已达对照水平,而感病及耐病棉株内的酶活性均有不同程度的变化。所测定的棉株内的酶活性及脯氨酸含量的变化与棉株的抗病性相关。     

陆地棉品种和骨干品系黄萎病抗性鉴定

选育和推广抗病品种是防治陆地棉黄萎病的主要措施,为了早日实现多类型、多区域大面积抗病品种的应用,本研究选取107份遗传背景差异较大的种质,利用河北省农林科学院棉花研究所小安舍试验站黄萎病病圃进行了3年黄萎病抗性重复鉴定。鉴定得到抗病品系8个,占7.5%;耐病品种(系)20个,占18.7%。本研究表明,当前被作为育种亲本的抗病品系还太少,需要深入开展抗病遗传机制,以及与其他经济性状协同改良的关系,为陆地棉抗病育种提供理论指导;达到抗病或接近抗病水平的大部分品种(系)来自于海陆野远缘后代,具有外源基因血统,证明了远缘杂交是陆地棉黄萎病抗性改良的有效手段。     

Molecular mapping of Verticillium wilt resistance QTL clustered on chromosomes D7 and D9 in upland cotton

Verticillium wilt is a destructive disease with international consequences for cotton production. Breeding broad-spectrum resistant cultivars is considered to be one of the most effective means for reducing crop losses. A resistant cotton cultivar, 60182, was crossed with a susceptible cultivar, Jun-mian 1, to identify markers for Verticillium resistance genes and validate the mode of its inheritance. Genetic segregation analysis for Verticillium wilt resistance was evaluated based upon infected leaf percentage in the seedling stage using major gene-polygene mixed inheritance models and joint analysis of P₁, P₂, F₁, B₁, B₂ and F₂ populations obtained from the cultivar cross. We found that resis-tance of upland cotton cultivar 60182 to isolates BP2, VD8 and T9, and their isoconcentration mixture was controlled by two major genes with additive-dominance-epistatic effects, and the inheritance of the major gene was dominant. Furthermore, a genetic linkage map was constructed using F₂ segregating population and resistance phenotypic data were obtained using F_2︰3 families inoculated with different isolates and detected in different developmental stages. The genetic linkage map with 139 loci was comprised of 31 linkage groups covering 1165 cM, with an average distance of 8.38 cM between two markers, or 25.89% of the cotton genome length. From 60182, we found 4 QTL on chromosome D7 and 4 QTL on D9 for BP2, 5 QTL on D7 and 9 QTL on D9 for VD8, 4 QTL on D7 and 5 QTL on D9 for T9 and 3 QTL on D7 and 7 QTL on D7 for mixed pathogens. The QTL mapping results revealed that QTL clusters with high contribution rates were screened simultaneously on chromosomes D9 and D7 by multiple interval mapping (CIM), whether from resistance phenotypic data from different developmental stages or for different isolates. The result is consistent with the genetic model of two major genes in 60182 and suggests broad-spectrum resistance to both defoliating isolates of V. dahliae and nondefoliating iso-lates. The markers associated with resistance QTL may facilitate the use of Verticillium wilt resistance genes in improving breeding programs for cotton.     

Detection of the defoliating and nondefoliating pathotypes of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> in artificial and natural soils by nested PCR

In Spain, Verticillium wilt, caused by Verticillium dahliae, is the most important disease of cotton and olive. Isolates of V. dahliae infecting these crops can be classified into highly virulent, defoliating (D), and mildly virulent, nondefoliating (ND), pathotypes. Infested soil is the primary source of inoculum for Verticillium wilt epidemics in cotton and olive, and severity of disease relates to the prevailing V.dahliae pathotype. In this work we have adapted the use of previously developed primer pairs specific for D and ND V. dahliae for the detection of these pathotypes by nested PCR in artificial and natural soils. Success in the detection procedure depends upon efficiency in extracting PCR-quality DNA from soil samples. We developed an efficient DNA extraction method from microsclerotia infesting the soil that includes the use of acid washed sand during the grinding process and skimmed milk to avoid co-purification of Taq-polymerase inhibitors with DNA. The specific nested-PCR procedure effectively detected 10 or more microsclerotia per gram of soil. The detection procedure has proven efficient when used with a naturally infested soil, thus demonstrating usefullness of the diagnostic method for rapid and accurate assessment of soil contamination by V. dahliae pathotypes.     

Arbuscular mycorrhizal fungi and biological control of Verticillium-wilted cotton plants

The interaction of arbuscular mycorrhizal fungi (Glomus etunicatum, Glomus intraradices, and Glomus versiforme) with a wilt-causing soil-borne pathogen, Verticillium dahliae, was studied in cotton. It was found that establishment by arbuscular mycorrhizal fungi reduced disease index. In diseased cotton plants colonised by G. etunicatum, the disease index was less than other diseased mycorrhizal and non-mycorrhizal ones. In diseased cotton plants, chlorophyll content was lower than others. Three Glomus species significantly increased content of sugar and protein in shoot and root. Pathogen-infected plants had higher proline concentration in shoot and root than healthy plants. On the other hand, the increased content of proline as stress sensor showed that Verticillium accelerates senescence and reduces yield. These results suggest that the beneficial effects of mycorrhiza can alleviate the pathogenesis effects of V. dahliae partly, and also there is a competitive interaction between the pathogenic and symbiotic fungi.     

Reaction of Cotton Cultivars and an F2 Population to Stem Inoculation with Isolates Verticillium dahliae

Four Verticillium dahliae isolates (V76, TS‐2, PH, and V44) were used in screening four cotton cultivars (Pima S‐7, Acala Prema, M‐315 and Acala 44). Pima S‐7 and Acala Prema gave the highest resistance reactions and Acala 44 was the most susceptible. Isolate V76 of V. dahliae was the most virulent. An interspecific cross between the resistant cv Pima S‐7 (Gossypium barbadense) and the susceptible cv. Acala 44 (G. hirsutum) was made and the F₂ population phenotyped for Verticillium wilt effect. Phenotyping of plant reaction to the disease was quantified by using a set of six growth parameters (number of healthy leaves, number of nodes, leaf weight, stem weight, leaf to stem ratio, and total shoot weight) measured 3 weeks after inoculation. The F₂ phenotypic distribution of these parameters suggests that distribution is towards resistance and polygenic. Transgressive segregation also was observed. The number of healthy leaves and total shoot weight were found to be the best indicators of resistance. Results obtained in this study will be useful to quantify resistance to V. dahliae and identify the best parameters to phenotype in genetic studies.     

Evaluation of the effectiveness of a consortium of three plant-growth promoting rhizobacteria for biocontrol of cotton Verticillium wilt

We investigated the biocontrol efficacy of a consortium of three rhizobacteria (hereafter BBS) against cotton Verticillium wilt, along with the effect on plant growth, in a series of greenhouse and field experiments. BBS treatment inhibited germination of Verticillium dahliae spores by 88.4%. Under greenhouse conditions, BBS reduced Verticillium wilt by 86.1% compared to the untreated control, and promoted plant growth by 49.9%. In field experiments, nine L/acre of BBS suspension reduced Verticillium wilt by 76.0% and increased cotton yields by 13.7%. Soil inoculation with BBS also improved the breaking tenacity and uniformity index of harvested cotton. Soil properties improved with BBS treatment in field experiments, including an increase in organic matter and the availability of nitrogen (N), phosphorus (P) and potassium (K). There was no significant difference in the biocontrol efficacy of BBS among eight tested cotton cultivars.     

Mapping and quantitative trait loci analysis of verticillium wilt resistance genes in cotton

Verticillium wilt is one of the most serious constraints to cotton production in almost all of the cotton-growing countries. In this study, ＂XinLuZaol＂ （XLZl）, a susceptible cultivar Gossypium hirsutum L. and ＂Hai7124＂ （H7124）, a resistant line G. barbadense, and their F2：3 families were used to map and study the disease index induced by verticillium wilt. A total of 430 SSR loci were mapped into 41 linkage groups; the map spanned 3 745.9 cM and the average distance between adjacent loci was 8.71 cM. Four and five quantitative trait loci （QTLs） were detected based on the disease index investigated on July 22 and August 24 in 2004, respectively. These nine QTLs explained 10.63-28.83% of the phenotypic variance, six of them were located on the D sub-genome. Two QTLs located in the same marker intervals may partly explain the significant correlation of the two traits. QTLs explaining large phenotypic variation were identified in this study, which may be quite useful in cotton anti-disease breeding.     

Cotton GhSSI2 isoforms from the stearoyl acyl carrier protein fatty acid desaturase family regulate Verticillium wilt resistance

Lipids are major and essential constituents of plant cells and provide energy for various metabolic processes. However, the function of the lipid signal in defence against Verticillium dahliae, a hemibiotrophic pathogen, remains unknown. Here, we characterized 19 conserved stearoyl-ACP desaturase family proteins from upland cotton (Gossypium hirsutum). We further confirmed that GhSSI2 isoforms, including GhSSI2-A, GhSSI2-B, and GhSSI2-C located on chromosomes A10, D10, and A12, respectively, played a dominant role to the cotton 18:1 (oleic acid) pool. Suppressing the expression of GhSSI2s reduced the 18:1 level, which autoactivated the hypersensitive response (HR) and enhanced cotton Verticillium wilt and Fusarium wilt resistance. We found that low 18:1 levels induced phenylalanine ammonia-lyase-mediated salicylic acid (SA) accumulation and activated a SA-independent defence response in GhSSI2s-silenced cotton, whereas suppressing expression of GhSSI2s affected PDF1.2-dependent jasmonic acid (JA) perception but not the biosynthesis and signalling cascade of JA. Further investigation showed that structurally divergent resistance-related genes and nitric oxide (NO) signal were activated in GhSSI2s-silenced cotton. Taken together, these results indicate that SA-independent defence response, multiple resistance-related proteins, and elevated NO level play an important role in GhSSI2s-regulated Verticillium wilt resistance. These findings broaden our knowledge regarding the lipid signal in disease resistance and provide novel insights into the molecular mechanism of cotton fungal disease resistance.