At Least Two Origins of Fungicide Resistance in Grapevine Downy Mildew Populations

Quinone outside inhibiting (QoI) fungicides represent one of the most widely used groups of fungicides used to control agriculturally important fungal pathogens. They inhibit the cytochrome bc₁ complex of mitochondrial respiration. Soon after their introduction onto the market in 1996, QoI fungicide-resistant isolates were detected in field plant pathogen populations of a large range of species. However, there is still little understanding of the processes driving the development of QoI fungicide resistance in plant pathogens. In particular, it is unknown whether fungicide resistance occurs independently in isolated populations or if it appears once and then spreads globally by migration. Here, we provide the first case study of the evolutionary processes that lead to the emergence of QoI fungicide resistance in the plant pathogen Plasmopara viticola. Sequence analysis of the complete cytochrome b gene showed that all resistant isolates carried a mutation resulting in the replacement of glycine by alanine at codon 143 (G143A). Phylogenetic analysis of a large mitochondrial DNA fragment including the cytochrome b gene (2,281 bp) across a wide range of European P. viticola isolates allowed the detection of four major haplotypes belonging to two distinct clades, each of which contains a different QoI fungicide resistance allele. This is the first demonstration that a selected substitution conferring resistance to a fungicide has occurred several times in a plant-pathogen system. Finally, a high population structure was found when the frequency of QoI fungicide resistance haplotypes was assessed in 17 French vineyards, indicating that pathogen populations might be under strong directional selection for local adaptation to fungicide pressure.     

山葡萄几丁质酶基因VCH3启动子的分离及鉴定

利用接头PCR技术首次分离了长度为1 216bp的双优山葡萄(Vitis amurensis Rupr.)classⅢ几丁质酶基因VCH3上游启动子序列(GenBank登录号AF441123),并用引物延伸方法鉴定了该启动子的转录起始位点,为5'-ATCAAGCAC-31序列中的第二个A.序列分析结果表明,代表真核基因启动子特征的CAAT盒和TATA盒分别位于VCH3启动子转录起始位点上游-122和-29处.另外,在转录起始位点上游-1 181 bp和-293 bp处各有一个水杨酸(SA)响应的顺式作用元件TGACG.为了鉴定该启动子的功能,将该启动子连接到β-葡糖苷酸酶基因(GUS)编码区的上游构建了VCH3启动子-GUS融合基因,并用农杆菌介导叶盘转化法将该融合基因转入烟草栽培品种NC89中.SA处理的转基因烟草根系和叶片GUS酶活性的荧光和组织化学检测结果表明VCH3启动子的驱动作用被SA诱导,因而该启动子在基因工程中将具有潜在的应用价值.     

Procyanidins from the seeds of Vitis amurensis

Three procyanidins, procyanidin B-5 3'-O-gallate, vitisinol, and amurensisin, were isolated from the seeds of Vitis amurensis, whose structures were elucidated on the basis of spectral and chemical evidence. Vitisinol and amurensisin contain a spiro-type biflavanyl linkage and a biphenyl-lactone partial structure, respectively.     

甜瓜抗霜霉病基因同源序列克隆与分析

采用RT—PCR扩增的方法,从高抗霜霉病甜瓜品种‘日本安农二号’中克隆到约3kb的cDNA片段（命名为MRGH-D,该基因是一个连续的通读编码框,编码1007个氨基酸。推测的蛋白质分子量为113．7kDa,等电点为7．88,蛋白质预测无跨膜区。根据推测的氨基酸序列,该基因属于TIR—NBS—LRR类抗病基因,具有TIR-NBS—LRR类抗病基因所有的保守结构域。核苷酸序列和氨基酸序列同源性分析结果显示,MRGH-J与甜瓜抗病基因的同源序列MRGHl2及抗霜霉病相关基因mp-19均具有高达99％的同源性,推测该基因可能在甜瓜抗霜霉病中起作用。     

Overexpression of an Oil Radish Superoxide Dismutase Gene in Broccoli Confers Resistance to Downy Mildew

Superoxide dismutases (SOD) play important roles in plant disease resistance. In this study, a manganese superoxide dismutase gene, designated RsrSOD, was isolated from oil radish (Raphanus sativus var. raphanistroides). RsrSOD was 696?bp in length predicted to encode 231 amino acids. The deduced amino acid sequence contained four putative Mn-binding sites. Under the control of the CaMV35S promoter, RsrSOD was introduced into broccoli (Brassica oleracea var. italica) via Agrobacterium tumefaciens-mediated transformation. Six transgenic lines were obtained out of 35 independent shoots. Both gene expression and enzyme activity of SOD increased significantly in transgenic lines when challenged with Hyaloperonospora parasitica. Three lines, L19, L23, and L25, exhibited the highest resistance against downy mildew with disease symptoms restricted completely. These highly resistant lines would serve as good broccoli breeding materials.     

Rust and Downy Mildew Resistance in Pearl Millet (Pennisetum glaucum) Mediated by Heterologous Expression of the afp Gene from Aspergillus giganteus

The cDNA encoding the antifungal protein AFP from the mould Aspergillus giganteus was introduced into two pearl millet (Pennisetum glaucum) genotypes by particle bombardment. Stable integration and expression of the afp gene was confirmed in two independent transgenic T₀ plants and their progeny using Southern blot and RT-PCR analysis. In vitro infection of detached leaves and in vivo inoculation of whole plants with the basidomycete Puccinia substriata, the causal agent of rust disease, and the oomycete Sclerospora graminicola, causal agent of downy mildew, resulted in a significant reduction of disease symptoms in comparison to wild type control plants. The disease resistance of pearl millet was increased by up to 90% when infected with two diverse, economically important pathogens. This is the first report of genetic enhancement of Pennisetum glaucum against fungal infections.     

Chromosomal Location and Comparative Genomics Analysis of Powdery Mildew Resistance Gene Pm51 in a Putative Wheat-Thinopyrum ponticum Introgression Line

Powdery mildew (PM) is a very destructive disease of wheat (Triticum aestivum L.). Wheat-Thinopyrum ponticum introgression line CH7086 was shown to possess powdery mildew resistance possibly originating from Th. ponticum. Genomic in situ hybridization and molecular characterization of the alien introgression failed to identify alien chromatin. To study the genetics of resistance, CH7086 was crossed with susceptible genotypes. Segregation in F₂ populations and F_2:3 lines tested with Chinese Bgt race E09 under controlled conditions indicated that CH7086 carries a single dominant gene for powdery mildew resistance. Fourteen SSR and EST-PCR markers linked with the locus were identified. The genetic distances between the locus and the two flanking markers were 1.5 and 3.2 cM, respectively. Based on the locations of the markers by nullisomic-tetrasomic and deletion lines of ‘Chinese Spring’, the resistance gene was located in deletion bin 2BL-0.89-1.00. Conserved orthologous marker analysis indicated that the genomic region flanking the resistance gene has a high level of collinearity to that of rice chromosome 4 and Brachypodium chromosome 5. Both resistance specificities and tests of allelism suggested the resistance gene in CH7086 was different from previously reported powdery mildew resistance genes on 2BL, and the gene was provisionally designated PmCH86. Molecular analysis of PmCH86 compared with other genes for resistance to Bgt in the 2BL-0.89-1.00 region suggested that PmCH86 may be a new PM resistance gene, and it was therefore designated as Pm51. The closely linked flanking markers could be useful in exploiting this putative wheat-Thinopyrum translocation line for rapid transfer of Pm51 to wheat breeding programs.     

黑麦6R抗白粉病基因向小麦的渗进与鉴定

为了将黑麦６Ｒ染色体上抗小麦白粉病的基因导入小麦,选用了一个６Ｒ／６Ｄ代换系Ｍ２４为亲本之一,分别与小麦栽培品种和第６部分同源群缺体系杂交,杂种出现６Ｒ或／或６Ａ,６Ｂ,６Ｄ单,双或三单体等各种情况,取其花药进行培养,共获得２４１个再生植株,对其中３２个抗白粉病的花粉植株经染色体计数,Ｃ－分带,基因组原位杂交,同工酶等电聚焦电泳和或／ＲＦＬＰ分子标记检测,发现有６株仍保持为６Ｒ／６Ｄ代换系,有１０     

Microsatellite Markers for Characterization of Native and Introduced Populations of Plasmopara viticola, the Causal Agent of Grapevine Downy Mildew

We reported 31 microsatellite markers that have been developed from microsatellite-enriched and direct shotgun pyrosequencing libraries of Plasmopara viticola, the causal agent of grapevine downy mildew. These markers were optimized for population genetics applications and used to characterize 96 P. viticola isolates from three European and three North American populations.     

山葡萄CBF基因表达载体构建及对拟南芥根生长的影响

为了研究山葡萄CBF基因调节植物对盐胁迫的应答机理,分别构建了山葡萄Va CBF1、Va CBF2和Va CBF3的植物过表达载体。经酶切及琼脂糖电泳检测证实3个基因均插入到p BASTA中,表明表达载体构建成功。然后,分别将3个植物过表达载体转入农杆菌EHA105中,并通过浸花法浸染拟南芥。利用除草剂筛选获得3个基因的拟南芥过表达株系。最后,对野生型拟南芥与转基因拟南芥进行盐胁迫处理,发现OE-CBF2转基因植株的主根伸长长度显著长于其它植株,3个转基因株系的侧根长度也明显长于野生型植株。上述结果表明山葡萄CBF基因可能在植物盐胁迫中对根部生长发育起到非常重要的调控作用。     

Pedigree Reconstruction of the Italian Grapevine Aglianico (Vitis vinifera L.) from Campania

A total of 41 accessions of Aglianico belonging to three different biotypes (Taburno, Taurasi, and Vulture) and 9 accessions of Sirica grapes were sampled from diverse areas of Campania (Italy). All accessions were first genotyped using 21 microsatellite markers (SSR) to evaluate possible homonymies, synonymies, and the genetic structure of each group. A larger dataset was then constructed adding Italian and International cultivars. On the basis of results obtained analyzing the first dataset, further investigations were carried out enlarging the number of investigated loci (up to 43). The addition of 22 SSRs was useful in the definition of likely genetic relationships linking Aglianico biotypes, Sirica and Syrah. According to their SSR allelic profiles, the monophyletic origin of the three Aglianico biotypes was confirmed. Among Aglianico Taburno accessions, eight samples (called Aglianico like-to-type) performed a different SSR allelic profile from Aglianico true-to-type. Sirica and Syrah proved to be synonyms. This work allowed to determine the genetic relationship between Aglianico and the cultivars supposed to be related. The parentage analysis was investigated. The most likely pedigree has been reconstructed; revealing a second-degree relationship between the worldwide cultivated Syrah from the Rhone Valley and Aglianico. Aglianico like-to-type appeared related to Aglianico in a parent-offspring fashion.     

Control of Downy Mildew, Plasmopara viticola (de Bary) Berlese et de Toni, on Grapevine Leaves through Water Extracts from Composted Organic Wastes

Watery extracts of composted manure-straw-soil mixtures induced increased resistance of grapevine leaves against downy mildew, Plasmopara viticola, if applied by dipping or spraying. The extracts had no direct fungicidal or fungitoxic effects.     

水杨酸诱导小白菜抗霜霉病的作用研究

以小白菜(Brassica campestris ssp.chinesis L.)为材料,采用叶面喷洒的方法施用不同浓度水杨酸(Salicylic acid,SA),研究了SA小白菜对霜霉病的抗性诱导,并对经SA诱导后小白菜植株体内相关酶活性进行了研究.结果发现,在0.2～2.0 mmol·L-1的浓度范围内,随着SA浓度的增加,SA的诱导防治效果先增后减,1.0 mmol·L-1浓度为诱导抗性最适浓度.经SA诱导后,小白菜植株体内SOD、POD和PPO等酶活性也明显增强.     

12个物种miR-302基因簇的生物信息学分析

很多microRNA (miRNA)基因在基因组上聚集排列形成miRNA基因簇.miRNA基因簇在进化上较为保守,有其特殊的生物学意义.miR-302基因簇在胚胎干细胞中特异表达,对胚胎干细胞的自我更新和多潜能维持有重要的作用.本文采用miRBase数据库查询和BLAST同源搜索方法共搜寻并分析了12个物种的miR-302基因簇.生物信息学分析表明,这些物种miR-302基因簇均位于LARP7蛋白基因的内含子区,但转录方向与LARP7基因转录方向相反.序列相似性分析显示,同一物种miR-302簇成员间以及不同物种相应miR-302簇成员间相似性均较高.进化分析表明,基因复制可能是miR-302基因簇进化的主要驱动力.     

Hybridization of Senecio squalidus and S. viscosus and Introgression of Genes from Diploid into Tetraploid Senecio Species

Natural triploid hybrids (Senecio x londinensis Lousley) betweenS. squalidus L. (2n = 20) and 5. viscosus L. (2n = 40) are fairlyfrequently found in Britain. Under glasshouse conditions bothnatural and artificial hybrids displayed very low levels ofseed fertility and gave rise to morphologically diverse F₂ plantsat about the triploid or pentaploid chromosome levels. By theF₄ generation, progeny of a F₂ pentaploid plant had somaticchromosome numbers near to the tetraploid level and considerablyincreased pollen and seed fertilities. Such fertile tetraploidsegregants of S. x londinensis permit the introgression of S.squalidus genes into S. viscosus, and may indicate the courseof introgression into other tetraploid species of Senecio. Senecio, hybridization, introgression     

The antimicrobial activity of compounds from the leaf and stem of Vitis amurensis against two oral pathogens

Nine compounds isolated from the leaf and stem of Vitis amurensis Rupr. (Vitaceae) were evaluated for their antimicrobial activity against two oral pathogens, Streptococcus mutans and Streptococcus sanguis, which are associated with caries and periodontal disease, respectively. The results of several antimicrobial tests, including MIC, MBC, and TBAI, showed that three compounds inhibited the growth of the test bacteria at concentrations ranging from 12.5 to 50 μg/mL. Among these compounds, compound 5, trans-ε-viniferin, displayed the strongest activity against S. mutans and S. sanguis with MIC values of 25 and 12.5 μg/mL, respectively. This is the first report on the antimicrobial activity of stilbenes and oligostilbenes isolated from the leaf and stem of V. amurensis. Thus, this result suggests that natural antimicrobial compounds derived from V. amurensis may benefit oral health as plaque-control agents for the prevention of dental caries and periodontal disease.     

Enhancement of Stilbene Biosynthesis in Grape-Cell Cultures by Natural Products Based on Endophytes of the Wild Grape Species Vitis amurensis RUPR.

Applied Biochemistry and Microbiology - A new approach based on endophyte products is proposed to enhance the biosynthesis of stilbenes (compounds with established health benefits) and to alter...     

Overexpression of VaCBF4, a Transcription Factor from Vitis amurensis, Improves Cold Tolerance Accompanying Increased Resistance to Drought and Salinity in Arabidopsis

Environmental stress has a great impact on fruit yield and quality in grapes. Understanding mechanisms underlying stress tolerance in plants is useful for grape breeding. Here, a CBF gene, designated VaCBF4, was identified in V. amurensis. The expression of VaCBF4 was induced by several abiotic stresses, including cold, exogenous abscisic acid (ABA), drought, salinity, and cold-drought conditions. A yeast one-hybrid assay demonstrated that VaCBF4 protein could bind to a conserved DRE cis-element, which contains the core sequence ACCGAC and regulates cold- and dehydration-responsive. Transgenic Arabidopsis seedlings overexpressing VaCBF4 showed enhanced tolerance to cold, drought, and salinity when compared with wild-type controls. LT₅₀, a chilling temperature required to cause 50 % electrolyte leakage in leaves, was 4 °C lower in transgenic Arabidopsis lines than that in non-cold-acclimated wild-type seedlings. Moreover, two stress-responsive genes, AtRD29A and AtCOR47, also showed higher levels of expression in the transgenic lines than in wild-type seedlings under normal growth condition. Taken together, all these results clearly indicate that VaCBF4 is involved in the response to abiotic stresses, and it may be a good candidate gene for genetic improvement to develop stress-tolerant varieties in grapes.     

Anti-inflammatory tetramers of resveratrol from the roots of Vitis amurensis and the conformations of the seven-membered ring in some oligostilbenes

Five resveratrol tetramers, amurensins I-M, were isolated from the roots of Vitis amurensis (Rupr.), together with five known resveratrol tetramers, (+)-hopeaphenol, isohopeaphenol, vitisin A, (+)-vitisifuran A, and heyneanol A. Their structures and stereochemistry were determined by chemical and spectroscopic methods, especially by use of 2D NMR analysis. Some of them had an ampelopsin A or a balanocarpol unit, in which the conformations of the seven-membered carbon ring were described for the first time. The anti-inflammatory activities of the tetramers were also tested. Among them, (+)-hopeaphenol, isohopeaphenol, vitisin A, (+)-vitisifuran A and heyneanol A showed potent inhibition on the biosynthesis of leukotriene B4 (LTB4), and amurensins I and L showed strong antagonism of the histamine acceptor.     

一个来自硬粒小麦的抗白粉病基因的鉴定和微卫星标记

在起源于硬粒小麦(TriticumdurumDesf.accessionDR147)和尾状山羊草(AegilopscaudataL.acc.Ae14)合成的双二倍体与普通小麦品种“莱州953”杂交组合衍生的BC3F2群体中鉴定了一个抗小麦白粉病基因。遗传分析表明,该基因为一个显性单基因。应用分离群体分组法(BSA),鉴定了两个与抗病基因紧密连锁的微卫星标记Xgwm311和Xgwm382,它们与抗病基因的遗传距离分别为5.9cM和4.9cM。对双二倍体亲本硬粒小麦DR147和尾状山羊草Ae14及轮回亲本“莱州953”的DNAPCR扩增结果表明,与抗病基因相关的微卫星标记Xgwm311和Xgwm382来源于硬粒小麦DR147。根据已发表的小麦微卫星图谱和对“中国春”缺-四体系DNA扩增结果,抗病基因被定位在小麦2A染色体的长臂末端。