Elimination of grapevine fleck virus and grapevine rupestris stem pitting-associated virus from Vitis vinifera 87-1 by ribavirin combined with thermotherapy

Vitis vinifera 87-1 plants infected by grapevine fleck virus(GFkV) and grapevine rupestris stem pitting-associated virus(GRSPaV) were used as the plant materials for virus elimination treatment. This study evaluated the effects of ribavirin at different concentrations(15 and 25 μg mL~(–1); R15 and R25, respectively), thermotherapy(37°C; T), and the combination of ribavirin and thermotherapy(R15+T and R25+T) on eliminating viruses from grapevine plants in vitro. Both R15 and R25 had phytotoxic effects and weakened plant growth. Thermotherapy positively affected the growth of grapevine plants. Plant height was significantly greater in T, R15+T, and R25+T than in CK, R15 and R25. The proportion of dead plants after T, R15+T, and R25+T was 51.4, 11.4, and 8.6%, respectively. The survival rates of regenerated plants after all treatments were 68.0%. Ribavirin concentration and treatment time were related to the regeneration of shoot tips and elimination efficiencies of the two viruses. The survival rates of plants after R15+T for 30, 40, and 50 days were 97.3, 90.7, and 74.4%, respectively. The elimination rates of GRSPaV from plants in the three time quantum were 55.6, 84.6, and 93.8%, respectively. The elimination rate of GFkV was 23.9% higher in R25(35/44) than in R15(25/45), and that of GRSPaV was 7.0% higher in R25 than in R15. The combination of thermotherapy and chemotherapy was found to have a positive effect on the eradication of GFkV and GRSPaV, and R25+T for 50 days was able to completely eliminate the two viruses from in vitro grapevines.     

Induced expression of sucrose synthase and alcohol dehydrogenase I genes in phytoplasma-infected grapevine plants grown in the field

Interaction between phytoplasma and grapevine at the physiological level is still poorly understood, as are plant defence mechanisms against the pathogen. This study investigates the level of gene expression of three selected genes in a large number of grapevine plants belonging to six disease/cultivar groups (healthy Chardonnay, Bois noir-infected Chardonnay, Flavescence dorée-infected Barbera and Prosecco, and recovered Barbera and Prosecco). All plants were grown in vineyards in uncontrolled conditions in order to represent the physiology of disease as accurately as possible. Sucrose synthase was significantly upregulated in infected plants of all cultivars with the lowest P -values in cvs Chardonnay and Prosecco ( P  < 0·001) and median fold-change around 2. This clearly indicates that carbohydrate metabolism changed in infected compared to healthy or recovered plants. Alcohol dehydrogenase I was significantly upregulated in infected relative to healthy Chardonnay plants ( P  < 0·05) indicating that alcoholic fermentation, a sign of hypoxic conditions, was induced in infected plants. Heat shock protein 70 was upregulated in infected compared to recovered plants only in cv. Prosecco. Linear discriminant analysis showed that classification of samples into disease status groups based on gene expression was highly accurate (82%), indicating that the response of field-grown plants to phytoplasma infection at the level of expression of selected genes was so intensive and uniform that it was possible to detect it in grapevine plants regardless of natural variables.     

Two Types of Grapevine Yellow Speckle Viroid 1 Isolated from Commercial Grapevine Had the Nucleotide Sequence of Yellow Speckle Symptom-inducing Type

Grapevine yellow speckle viroid 1 (GYSVd 1) was first detected in Japan from commercial grapevines. The viroid seems to be spreading in Japanese commercial vineyards but without causing noticeable disease symptoms. Two Japanese GYSVd 1 isolates from the cultivars Campbell and Steuben were sequenced. The Campbell isolate is included in the type 2 variant of GYSVd 1, and the Steuben isolate is included in the type 3. Both of the isolates were grouped into the yellow speckle-inducing variants in the nucleotide sequence ; however, the etiological importance of this viroid on the viticulture of Japan has not yet been determined. Received 14 June 1999/ Accepted in revised form 25 August 1999     

日光温室种植环境对葡萄液流特征的影响

为了探究我国东北地区日光温室种植条件下葡萄的液流特性及主要影响因素，对温室主棚（保温）与副棚（无保温措施）葡萄全生育期液流、生长指标及环境指标进行动态监测和系统分析。结果表明：（1）温室主棚与副棚葡萄液流的日内变化呈现单峰或多峰变化趋势，峰值出现在12∶00左右，液流基本停止时间为21∶00，夜间仍有微弱液流产生。不同月份液流强度不同，8月份液流速率最大，其次为6、10月。（2）全生育期下，主棚内相对液流速率（SF_R）主要受光能驱动的影响，光合有效辐射（PAR）是其主要影响因子，副棚的相对液流速率主要受到光能驱动和气孔导度的综合影响，饱和水汽压亏缺（VPD）和PAR均是其主要影响因子。气象因子与SF_R响应呈现明显的时间变异性，其中8月份SF_R与环境因子的相关性最为密切。（3）主棚的相对液流与影响因子的决定系数均大于副棚，且双因子组合模型预测精度高于单因素。（4）不同种植环境下葡萄的夜间液流有一定的占比，主棚的夜间液流占比略小于副棚的夜间液流占比，随着生育期的推进，葡萄植株夜间液流占比呈现先减小后增大的趋势。     

Assessment of Pierce's disease susceptibility in Vitis vinifera cultivars with different pedigrees

Pierce's disease (PD) of grapevine is caused by the bacterium Xylella fastidiosa. In this study, an integrated approach was applied to assess PD susceptibility among different Vitis vinifera cultivars that incorporated disease severity, bacterial pathogen abundance and loss of stem xylem hydraulic conductivity. It was hypothesized that levels of PD susceptibility in V. vinifera can be attributed in part to the host anatomical features that are shaped by its pedigree background. Two popular wine grape cultivars were initially selected from the occidentalis group, Merlot and Cabernet Sauvignon, and one from the orientalis group, Thompson Seedless. The more recently bred table grape cultivar Scarlet Royal, that has mixed pedigree parentage, was also included. PD susceptibility was compared to the known PD resistant b43-17 V. arizonica/candicans wild grape species from North America. The data indicated that Thompson Seedless was ranked as the most susceptible to PD because it significantly exhibited the most severe disease symptoms at 12 weeks post-inoculation and hosted the highest X. fastidiosa titre of the cultivars, and lost over 90% of its stem hydraulic conductivity. In contrast, the other three cultivars displayed less susceptibility to PD. The way in which the xylem anatomy could impact PD susceptibility in V. vinifera cultivars is discussed, together with how grape pedigrees and their cognate centre of domestication may have influenced xylem anatomical features. This work provides a reference framework to further test the hypothesis that V. vinifera cultivars with wide xylem vessels may be more prone to PD decline.     

葡萄miR159及其靶基因VvGAMYB在花发育过程中的作用分析

以‘魏可’葡萄(Vitis vinifera L.‘Wink’)为试材,克隆得到一个葡萄赤霉素信号途径中的MYB转录因子基因Vv GAMYB。该基因开放阅读框(ORF)长1 680 bp,编码559个氨基酸,该蛋白序列含有GAMYB基因家族保守的R2R3 DNA结合域以及Box1、Box2、Box3保守域。洋葱表皮细胞的瞬时表达结果显示,VvGAMYB定位于细胞核中。VvGAMYB和其下游基因Vv LFY在葡萄花发育过程中的表达都呈现先上升后下降的趋势,而赤霉素处理促进了VvGAMYB和VvLFY的表达,从而使开花提前。VvGAMYB是miR159的靶基因,其作用受到miR159的调控。miR159和VvGAMYB的表达负相关,赤霉素处理下调miR159表达从而也降低了对VvGAMYB的裂解作用。由上述结果可以看出,VvGAMYB通过赤霉素开花途径参与葡萄的开花过程。在这一过程中,赤霉素抑制miR159裂解VvGAMYB的作用,从而上调VvLFY的基因表达,参与了调控葡萄开花过程。     

自动避障式葡萄藤防寒土清土机研制

针对目前葡萄藤防寒土清土机自动化程度低、清土不彻底和易伤藤等问题,该研究基于多体动力学软件RecurDyn和离散元软件EDEM耦合仿真的方法,面向中国北方砂壤土地区葡萄园内的葡萄藤防寒土清除,提出采用柔性刷子与橡胶组合的清土作业部件,并设计了一种自动避障式葡萄藤防寒土清土机,主要由机架、避障摆动机构、避障信号采集机构、控制器、清土部件、挡土板、传动部件和限深轮等部分组成,在机具前进作业过程中,通过触杆感知自动避开水泥柱,并将葡萄藤上覆盖的防寒土清除干净。采用耦合仿真试验,模拟整机避障清土的作业过程,以机具前进速度、避障油缸速度、清土部件转速和触杆转动角度阈值为试验因素,以土壤清除率为试验指标,进行四因素二次回归正交旋转中心组合模拟试验,采用Design-Expert软件对试验数据进行回归显著性分析,确定了对试验结果影响显著的因素,以土壤清除率最大为优化目标,获得了机具的最佳作业参数组合:机具前进速度0.43 m/s、避障油缸速度60 mm/s、清土部件转速550 r/min、触杆转动角度阈值10°,此时仿真优化的土壤清除率为54.65%。加工物理样机并进行田间验证试验,得到田间试验的土壤清除率为59.73%,与仿真试验的土壤清除率相对误差约为8.50%,田间试验结果与仿真优化结果基本一致,满足自动避障式葡萄藤防寒土清土作业要求。研究结果可为触土机具避障机构的整体设计及优化提供理论依据和技术支撑。     

Multilocus sequence typing reveals the presence of three distinct flavescence dorée phytoplasma genetic clusters in Croatian vineyards

Flavescence dorée phytoplasma (FDp) is a quarantine pathogen associated with a severe and epidemic grapevine yellows disease representing a great threat for grapevine cultivation in Europe. An increase in disease spread prompted efforts to identify FDp strains in Croatia. Over 800 samples of grapevine together with presumed reservoir plants and almost 400 samples of Scaphoideus titanus and other potential vectors were collected countrywide and analysed. FDp isolates were characterized by multilocus sequence typing (MLST) of map, secY and uvrB-degV genes in order to determine genetic diversity and structure of FDp populations, and to trace transmission pathways. FD-related phytoplasmas were found in Croatia for the first time in alder, the invasive tree species Ailanthus altissima and leafhopper Phlogotettix cyclops. Phylogenetic analysis revealed the presence of three mapFD strain clusters: mapFD1, mapFD2 and mapFD3, and for the first time in Croatia a case of Palatinate grapevine yellows strain A (PGY-A). In total, 7 different map, 10 secY and 11 uvrB-degV genotypes were detected. The identification of 15 comprehensive FDp genotypes based on MLST suggests separate routes for disease introduction and propagation origins in Croatia. Moreover, high genetic variability of Croatian isolates indicates a complex ecological cycle of FDp involving various hosts.     

葡萄微茎尖培养及葡萄扇叶病毒的ELISA和探针检测

在建立‘藤稔’等５个葡萄品种离体培养快速繁殖体系的基础上，通过热处理和微茎尖脱毒培养获得了再生植株；并经过ＰＡＳ－ＥＬＩＳＡ和长臂光敏生物素标记ＧＦＬＶ－ＣＰ基因ｃＤＮＡ探针检测．结果表明白天３８℃夜晚３５℃处理脱除ＧＦＬＶ效果最佳；多次继代培养也有脱除ＧＦＬＶ的作用．从而首次获得了‘藤稔’、‘红地球’、‘９３０７’３个葡萄品种的脱除ＧＦＬＶ的试管苗．ＰＡＳ－ＥＬＩＳＡ与探针平行检测的结果还表明，ＰＡＳ－ＥＬＩＳＡ检测以Ｐ／Ｎ＜１作为阴性标准更为可靠