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.     

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.     

Biological and integrated control of grey mould of grapevine: results in Italy1

Three experimental trials were carried out in Northern Italy during 1985 and 1986 in order to control grey mould of grapevine (Botrytis cinerea) by using isolates of Trichoderma spp. resistant to several fungicides commonly sprayed against grapevine pathogens, alone or in alternation with benzimidazoles or dicarboximides, in vineyards where fungicide-resistant strains of B. cinerea are frequent. The antagonists alone partially controlled the pathogen on cv. Moscato ?Asti. In one case, the integration of chemical and biological control measures showed slightly better results than for the fungicide alone (for benomyl but not for vinclozolin), but further trials are needed to investigate the full potential for using fungicide-resistant Trichoderma in alternation with fungicides. Trichoderma spp. performed very poorly on cv. Barbera.     

Understanding the effects of multiple sources of seasonality on the risk of pathogen spread to vineyards: vector pressure,natural infectivity,and host recovery

Seasonality plays an important role in the dynamics of infectious disease. For vector‐borne pathogens, the effects of seasonality may be manifested in the variability in vector abundance, vector infectiousness, and host‐infection dynamics over the year. The relative importance of multiple sources of seasonality on the spread of a plant pathogen, Xylella fastidiosa, into vineyards was explored. Observed seasonal population densities of the primary leafhopper vector, Graphocephala atropunctata, from 8 years of surveys in northern California were incorporated into a model of primary spread to estimate the risk of pathogen infection under different scenarios regarding seasonality in vector natural infectivity (i.e. constant or increasing over the season) and grapevine recovery from infection (i.e. none or seasonal recovery). The extent to which local climatic conditions affect risk estimates via differences in vector abundance was investigated. Seasonal natural infectivity, seasonal recovery, and especially the combination, reduced (up to 8‐fold on average) within‐season and cumulative yearly estimates of pathogen spread. Estimated risk of infection also differed greatly among years due to large differences in vector abundance, with wet and moderate winter and spring conditions favouring higher G. atropunctata abundance. Seasonal variation of the pathogen–vector interaction may play an important role in the dynamics of disease in vineyards, reducing the potential prevalence from what it could be in their absence. Moreover, climate, by affecting sharpshooter leafhopper abundance or activity, may influence Pierce’s disease dynamics.     

Formation of overwintering structures of Uncinula necator and colonization of grapevine under field conditions

Developing shoots of grapevine ( Vitis vinifera cv. Kerner) were inoculated with conidia of the powdery mildew, Uncinula necator , at well-defined phenological stages of the host to provoke the development of flag shoots in the field and to investigate these shoots as early as possible in the following growing season for the presence of the pathogen. The disease progress was monitored and fungal growth and development on samples from a field trial were analysed by means of low-temperature scanning electron microscopy (LT–SEM). Mycelium was detected on the surface and in the interior parts of buds. The suitability of the field plot to analyse flag shoots was proven by the occurrence of such shoots in the following spring. Furthermore, early stages of cleistothecia development on berries were described for the first time. Establishment of U. necator in dormant buds of grapevine, giving rise to flag shoots in the following spring, is considered to play a significant role in overwintering of the pathogen in the vineyards of southern Germany.     

Characterization of resistance mechanisms activated by Trichoderma harzianum T39 and benzothiadiazole to downy mildew in different grapevine cultivars

Downy mildew, caused by Plasmopara viticola, is one of the most destructive diseases of grapevine and is controlled with intense application of chemical fungicides. Treatment with Trichoderma harzianum T39 (T39) or benzothiadiazole‐7‐carbothioic acid S‐methyl ester (BTH) has been previously shown to activate grapevine resistance to downy mildew and reduce disease symptoms in the Pinot noir cultivar. However, enhancement of plant resistance can be affected by several factors, including plant genotype. In order to further extend the use of resistance inducers against downy mildew, the physiological and molecular properties of T39‐ and BTH‐activated resistance in different cultivars of table and wine grapes were characterized under greenhouse conditions. T39 treatment reduced downy mildew symptoms, but the degree of efficacy differed significantly among grapevine cultivars. However, efficacy of BTH‐activated resistance was consistently high in the different cultivars. Expression profiles of defence‐related genes differed among cultivars in response to resistance inducers and to pathogen inoculation. T39 treatment enhanced the expression of defence‐related genes in the responsive cultivars, before and after P. viticola inoculation. A positive correlation between the efficacy of T39 and the expression level of defence‐related genes was found in Primitivo and Pinot noir plants, while different genes or more complex processes were probably activated in Sugraone and Negroamaro. The data reported here suggest that the use of a responsive cultivar is particularly important to maximize the efficacy of resistance inducers and new natural inducers should be explored for the less responsive cultivars.     

Experimental and molecular evidence of Reptalus panzeri as a natural vector of bois noir

Bois noir (BN) is an economically important grapevine yellows disease induced by the stolbur phytoplasma and principally vectored by the cixiid Hyalesthes obsoletus. This study addresses the involvement of other planthoppers and/or leafhoppers in BN epidemics in the South Banat district of northeastern Serbia, by performing transmission experiments and multilocus typing of stolbur phytoplasma isolates to determine the vector‐related characteristics of the disease. Transmission trials were conducted with adults of two cixiid congeners, Reptalus panzeri and R. quinquecostatus, which were found to harbour stolbur phytoplasma in the vineyards under study. A molecular characterization of stolbur phytoplasma isolates was performed by sequence analysis and/or RFLP typing of the two housekeeping genes tuf and secY and the two membrane proteins stamp and vmp1. Transmission trials with naturally infected R. panzeri adults from either the BN‐infected vineyards or maize redness (MR)‐affected maize fields revealed a high stolbur phytoplasma transmission efficiency to grapevines. In contrast, experiments conducted with stolbur‐positive R. quinquecostatus originating from BN‐infected vineyards, provided no evidence for a vector role of this species. Seven stolbur phytoplasma genotypes, all of which were tuf‐b types, were detected among the grapevine‐ and insect‐associated field samples according to the tuf/secY/vmp1/stamp typing. STOLg was the genotype most frequently found in naturally infected grapevine (42%), as well as R. panzeri originating from the vineyards (85%) and maize fields (98%). The same genotype was found in all experimental plants inoculated by R. panzeri, confirming its vectorship of the disease.     

我国北方葡萄根癌病的发生规律及药剂防治

葡萄根癌病在我国北方地区发生分布广泛,部份葡萄园发病严重,感病品种病株率高达100％。葡萄的根、根颈、枝蔓均能发病,以根颈部冠瘿瘤最多。在受冷伤害严重的葡萄园,枝蔓上发病也重,且成为植株死亡的主要原因。在内蒙古包头市一带,5月下旬发病,6月中旬至8月为发病盛期,9月下旬停止发病。温度、湿度及降水与病害关系密切,葡萄受霜冻和冬季低温的影响,增加了病菌新的侵染点,加快病害的发生发展。葡萄品种抗病性差异显著,玫瑰香、巨峰等高度感病,罗也尔玫瑰、康拜尔、贝达等抗病性强。室内药效测定和田间防治试验,抗菌剂401、402,福美砷和石硫合剂渣子等抑菌力强,并有较好的防治效果。     

实时荧光定量PCR(SYBR Green I)检测不同抗枣疯病枣树品种嫁接接穗中的植原体浓度

在枣疯植原体16S r DNA片段内设计并合成引物对DZ16SF-2/DZ16SR-2(扩增190 bp片段),用重组质粒p MD18-DZ16S为模板建立标准曲线,并建立枣疯植原体实时荧光定量PCR(SYBR Green I)检测体系。溶解曲线分析显示:该引物特异性好,该体系对质粒模板的检测灵敏度达102copies/μL。利用该体系对不同月份、不同发病级别的嫁接接种枣树接穗样品进行植原体含量测定。通过对高抗品种‘星光’,中抗品种‘黑腰子枣’、‘嘎嘎枣’和‘葫芦枣’,感病品种‘月光枣’、‘马牙枣’、‘莲蓬子’、‘北流西冬枣’和高感品种‘大红袍枣’嫁接到发病‘冬枣’砧木上所表现出的不同病级、病情指数和病原浓度比较结果发现,表现无症状的样品中植原体的含量在每克鲜重105~106个,发病Ⅰ~Ⅱ级枝条中植原体浓度在106~107个/g,Ⅲ~Ⅳ级发病枝条含量在107~108个/g鲜重,而V级发病枝条达到每克鲜重109个,表明接种发病的接穗症状严重度与其体内植原体浓度积累密切相关。高抗枣疯病‘星光’品种嫁接接穗体内病原浓度明显低于感病品种,而且有较多‘星光’接穗嫁接的发病砧木症状有所减轻,植原体浓度也有所降低。本研究可为探索枣树品种的抗病机制、抗病性鉴定及枣疯病植原体的防治提供参考。     

Co‐infection by Botryosphaeriaceae and Ilyonectria spp. fungi during propagation causes decline of young grafted grapevines

Decline of newly planted, grafted grapevines is a serious viticultural problem worldwide. In the Riverina (New South Wales, Australia), characteristic symptoms include low fruit yields, very short shoots and severely stunted roots with black, sunken, necrotic lesions. To determine the cause, roots and wood tissue from affected plants in 20 vineyards (Vitis vinifera cv. Chardonnay grafted to V. champini cv. Ramsey rootstock) were assayed for microbial pathogens. Ilyonectria spp. (I. macrodidyma or I. liriodendra, producers of phytotoxin brefeldin A, BFA, and cause of black foot disease of grapevines) and Botryosphaeriaceae spp. (predominantly Diplodia seriata) were isolated from rootstocks of 100 and 95% of the plants, respectively. Togninia minima and Phaeomoniella chlamydospora (cause of grapevine Petri disease) were isolated from 13 and 7% of affected plants, respectively. All Ramsey rootstock stems of grafted plants sampled from a supplier nursery were infected with Ilyonectria spp. and D. seriata. Diplodia seriata, but not Ilyonectria spp., was also isolated from 25% of canes sampled from the rootstock source block. Root inoculation of potted, disease‐free Chardonnay plants with Ilyonectria isolates from diseased vineyards caused typical disease symptoms, while co‐inoculation with Botryosphaeriaceae spp. increased disease severity. This is the first study to show that a major cause of young grapevine decline can be sequential infection by Botryosphaeriaceae from rootstock cuttings and Ilyonectria spp. from nursery soil. Although the Petri disease fungi were less common in young declining grafted grapevines in the Riverina, they are likely to contribute to the decline of surviving plants as they mature.     

Phloem and xylem modifications of Vitis vinifera stems in response to flavescence dorée phytoplasma infection

Anatomical modifications of xylem and phloem tissues of grapevine (Vitis vinifera) stems of shoots infected by the flavescence dorée phytoplasma (FDp) were first observed and described in the 1960s, but never quantified in detail. In this paper, we describe and quantify the impact of FDp on grapevine stem tissues, and relate it to the level of expression of symptoms and to cultivar-specific FDp susceptibility. For this purpose, we measured and quantified the anatomical parameters of xylem and phloem tissues of a tolerant (Merlot) and a susceptible (Chardonnay) cultivar. For each cultivar, thin sections of eight shoots with symptoms from FDp-infected grapevines, eight symptomless shoots from the same FDp-infected grapevines, and eight symptomless shoots from symptomless grapevines (control) were compared. Results showed general inhibition of xylem growth and proliferation of phloem tissues (hyperplasia) with lack or irregular arrangement of the fibre-sclereids in the axial phloem of the stems from shoots with symptoms, irrespective of the cultivar. Xylem vessels of infected Merlot shoots were partly occluded by tyloses and a higher number of smaller vessels were produced than in control plants. Thus, the anatomical responses confirmed the detrimental effect of FDp on stems of infected grapevine shoots, including impaired stem development and lack of periderm formation. Statistically significant differences were found between the two cultivars with different levels of susceptibility to FDp infection.     

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.     

Site-specific field resistance of grapevine to <Emphasis Type="Italic">Plasmopara viticola</Emphasis> correlates to altered gene expression and was not modulated by the application of organic amendments

The influence of site on resistance of grapevine (cv. Chasselas) to Plasmopara viticola was evaluated. Grapevine leaves from three vineyards in the region of Lake Neuchatel (Switzerland) were tested for their susceptibility to P. viticola in the lab in five successive years (2004–2008), and the expression levels of four selected defence-related genes (Glucanase, Lipoxygenase 9, 9-cis epoxycarotenoid dioxygenase, Stilbene synthase) were studied in 1 year. In all 5 years of examination, differences between sites were substantial. In four out of 5 years, plants from site Hauvernier were much less susceptible to P. viticola than plants from site Auvernier. In another year, differences were less pronounced but still significant for one leaf age. Susceptibility of plants from a third site (Concise) varied from year to year. Differences in the genetic background were excluded by microsatellite analysis. Differences in susceptibility were mirrored in the constitutive expression pattern of four defence-related genes, with samples from the Hauterive site clearly separated from samples of the other two sites in redundancy analysis. Furthermore, it was evaluated whether site-specific resistance can be modulated by agronomic practices such as the application of organic amendments. In two commercial vineyards (cv. Pinot noir), soils had either not (control) or yearly (compost) been amended with a compost for the last 9 years. Leaves from plants grown in any of the two treatments did not differ in their susceptibility to P. viticola in both years of examination. Additionally, under controlled conditions, none of 19 different composts amended to the substrate of grapevine seedlings or cuttings affected their susceptibility to P. viticola, but 8 out of 19 composts reduced severity in the control bioassay Arabidopsis thaliana—Hyaloperonospora arabidopsidis, indicating that a modulation of site-specific susceptibility of grapevine plants by organic amendments is at the very least, difficult.     

A yellows disease system with differing principal host plants for the obligatory pathogen and its vector

A stolbur‐type phytoplasma is the putative pathogen of grapevine yellows disease that causes economic damage to vineyards in most growing areas around the world. The pathogen is known to be transmitted to vines by two planthoppers, Hyalesthes obsoletus and Reptalus panzer; the latter is found in Europe but has not yet been observed in Israel. The establishment of a vector–pathogen–plant relationship requires that the pathogen and the vector meet on a shared host plant. This does not happen in the ecosystem examined here, where two different principal host plants for the obligate pathogen and its vector exist: the pathogen is established on vines, while its vector, H. obsoletus, develops on Vitex agnus‐castus. The present study verified that: (i) the vector cannot complete its life cycle on vines; (ii) V. agnus‐castus does not grow in the immediate vicinity of vines, and does not harbour the pathogen; and (iii) the pathogen is not vertically transmitted from mother to offspring. Moreover, in a thorough search of plants in vine growing areas, no other plants were found that host both the vector and the pathogen. However, it was found that the planthopper can acquire the phytoplasma from infected vines. Nonetheless, this does not prove the ability of the planthopper to further transmit the pathogen to vines and does not explain the presence of the vector on the non‐preferred vines. Thus, the enigma of the pathogen–vector–host triangle in this system remains unresolved.     

Recent research progress on Xanthomonas ampelina1

Bacterial blight of grapevine (caused by Xanthomonas ampelina) occurs in Greece, France, Spain, Italy, Turkey, Portugal, USSR and South Africa. It is a serious, chronic vascular disease which is widespread in some areas of grape production, affecting commercially important cultivars. There is evidence that the disease is presently spreading into new areas in some countries. The actual distribution of blight may be much wider, since its symptoms may be confused with those of other diseases. It is therefore essential to confirm the presence of X. ampelina by isolation and identification. X. ampelina is an anomalous member of the genus Xanthomonas which is readily distinguished on the basis of a few tests. The pathogen may also be rapidly detected in grapevine sap using the indirect immunofluorescence technique. The susceptibility of shoots to infections, under Cretan conditions, is highest from November to late January and very low during February and March. It has been found that up to 50% of apparently healthy canes obtained from diseased vineyards were latently infected. Transmission of the pathogen through soil and roots during the control of Daktulosphaira vitifolii or frost damage by flooding has been demonstrated in France. Several of the more widely grown cultivars were found to be very susceptible to the pathogen while no completely resistant cultivar has been detected. Control trials based on four sprays with a number of chemicals were unsuccessful. Control policy should be directed: (a) to preventing spread of the disease, by exclusive use of certified material; (b) to minimizing the disease in affected areas by suitable sanitary, cultural and preventive measures.     

Temporal association of potato tuber development with susceptibility to common scab and Streptomyces scabiei‐induced responses in the potato periderm

Using hydroponics and novel non‐destructive pot culture systems which enable inoculation at specific tuber development stages, the dynamics of common scab infection patterns in potato were studied in order to provide more precise identification of tuber physiological factors associated with susceptibility. At the whole‐tuber level, infection percentages were greatest when Streptomyces scabiei inoculation occurred early; at 2 weeks after tuberization (WAT) 68% of tubers became infected, contrasting with late inoculation (8 WAT), when only 4% infection occurred. The first‐formed internodes were most susceptible to infection, whilst later‐forming and slower‐expanding internodes were less susceptible. Detailed tuber physiological examination of internode 2 showed that pathogen‐induced changes, including increased phellem (periderm) thickness, cell layers and phellem suberization (key physiological features believed critical to S. scabiei infection) were promoted through S. scabiei inoculation. Sequential harvesting showed enhanced phellem suberization (28% greater than the control) within 7 days of pathogen exposure, while phellem thickness and layer responses were also initiated early in the infection process (10–14 days after pathogen exposure) and these responses were independent of symptom expression. Differences in cultivar response were observed, with greater phellem suberization observed 10 days after tuberization (DAT) in the common‐scab‐tolerant cv. Russet Burbank than in the susceptible cv. Desiree. Likewise, Russet Burbank had thicker and more numerous cell layers in the phellem (up to eight cell layers) during early tuber growth (20–30 DAT) than Desiree (up to six cell layers).     

Insect injection and artificial feeding bioassays to test the vector specificity of flavescence dorée phytoplasma

ABSTRACT The specificity of vector transmission of Flavescence dorée phytoplasma (FDP) was tested by injecting FDP, extracted from laboratory-reared infective Euscelidius variegatus, into specimens of 15 other hemipteran insect species collected in European vineyards. Concentrations of viable phytoplasma extracts and latency in vectors were monitored by injection of healthy-reared E. variegatus leafhoppers. Based on these preliminary results, insects were injected by using phytoplasma extracts that ensured the highest rate of FDP acquisition and transmission by E. variegatus. Transmission into an artificial diet through a Parafilm membrane about 3 weeks after insect injection was attempted. FDP-injected insects that belonged to 15 hemipteran species were confined in cages and fed through the membrane for a 4- to 5-day inoculation access period. FDP DNA was detected by polymerase chain reaction (PCR) in the feeding buffer fed upon by Anoplotettix fuscovenosus, Aphrodes makarovi,E. variegatus, and Euscelis incisus. PCR amplification with specific primers detected FDP DNA in injected insects of all test insect species. Band intensity was positively correlated with the transmissibility of FDP. Transmission of FDP to plants by feeding was confirmed for Anoplotettix fuscovenosus, E. variegatus, and Euscelis incisus, but not for Aphrodes makarovi. Our results suggest that vector competency of FDP is restricted to specimens belonging to the family Cicadellidae, subfamily Deltocephalinae.     

Association between genetic variability and titre of Grapevine Pinot gris virus with disease symptoms

The present work was carried out in order to verify the possible association between a new grapevine disease, characterized by leaf mottling and deformation, and the genetic variability and concentration of Grapevine Pinot gris virus (GPGV), a recently identified virus tentatively associated with the pathology. After vineyard surveys and the establishment of real‐time qPCR assays, characterization of GPGV isolates and evaluation of GPGV titre were assessed in more than 100 samples of grapevine Glera, collected from plants regardless of whether or not they showed the symptomatology. Results showed that there was an important association between the GPGV variants and manifestation of the symptoms, and that grapevines with symptoms harboured significantly higher GPGV titre than symptomless vines. Moreover, an interesting relationship among the phylogenetic clustering of the isolates originating from plants with symptoms and some epidemiological characteristics of the disease was found. The current study confirmed the role of GPGV in the emergent disease characterized by grapevine leaf mottling and deformation.