葡萄白粉病抗性鉴定方法优化及品种抗性评价

本研究通过对叶盘保湿方法的改进, 不同菌源接种后发病严重度的分析, 抗?感病对照品种的确定, 建立了一种更为便捷?可靠?稳定的葡萄白粉病室内抗性鉴定方法:取葡萄藤自上而下第2~4片幼嫩叶片, 打取直径15 mm的叶盘置于1.5%水琼脂平板进行保湿, 采集田间菌源直接制备孢子浓度为2×105个/mL的孢子悬浮液接种, 选定抗?感病葡萄品种做对照, 14 d即可完成抗性鉴定?采用此方法对11个葡萄品种(品系)进行了葡萄白粉病抗性评价, 结果表明:供试的11个品种(品系)对葡萄白粉病的抗性存在明显差异, 其中高感品种为‘巨峰’‘夏黑’和‘阳光玫瑰’等, 感病品种为‘玫瑰香’和‘马瑟兰’, 抗病品种为‘瑞都科美’和‘无核翠宝’, 高抗品种为‘北冰红’?试验结果为葡萄抗病品种的选育及利用提供了方法和基础参考数据?     

Spatiotemporal distribution of flavescence dorée phytoplasma in grapevine

In this study quantitative real‐time PCR was used to follow the seasonal changes of flavescence dorée phytoplasma (FDp) titre in grapevines of cv. Modra frankinja (syn. Blaufränkisch) and cv. Refo?k (syn. Refosco'd'Istria) from two vineyards located in climatically different vine‐growing regions of Slovenia. Besides its known presence in the leaf veins, FDp was also detected in flowers, berry tissues and tendrils. In plants with high concentrations of FDp in tissues with symptoms, phytoplasma was also detected in symptomless tissues. A trend of decreasing FDp titre in all examined symptomless tissues from June to July and an increasing one throughout the growing season in tissues with symptoms was recorded. Accordingly, FDp was present in detectable amounts in flowers, petioles and veins of almost all infected plants in the late spring, and was detected in all examined tissue types in summer, with the highest titre in berries in August. The study showed that in the absence of plant health measurements an FDp infection may spread exponentially by a factor of 40 per year.     

The spatiotemporal spread of esca disease in a Cabernet Sauvignon vineyard: a statistical analysis of field data

The occurrence and spread of plants showing esca symptoms were assessed in a vineyard located on the plains of a northern Italian wine‐growing region. Esca disease symptoms were assessed over 16 consecutive years, beginning one year after planting. The number of plants with symptoms was recorded over time, considering both vines with foliar symptoms in the year of assessment (manifest esca) and vines with foliar symptoms in previous years (hidden esca). The sum of manifest and hidden esca was indicated as cumulative esca. The first symptoms of esca appeared in the sixth year of cultivation, with the incidence of manifest esca increasing to approximately 3% nine years after planting. The number of cumulative plants with symptoms increased exponentially in the final period of observation. The aim of this work was to investigate the spatiotemporal spread of esca infection throughout the vineyard and to assess the distribution pattern of plants with symptoms using Bayesian spatiotemporal models. The research results seem to support a higher probability of infection along rows rather than among adjacent rows. This observation may have implications for the technical management of the vineyard.     

Effect of High Nickel and Chromium Background Levels in Serpentine Soil on Their Accumulation in Organs of a Perennial Plant

The effect of high concentrations of nickel (Ni) and chromium (Cr) in alkaline serpentine Fluvisol (FL 1) on their uptake by grapevine as a perennial plant was compared to their accumulation on alkaline Fluvisol (FL 2) and an acid Cambisol (CM). The FL 1 revealed high pseudo total Ni (900–1737 mg kg^?1) and Cr (263–775 mg kg^?1) concentrations, whereas those in FL 2 and CM were low. Diethylenetriaminepentaacetic acid (DTPA)–extractable Ni was greatest in FL 1; DTPA‐extractable Cr was less than the detection limit. Concentrations of metals in grapevines revealed the pattern root > leaves > shoots > grapes. At FL 1, high Ni and Cr concentrations (40.7–68.8; 23.3–41.3 mg kg^?1) in roots were measured. In grapes, these concentrations were low (Ni 0.4–0.9; Cr 0.1–0.6 mg kg^?1), whereas those on FL 1 do not differ significantly from others, indicating that alkaline serpentine soils may be used for grapevine or other perennial plant growth.     

Phylogenetic and serological analyses reveal genetic diversity of Agrobacterium vitis strains in Japan

Twenty-eight strains of Agrobacterium vitis , including both tumorigenic and nonpathogenic phenotypes involving 26 isolated in Japan and strains NCPPB 3554^T and NCPPB 2562 isolated in Australia and Greece, respectively, were characterized by means of a slide agglutination test (SAT) using antisera prepared against somatic antigens. Phylogenetic analyses were also carried out, using the results of repetitive sequence-based polymerase chain reaction and the partial nucleotide sequences of pyrG , recA and rpoD . The A. vitis strains separated into four serogroups (A to D) based on the SAT reactivity. The phylogenetic analyses showed A. vitis strains separated into four tumorigenic groups (A to D) and one nonpathogenic group (E). Serogroups A to C corresponded exactly to genetic groups A to C, respectively, whereas serogroup D further divided into two distinct genetic groups, D and E. Genetic group E was isolated in Okayama Prefecture, Japan, and all strains of it have been found to coexist with tumorigenic strains belonging to the other groups within the same galled grapevine tissues. These results suggest that A. vitis strains are genetically heterogeneous and can be separated into several genetic groups. The differences between the nucleotide sequences of pyrG , recA and rpoD of the genetic groups will enable the development of a simple and convenient monitoring method, which will increase understanding of the dynamics of each genetic group of A. vitis in the natural environment.     

干旱胁迫对酿酒葡萄赤霞珠光合特性的影响

应用温室盆栽试验方法,研究了两年生酿酒葡萄赤霞珠(Cabernet Sauvignon)受不同梯度干旱胁迫后,其形态指标、光合生理、光合色素含量等的变化.研究结果表明:①干旱胁迫可以显著降低赤霞珠葡萄的植株生物量,且胁迫程度越深,对植株生长影响越大;②随着干旱胁迫程度的加深,净光合速率(Pn)、气孔导度(Gs)和蒸腾速率(Tr)均有显著降低,胞间CO2浓度(Ci)呈现先降低、但最终上升而累积的现象,葡萄光合作用能力随胁迫程度的加重而下降;③叶绿素含量随胁迫程度加深而降低,叶绿素a/b的值则较稳定.     

生物技术与葡萄遗传育种

 生物技术的发展和应用已渗透到葡萄遗传育种的各个领域。DNA分子标记为研究葡萄品种起源和育成新品种提供了强有力的工具,特别是SSR标记在葡萄品种鉴别、系谱分析和遗传图谱构建方面已得到广泛应用。基于DNA分子标记遗传图的构建加速了杂种的早期选择,遗传图和物理图的结合可以克隆控制重要性状的功能基因,遗传转化技术的应用可以改良现有的葡萄品种。因此,生物技术正深刻改变着传统的葡萄育种方法。     

葡萄枝条中多酚类物质的超声波辅助提取

【目的】探讨利用超声辅助技术从葡萄枝条中提取多酚类物质的最佳工艺条件,并比较8个酿酒葡萄品种枝条中多酚类物质含量的差异。【方法】对影响葡萄枝条中多酚类物质提取率的乙醇溶液体积分数、浸提温度、料液比(质量(g)与体积(mL)比)、提取时间和提取次数进行单因素试验,并在此基础上设计4因素3水平的正交试验,以确定超声波提取的最佳工艺条件;利用最佳工艺条件提取8个葡萄品种枝条中的多酚类物质,并对其含量进行测定。【结果】超声波辅助提取葡萄枝条中多酚类物质的最佳条件为:以体积分数60%乙醇溶液为提取液,料液比为1∶12,超声提取时间为30 min,于20℃下浸提2次。在最佳工艺条件下,多酚类物质的提取率为97.38%。8个酿酒葡萄品种枝条中的多酚类物质含量均大于20 mg/g,其中品丽珠含量最高(32.855 4 mg/g),其次为梅尔诺(31.967 7 mg/g),以8804最低(20.815 6 mg/g)。【结论】确定了超声波辅助提取葡萄枝条中多酚类物质的最佳工艺条件,在此工艺条件下提取率最高可达97.38%。     

葡萄类钙调磷酸酶B亚基互作蛋白激酶VvCIPK10的特性与表达

【目的】在葡萄中克隆丝苏氨酸蛋白激酶Vv CIPK10,分析其激酶特性和在逆境胁迫下的表达模式,为进一步研究该基因参与逆境胁迫的分子功能,探讨葡萄抗逆分子机制提供理论依据。【方法】利用电子克隆技术获得Vv CIPK10序列,设计特异引物进行RT-PCR反应,对克隆到的序列进行开放阅读框和保守结构域分析;构建原核表达载体,转化表达菌株后用IPTG进行诱导表达,收集菌体后裂解细胞,制备蛋白上样液,SDS-PAGE电泳对表达产物进行分析,同时对融合蛋白进行可溶性分析;IPTG大量诱导表达融合蛋白,收集菌体后进行超声破碎细胞,用麦芽糖结合蛋白纯化柱纯化MBP-Vv CIPK10融合蛋白,SDS-PAGE电泳进行分析;纯化后的融合蛋白与体外自磷酸化缓冲液进行自磷酸化反应,反应后SDS-PAGE电泳,压磷屏检测体外自磷酸化反应;构建重组瞬时表达载体p BI221-GFP/Vv CIPK10;分离拟南芥原生质体,通过PEG介导的瞬时转化方法将重组表达载体p BI221-GFP/Vv CIPK10转化至原生质体;通过基因枪介导的转化方法将重组表达载体p BI221-GFP/Vv CIPK10转化至洋葱表皮细胞,培养16 h后用激光共聚焦显微镜进行荧光信号检测;选择生长相对一致且健壮的葡萄植株,于干旱、低温和盐胁迫处理后不同时间取样,同时在田间取葡萄不同组织样品,实时荧光定量PCR检测Vv CIPK10在葡萄不同组织中的表达以及在不同逆境胁迫下的表达模式。【结果】PCR克隆获得葡萄Vv CIPK10全长为1 357 bp,5′端非编码区为30 bp,3′端非编码区为156 bp,开放阅读框为1 171 bp,编码436个氨基酸,理论等电点为8.59,分子量为48.7 k Da。保守结构域预测分析显示该蛋白5′端具有一个激酶结构域,3′末端具有一个PPI结构域和一个NAF结构域。BLSATP分析表明葡萄Vv CIPK10与桃树CIPK(XP_007205151)一致性最高(74%)。重组表达载体p MAL-C5X/Vv CIPK10在大肠杆菌中经诱导表达获得与理论分子量(43 k Da+48.7 k Da)相一致的融合蛋白。MBP-Vv CIPK10融合蛋白经柱纯化后获得单一的蛋白条带,Vv CIPK10的自磷酸化活性依赖于Mn~(2+),不依赖于Mg~(2+)和Ca~(2+),EDTA可以抑制Vv CIPK10的自磷酸化活性。亚细胞定位结果显示,Vv CIPK10定位在细胞核、细胞膜和细胞质。Vv CIPK10在葡萄各个组织中均有表达,主要在葡萄根和叶片中大量表达,葡萄茎、花序、果实和卷须中的表达量较低。在干旱、低温和盐胁迫处理后,Vv CIPK10呈现受诱导表达模式。Vv CIPK10的表达在低温胁迫后6 h达到峰值,干旱和盐胁迫后2 h即达到峰值。【结论】葡萄Vv CIPK10能够响应干旱、低温和盐胁迫,推测Vv CIPK10在葡萄抗非生物逆境胁迫中具有重要作用。