Pathogenesis of Eutypa lata in grapevine: identification of virulence factors and biochemical characterization of cordon dieback

Eutypa lata is a vascular pathogen of woody plants. In the present study we (i) determined which component(s) of the cell wall polymers were degraded in naturally infected grapevines and in artificially inoculated grape wood blocks; (ii) compared the pattern of wood decay in the tolerant grape cv. Merlot versus the susceptible cv. Cabernet Sauvignon; and (iii) identified secondary metabolites and hydrolytic enzymes expressed by E. lata during wood degradation. Biochemical analyses and a cytochemical study indicated that glucose-rich polymers were primary targets of E. lata. Structural glucose and xylose of the hemicellulose fraction of the plant cell wall and starch were depleted in infected woods identically in both cultivars. Moreover, the more tolerant cv. Merlot always had more lignin in the wood than the susceptible cv. Cabernet Sauvignon, indicating that this polymer may play a role in disease resistance. In vitro assays demonstrated the production by E. lata of oxidases, glycosidases and starch degrading enzymes. Phytotoxic secondary metabolites were also produced but our data suggest that they may bind to the wood. Finally, we demonstrated that free glucose in liquid cultures repressed primary but not secondary metabolism.     

PP 1/303 (1) Eutypa lata on grapevine

Specific scope

This Standard describes the conduct of trials for the efficacy evaluation of plant protection products against Eutypa lata causing eutypa dieback on grapevine.

Specific approval and amendment

First approved in 2017‐09.     

Genetic structure of the fungal grapevine pathogen Eutypa lata from four continents

The generalist ascomycete fungus Eutypa lata causes Eutypa dieback of grapevine (Vitis vinifera) worldwide. To decipher the cosmopolitan distribution of this fungus, the population genetic structure of 17 geographic samples was investigated from four continental regions (Australia, California, Europe and South Africa), based on analysis of 293 isolates genotyped with nine microsatellite markers. High levels of haplotypic richness (R = 0·91–1) and absence of multilocus linkage disequilibrium among loci supported the preponderance of sexual reproduction in all regions examined. Nonetheless, the identification of identical multilocus haplotypes with identical vegetative compatibility groups, in some vineyards in California and South Africa, suggests that asexual dispersal of the fungus among neighbouring plants could be a rare means of disease spread. The greatest levels of allelic richness (A = 4·89–4·97) and gene diversity (H = 0·66–0·69) were found in Europe among geographic samples from coastal areas surrounding the Mediterranean Sea, whereas the lowest genetic diversity was found in South Africa and Australia (A = 2·78–3·74; H = 0·49–0·57). Samples from California, Australia and South Africa, which had lower genetic diversity than those of Europe, were also characterized by demographic disequilibrium and, thus, may represent founding populations of the pathogen. Low but significant levels of genetic differentiation among all samples (D_EST = 0·12, P = 0·001; F_ST = 0·03, P = 0·001) are consistent with historical gene flow preventing differentiation at continental scales. These findings suggest that global, human‐mediated spread of the fungus may have resulted in its current global distribution.     

Variation in susceptibility of grapevine pruning wound to infection by Eutypa lata in south-western France

Grapevine cultivars Cabernet Sauvignon (susceptible to Eutypa dieback), and Merlot (tolerant), were pruned three times during the dormant season (mid-December, mid-January and mid-February) and wounds on the 1-year-old canes were inoculated weekly with ascospores of Eutypa lata after pruning. No differences in susceptibility to infection were observed between cultivars, although in the vineyard they differed in symptom expression. Infection by E. lata varied with pruning date and the age of the pruning wound, and was higher and of longer duration with early pruning (December). At low temperature, infection of the pruning wounds by E. lata was increased, whereas the growth of other microorganisms was reduced. Moderate temperatures encouraged the growth of other microorganisms, notably Rhodotorula sp. This natural colonizer of grapevine pruning wounds was able to reduce the infection capacity of E. lata . It was more effective when inoculation with E. lata was carried out with low numbers of ascospores and when it was delayed until 14 days after application of the wound colonizer, infection being reduced by 95–100%.     

Identification of grapevine marker genes for early,non‐destructive Eutypa lata infection diagnosis

Eutypa lata is the causal agent of eutypa dieback, a highly damaging trunk disease affecting all grape‐growing areas, with currently neither an efficient curative treatment nor an early non‐destructive diagnostic method. The present work was carried out to discover grapevine genes expressed in response to the presence of E. lata that could be useful to develop an early (before visible foliar symptoms) and non‐destructive (using grapevine leaves) diagnostic tool. Microarray analyses were carried out from (i) infected plants showing characteristic E. lata foliar and vascular symptoms and positive pathogen recovery from vascular lesions (S⁺R⁺), (ii) infected plants showing no symptoms (S^?R⁺), and (iii) symptomless plants with negative pathogen recovery (S^?R^?). Vineyard and greenhouse‐grown plants, naturally or artificially infected respectively, and uninoculated controls were characterized and leaf RNA was hybridized with 15k operon grapevine oligonucleotide microarrays. Among the grapevine genes differentially expressed between S^?R⁺ and S^?R^? plants in greenhouse and vineyard conditions, 10 were highlighted as robust candidate genes for diagnosis: seven were specifically involved in response to infection and three were associated with symptom absence. Five were confirmed to be effective diagnostic marker genes usable in a qRT‐PCR‐based test performed on RNA extracted from grapevine leaves cultivated in either greenhouse or vineyard conditions. Furthermore, their expression profiles in response to infection with E. lata or other major grapevine fungi (Erysiphe necator, Plasmopara viticola, Botrytis cinerea) could be distinguished. The usefulness of these genes to develop an early and non‐destructive method for diagnosis of E. lata infection is discussed with regard to the advantages and drawbacks of previous E. lata diagnostic studies.     

A Reassessment of the Species Concept in Eutypa lata, the Causal Agent of Eutypa Dieback of Grapevine

ABSTRACT Eutypa dieback is a vascular disease of several cultivated crops and trees worldwide. The attribution of the name to the agent responsible for branch dieback is ambiguous. Pathogenicity of Eutypa sp. first was reported on apricot and the causal agent was named E. armeniacae. However, no morphological differences were reported with the previously described E. lata, and some authors considered both species synonymous. Others regarded them as distinct species on the basis of pathogenesis and molecular analysis. We further investigated the relatedness of both species by phylogenetic analyses of the internal transcribed spacer region and beta-tubulin gene. These analyses included several other taxa placed in the same family (Diatrypaceae), and yielded three groups. The isolates referred to as E. lata in previous work clustered with Diatrype stigma in one group. Isolates of E. armeniacae and E. lata clustered in a second group, supporting the synonymy of these species. The third group included other Eutypa spp. supporting the polyphyletic origin of this genus. Measurements of conidia length and secondary metabolite production of isolates supported the phylogenetic analyses. Secondary metabolites appeared to be a synapomorphic character shared by several taxa including E. lata, E. armeniacae, E. laevata, and E. petrakii var. petrakii.     

Dieback symptoms on olive trees caused by the fungus Eutypa lata1

The fungus Eutypa lata was isolated from olive trees for the first time in 1988 from two young 3-year-old trees. It was evident that the infection was initiated from the graft union. The trees were also infected by Armillaria mellea. In a second case in 1991, extensive cankers were observed along the branches which often were girdled and died. The infected area extended from the bark into the sapwood and even into the heartwood. Cultures made on potatodextrose agar from the margin of necrotic and healthy tissues resulted in typical colonies of the fungus E. lata, as well as of Verticillium dahliae. The colonies of E. lata produced the characteristic pycnidia of the anamorph Libertella blepharis 3–4 weeks later. Pathogenicity tests made on olive, apricot, almond and walnut trees resulted in the death of the inoculated twigs or in canker formation and extensive brown discoloration of the wood.     

基于形态与分子数据鉴定海榄雌瘤斑螟

海榄雌瘤斑螟目前是红树林植物白骨壤的重要害虫,其种类在国内存在争议。本研究对福田红树林自然保护区内采集到的螟蛾标本进行了形态学鉴定,同时应用DNA条形码技术进行了进一步确认。本文将海榄雌瘤斑螟样本的线粒体COI序列与数据库中已有序列比对,结果显示海榄雌瘤斑螟COI序列与GenBank数据库中3条P. synataractis和BOLD系统中的32条P. syntaractis序列同源性达98%以上;构建的系统发育树显示福田红树林自然保护区内采集的标本与P. syntaractis聚为一支。综合形态和分子数据确定海榄雌瘤斑螟为P. syntaractis (Turner,1904)。     

Variation in Pathogenicity and Genetic Structure in the Eutypa lata Population of a Single Vineyard

ABSTRACT Fifty-five isolates of Eutypa lata were collected in 1994 from a single vineyard, each from a different vine that showed either shoot and foliar symptoms of Eutypa dieback or only abnormalities during the period of 1990 to 1994. These isolates showed a large variation in pathogenicity after inoculation on cuttings in the greenhouse. Variability also was observed for cultural traits and radial growth rate on potato dextrose agar (PDA) medium, but no relation was found between these characteristics and pathogenicity. The isolates were paired on PDA medium, and a barrage reaction was observed for all pairings, indicating the isolates were vegetatively incompatible. Thirty-two random amplified polymorphic DNA (RAPD) markers were used to study the genetic relatedness of the isolates and the genetic structure of the population. Fifty-five different RAPD patterns were observed, confirming the genetic uniqueness of each isolate. Gametic disequilibrium values were calculated among all pairs of the 32 putative RAPD loci, and only one value was significant. Unweighted pair-group method with arithmetic averages analysis of distance data, as well as a heterogeneity statistic (Fst), did not indicate any population substructure. The results strongly suggest that isolates originated from a random-mating population and that the disease was propagated in this vineyard by ascospores produced from diverse outside sources. Southern hybridization performed for five markers indicated that the two-allele assumption made to interpret RAPD data may be violated for markers that are similar in size. However, the exclusion of such markers did not change the conclusions of the study.     

Detection of Eutypa lata and Eutypella vitis in Grapevine by Nested Multiplex Polymerase Chain Reaction

ABSTRACT Two fungi were isolated from grapevines in Michigan vineyards with Eutypa dieback symptoms: Eutypa lata and Eutypella vitis. These fungi are difficult to distinguish morphologically but are genetically distinct as determined by sequencing of the internal transcribed spacer (ITS) regions. The ITS regions of 25 Eutypa lata and 15 Eutypella vitis isolates were sequenced. Eutypa lata sequences were more variable than those of Eutypella vitis. Polymerase chain reaction (PCR) primers were designed for each species and evaluated against isolates of both fungi as well as 11 closely related Diatrypaceous fungi and 23 isolates of other fungi representing various pathogenic, saprophytic, and endophytic genera on grape and other small fruit crops. The primers were specific for their intended species. A nested multiplex PCR protocol was developed and used to successfully detect these fungi in wood samples from cankers with and without stromata from naturally infected vines as well as in artificially inoculated, potted canes. The primers developed in this study will assist in our abilities to diagnose and study the roles of Eutypa lata and Eutypella vitis in Eutypa dieback development.     

Genetic Structure and Variation in Aggressiveness in European and Australian Populations of the Grapevine Dieback Fungus, Eutypa lata

Eutypa lata is an ascomycete fungus causing a severe dieback in grapevine. The genetic structure of populations of E. lata from seven regions in Australia, France, Italy and Spain was examined using 20 random amplified polymorphic DNA (RAPD) markers. In some regions, populations were subdivided and a total of 14 samples were analysed. A total of 231 RAPD haplotypes were found among the 240 isolates. Vegetative compatibility testing further demonstrated that isolates of the same haplotype were genetically distinct. Gene diversity was the highest in the population from northern Italy and lowest in the Alsace region in France. Linkage disequilibrium between pairs of putative loci was very low and most of the multilocus analyses were consistent with the hypothesis of random association of the loci. This suggests that random mating occurred in every population and that the sexual stage shapes the genetic structure of E. lata populations in the regions sampled. Only 6% of the total variability was attributable to differences between populations. Nevertheless, significant differences in allele frequency appeared with respect to six RAPD markers indicating some genetic differentiation between populations. This differentiation appeared attributable to differences between the Italian and Spanish populations and the other populations. We thus hypothesize that a restriction of gene flow exists within Europe. The population from Australia was genetically closer to the French and Spanish populations than to that from Italy. Genetic diversity is associated with considerable variation in aggressiveness, which was assessed on cuttings in the greenhouse in six populations. All populations included a range of isolates differing in aggressiveness, but the Italian population seemed to have more isolates with low aggressiveness.     

无花果树苗中截获的无花果孢囊线虫形态和分子鉴定

上海口岸从旅客携带进境的树苗中分离到1种孢囊线虫属的2龄幼虫,通过形态特征和分子特征分析,鉴定为无花果孢囊线虫(Heterodera fici Kirjanova,1954)。该2龄幼虫的主要形态特征为:体长409.9~454.4μm;唇环3个;口针发达,长22.6~24.0μm,基部球圆形略向前倾斜;侧线4条;尾长圆锥形,末端细圆,尾部透明区长24.7~30.5μm,约占尾长的1/2。序列分析显示其ITS区序列与GenBank中无花果孢囊线虫的序列相似性为99.48%~99.90%,序列差异为1~5 bp。系统发育分析显示该线虫与无花果孢囊线虫处于一个聚类组内。这是我国口岸首次报道截获无花果孢囊线虫。     

矮化核桃枝枯病病原菌的形态与分子鉴定

为了明确河南省洛阳地区核桃枝条一种新病害的病原种类,对采自田间的典型症状枝条进行了常规组织分离,对单孢菌株进行了形态鉴定、rDNA ITS序列分析及致病性测定。结果表明,病原菌在PDA培养基上菌落初为白色,随着色素的积累,由中心向边缘变为灰绿色,再变成墨绿色,最终全部变成黑色;气生菌丝棉絮毛状,菌落边缘不整齐,近圆形或不规则形。分生孢子器散生或聚生,呈黑色小粒状,分生孢子器球形或近球形,深褐色,具孔口;分生孢子梗短缺,无色,杆状,末端产生分生孢子;分生孢子梭形、纺锤形,无色,无隔,基部钝圆,顶部稍尖,单孢(24.5～29.0)μm×(4.8～7.5)μm。离体枝条接种结果表明,该菌能侵染核桃枝条引起与田间相同的症状。据此将分离物鉴定为葡萄座腔菌Botryosphaeria dothidea。     

Morphological and molecular identification of Neopestalotiopsis mesopotamica causing tomato fruit rot

Journal of Plant Diseases and Protection - Severe fruit rot symptoms were observed on tomato crop (Lycopersicon esculentum) located in Hamedan province of Iran, in spring 2014. Affected fruits...     

鸢尾种苗中截获的草莓滑刃线虫形态和分子鉴定

草莓滑刃线虫是一种重要的叶和芽寄生线虫,可侵染多种重要花卉、经济作物,被列入我国检疫性有害生物名录。目前,草莓滑刃线虫的鉴定以形态学鉴定为主,但一线口岸常分离不到雌虫,鉴定困难。本研究采用形态特征和分子特征相结合的方法对草莓滑刃线虫进行鉴定:对鸢尾种苗中截获的滑刃属线虫群体雌虫、雄虫进行形态观察和形态测计并与已报道文献进行比较;PCR扩增该群体的rDNA-ITS区并测序,构建基于ITS序列的系统发育进化树;采用草莓滑刃线虫特异引物对该群体DNA样本进行扩增。结果表明:该群体与草莓滑刃线虫俄罗斯种群的测量值相接近;系统进化关系分析显示,该群体与NCBI公布的草莓滑刃线虫均处于同一进化支;采用草莓滑刃线虫特异引物PCR扩增可产生片段大小为169 bp的目的条带。     

日本鸡爪槭中旱稻胞囊线虫形态和分子鉴定

从宁波口岸进境的日本鸡爪槭(Acer palmatum)景观树根际介质中分离到1种胞囊线虫属的2龄幼虫,通过形态鉴定和分子特征分析,鉴定其为旱稻胞囊线虫(Heterodera elachista Ohshima,1974)。该日本群体的2龄幼虫体长374~421μm;唇区有3个唇环;口针粗壮,长16.5~19.2μm,基部球圆形或前端略凹陷;侧线3条;尾长圆锥形,末端细圆,透明尾长22.9~30.7μm,约占尾长的50%。基于核糖体DNA的28S-D2/D3区和ITS区序列构建的系统发育树以及ITS序列酶切分析证实该鉴定结论,这是我国首次截获旱稻胞囊线虫。     

Grapevine rupestris stem pitting virus: a new pathogen of grapevine in Serbia

Journal of Plant Diseases and Protection - Grapevine rupestris stem pitting-associated virus (GRSPaV), a member of the genus Foveavirus, is a commonly detected grapevine virus around the world....     

西瓜种传镰刀菌形态和分子鉴定及其对种子发芽的影响

 采用洗涤检验法和PDA培养基法,从京欣一号和新京欣一号西瓜种子的种壳上分离得到镰刀菌,通过形态学观察和分子检测进行了鉴定,并研究了其对种子发芽的影响。通过菌落形态、色素颜色、菌落生长速率、大型分生孢子、小型分生孢子、厚垣孢子以及分生孢子梗等形态学观察,初步认为其为尖孢镰刀菌;采用真菌rDNA ITS区的通用引物、镰刀菌属特异性引物和尖孢镰刀菌特异性引物对其DNA进行PCR扩增,PCR产物经连接转化并验证后测序,将测序结果登录GenBank进行BLAST分析,分子检测结果与形态学观察结果一致,表明西瓜种子种壳携带的镰刀菌为尖孢镰刀菌,此结果为国内首次报道。用种传尖孢镰刀菌孢子悬浮液处理西瓜种子后,发芽势、发芽率和发芽指数均显著降低。     

危害红掌的穿孔线虫形态和分子鉴定

对一批引进后隔离培养中患病的红掌种苗,进行了根系线虫检测,通过病症分析、线虫形态测量、rDNA-ITS区序列分析等鉴定,结果表明:检测的线虫形态和香蕉穿孔线虫基本一致,rDNA-ITS区测序结果序列一致性为98％～100％.本报道再次证明香蕉穿孔线虫在红掌上的分布和危害,应该对种苗的跨区引进和种植过程进行严格的检疫.