Cryptic species and population genetic structure of Plasmopara viticola in São Paulo State,Brazil

Downy mildew (Plasmopara viticola) is one of the most important diseases in grape-growing areas worldwide, including Brazil. To examine pathogen population biology and structure, P. viticola was sampled during the 2015/16 growing season from 516 lesions on nine grape cultivars in 11 locations in subtropical areas of São Paulo State, Brazil. For identification of cryptic species, a subsample of 130 isolates was subjected to cleaved amplified polymorphic sequence (CAPS) analysis, and for 91 of these isolates the ITS1 region was sequenced. These analyses suggest that the population of P. viticola in São Paulo State consists of a single cryptic species, P. viticola clade aestivalis. Seven microsatellite markers were used to determine the genetic structure of all 516 P. viticola isolates, identifying 23 alleles and 55 multilocus genotypes (MLGs). Among these MLGs, 34.5% were clonal and represented 93% of the isolates sampled. Four dominant genotypes were present in at least five different locations, corresponding to 65.7% of the isolates sampled. Genotypic diversity (Ĝ = 0.21–0.89) and clonal fraction (0.58–0.96) varied among locations (populations). Most populations showed significant deviation from Hardy–Weinberg expectations; in addition, excess of heterozygosity was verified for many loci. However, principal coordinate analysis revealed no clusters among locations and no significant isolation by distance was found, suggesting high levels of migration. The results indicate that downy mildew epidemics result from multiple clonal infections caused by a few genotypes of P. viticola, and reproduction of P. viticola in São Paulo State is predominantly asexual.     

Role of basidiomycete fungi in the grapevine trunk disease esca

Phaeomoniella chlamydospora and wood-rotting basidiomycete fungi, namely Fomitiporia spp., are known esca pathogens. However, the effect of their mixed infections and the sequence of infection on disease development is unclear. To determine the effects of single and co-inoculations on symptoms, potted Vitis vinifera ‘Crimson Seedless’ was inoculated with P. chlamydospora either alone or in combination with one of four basidiomycetes: Coprinellus radians, Fomitiporia langloisii, F. polymorpha and the novel species Tropicoporus texanus. Basidiomycetes were isolated from vines with foliar symptoms of esca in California and Texas. In sequential co-inoculations, the effects of different sequences of infection (P. chlamydospora first, basidiomycete 6 months later; and vice versa) were tested, compared to simultaneous co-inoculations. Plants inoculated with P. chlamydospora either alone or in combination with a basidiomycete (in any sequence) did not differ significantly in the length of black-streaking lesions. In plants inoculated only with a basidiomycete, the appearance of large brown lesions, coupled with the absence of this wood symptom from control plants, suggests that C. radians, F. langloisii and T. texanus are pathogenic. Foliar symptoms resembling those of esca in the field (marginal and/or interveinal scorching, combined with red and/or yellow discoloration) were statistically more frequent among plants inoculated with F. polymorpha or T. texanus, either simultaneously or following P. chlamydospora, compared to single inoculations. Sequential co-inoculations of a basidiomycete before or after P. chlamydospora were associated with similar lesion lengths, suggesting that basidiomycetes may not require infection by P. chlamydospora in order to extensively colonize the wood.     

Temporal disease dynamics and relative importance of sexual and asexual reproduction of grape downy mildew (Plasmopara viticola) in an isolated vineyard in the North Georgia Mountains,USA

The North Georgia Mountains are the southernmost region along the United States East Coast where European wine grapes (Vitis vinifera) are grown commercially. Epidemics of downy mildew, caused by Plasmopara viticola, are frequent and severe, but little is known about the epidemiology and population biology of the pathogen in this region. Disease monitoring in an experimental vineyard from 2015 to 2017 indicated that times of disease onset and progress rates were highly variable across years and cultivars. Oospores were observed microscopically, and simulation with a process-based model indicated presence of conditions favourable for oospore germination in the spring and early summer each year. A total of 409 P. viticola isolates collected over three  years were genotyped with seven microsatellite markers, revealing very high genotypic diversity, which when combined with the observation of oospores is indicative of a sexually reproducing population. Among the 409 isolates, 225 multilocus genotypes (MLGs) were identified, of which 164 were detected only once and 61 were repeated (clonal). Eight MLGs (represented by 28 isolates) were detected across years, suggesting the possibility of asexual overwintering of P. viticola in this region. Across sampling dates, the percentage of isolates belonging to nonrepeated (unique) MLGs ranged from 27.3% to 63.2%. Even towards the end of the annual epidemic, the percentage of isolates in nonrepeated MLGs was still relatively high, around 30%. These MLGs may have originated from oospores germinating late during the growing season, although incomplete sampling at earlier dates and contribution by immigration cannot be fully excluded.     

山葡萄VaCIPK18原核表达与多克隆抗体制备

类钙调磷酸酶B亚基蛋白(CBLs)互作蛋白(CIPKs) 作为类丝氨酸/苏氨酸蛋白激酶,在植物响应非生物胁迫信号转导中起着重要作用。基于前期山葡萄响应低温胁迫转录组测序结果,发现低温胁迫引发的早期伤害及感知阶段的激酶基因中涉及CBL-CIPK 信号通路的VaCIPK18表达显著上调。为进一步研究山葡萄(Vitis amurensis)VaCIPK18激酶参与低温胁迫的功能,采用同源克隆获得了VaCIPK18基因,其开放阅读框为1 320 bp,编码439个氨基酸。基于对VaCIPK18蛋白生物信息学分析,获取胞外结构域中抗原表位丰富的肽段,并将其C端调控结构域(230~439 aa)构建到原核表达载体pET28a-SUMO。将重组表达载体转化至大肠杆菌(E. coli Rosetta)中,经0.8 mmol·L^-1 IPTG、37℃诱导4 h表达出大小为42 kDa的包涵体蛋白。将重组蛋白作为抗原免疫日本大耳白兔,获得anti-VaCIPK18多克隆抗体,经检测具有高效价及特异性。Western Blot结果表明,该抗体可以与葡萄内源CIPK18特异性结合,且在50 kDa位置出现与预期一致的条带。同时,CIPK18在低温胁迫后葡萄叶片中蛋白表达水平与室温下相一致,但两种状态下均存在可能的磷酸化与泛素化修饰现象。本研究结果为进一步探究VaCIPK18的蛋白定位、表达及其功能奠定了基础。     

盐胁迫对刺榆种子萌发及幼苗生理特征的影响

为了研究刺榆种子及幼苗对盐胁迫的响应，采用培养皿纸上发芽法进行种子萌发并采用盆栽法进行幼苗培养，测定刺榆种子在不同浓度NaCl、Na2SO4、Na2CO3和NaHCO3处理下的发芽率、发芽势、发芽指数、活力指数、相对盐害率，及幼苗丙二醛（malondialdehyde，MDA）、超氧化物歧化酶（superoxide dismutase，SOD）及过氧化物酶（peroxidase，POD）变化规律。结果显示：①在试验设置盐浓度范围内，刺榆种子在NaCl＜30 mmol·L-1、Na2CO3＜10 mmol·L-1、Na2SO4＜20 mmol·L-1、NaHCO3＜20 mmol·L-1的盐胁迫下能够正常发芽，碱性盐（NaHCO3和Na2CO3）的危害大于中性盐（NaCl和Na2SO4）。②刺榆种子的相对盐害率均为正值，4种盐均存在不同程度的种子萌发抑制，不同种类盐分对刺榆种子萌发抑制表现为NaCl＜Na2SO4＜NaHCO3＜Na2CO3。③幼苗生长中，不同处理幼苗叶片内MDA含量呈逐渐上升的趋势，SOD活性和POD活性随浓度的增加呈先升高后降低的趋势。但当NaCl＜30 mmol·L-1、Na2CO3＜10 mmol·L-1、Na2SO4＜10 mmol·L-1、NaHCO3＜20 mmol·L-1时，刺榆幼苗内MDA含量和保护酶活性未发生显著变化。研究表明刺榆种子萌发与幼苗生长对低浓度盐环境具有一定的耐受性，高浓度盐环境会使种子丧失发芽能力阻碍幼苗生长。结果为刺榆在盐碱地的种植和推广提供理论依据。     

中国野生毛葡萄芪合酶基因VqSTS11和VqSTS23调控抗白粉病的研究

利用同源克隆法得到抗白粉病的中国野生毛葡萄‘丹凤–2’芪合酶基因VqSTS11和VqSTS23的开放阅读框，并构建过表达载体；通过器官发生途径诱导不抗白粉病的欧洲葡萄‘无核白’分生愈伤组织并采用农杆菌介导法对其进行转化；经过PCR检测和Western blot鉴定，获得了5株VqSTS11超量表达植株和3株VqSTS23超量表达植株；人工接种白粉菌发现，转基因植株中白粉菌菌丝生长速度慢，孢子萌发受到一定的抑制，并且转基因植株中芪合酶基因相对表达量升高，抗病相关基因表达上调，芪类物质含量积累增多。本研究结果进一步证明中国野生毛葡萄‘丹凤–2’芪合酶基因VqSTS11和VqSTS23对白粉菌具有抗性，‘丹凤–2’可以为改良欧洲葡萄品种抗病性提供抗病基因，作为抗病育种的资源。     

酿酒山葡萄新品种‘紫晶甘露’

‘紫晶甘露’是以优质酿酒山葡萄酒品种‘左山二’为母本，‘哈桑’为父本杂交选育而成的能够单品种酿造干红酒的山葡萄新品种。果穗圆锥形，有岐肩或副穗。果实圆球形，果皮黑色，果肉绿色，无肉囊，种子2 ~ 4粒。生长势强，抗寒、抗病性极强，4年丰产，4 ~ 6年平均产量1 876 kg • km-2。果实可溶性固形物含量达到21.0%，总酸11.6 g • L-1，出汁率65.5%。     

欧洲葡萄栽培和酿酒在中国的传播

欧洲葡萄(Vitis vinifera L．)栽培和酿酒引入中国西部的时间是在西汉以前,引入中部地区的时间是在西汉。引入的路线是从中亚到汉代西域,再至都城长安,然后逐渐向东部地区传播,先至河南、山西,之后扩散至河北、山东以及其他地区。中国古代人民培育出许多著名的葡萄品种,采用了独特的葡萄栽培方式。自然发酵的葡萄酒基本上是在西域地区生产,广大的中东部地区很少采用这种技术。自然发酵在东部广泛应用的时期是元代,中国现代葡萄栽培和酿酒技术始于1892年。目前,欧洲葡萄栽培和酿酒已成为我国新疆、山东、河北等地农业和轻工业中的重要组成部分。