Seasonal dynamics and virus translocation of Grapevine leafroll‐associated virus 3 in grapevine cultivars

Grapevine leafroll‐associated virus 3 (GLRaV‐3) is associated with grapevine leafroll disease, one of the most economically important viral diseases of grapevines. This disease impacts on both vine health and grape quality; reduction in yield, brix and wine colour are among its detrimental effects. Many methods, including serological and molecular procedures, have been developed for the detection of GLRaV‐3; however, there is no PCR‐based assay available to quantify virus populations within plant tissues. A real‐time RT‐PCR assay with TaqMan probe was developed for specific and reliable quantitative detection of GLRaV‐3 in infected tissues. The designed primers and probes target the conserved sequence in the RNA‐dependent RNA polymerase (RdRp) domain of the viral genome to prevent amplification of most subgenomic and defective RNAs. This protocol was used to examine the seasonal dynamics and translocation of GLRaV‐3 in field‐grown grapevines. The results showed that the virus spread quickly from trunks to new growing shoots and leaves early in the growing season, and most samples still harboured detectable virus during late summer and autumn. The seasonal progress of one GLRaV‐3 isolate was compared in four grapevine cultivars (Chardonnay, Cabernet Sauvignon, Italia and Thompson Seedless). Within cultivars there was little variability in the distribution and translocation of GLRaV‐3, except for in Thompson Seedless. This quantitative detection assay will be a valuable tool for GLRaV‐3 diagnosis, disease monitoring and population ecology studies.     

Endophytic Beauveria bassiana activates expression of defence genes in grapevine and prevents infections by grapevine downy mildew Plasmopara viticola

Fungal entomopathogens like Beauveria bassiana (Ascomycota: Hypocreales) are known as antagonists of insects with multiple functional and ecological roles, and have attracted increased attention as biocontrol agents in integrated pest management programmes. For some crop plants, it has been proven that endophytic B. bassiana, besides its entomopathogenic habit, can provide protection against plant pathogens or limit their damaging effects. However, for grapevine, limited knowledge is available on the influence of endophytic B. bassiana on fungal pathogens and about the mechanisms underlying putative protection effects. This study assessed the protective potential of endophytic B. bassiana against grapevine downy mildew Plasmopara viticola in greenhouse experiments. Three and seven days after a B. bassiana treatment, potted grapevine plants were inoculated with P. viticola and the evolving disease severity was assessed. Disease severity was significantly reduced in B. bassiana-treated plants compared to control plants, depending on the age of leaves. Furthermore, a microarray and an RT-qPCR analysis were performed to work out fundamental aspects of genes involved in the interaction between grapevine and B. bassiana. The results indicate an up-regulation of diverse defence-related genes in grapevine as a response to endophytic establishment of B. bassiana. Thus, endophytic establishment of an entomopathogenic fungus such as B. bassiana in grapevine plants would represent an alternative and sustainable plant protection strategy, with the potential for reducing pesticide applications in viticulture.     

The <Emphasis Type="Italic">Foc</Emphasis> gene governs resistance to race 3 of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">cucumerinum</Emphasis> in the cucumber cv. SMR-18

Bois noir (BN) is the most widespread European grapevine yellows disease caused by ‘Candidatus Phytoplasma solani’. Although our knowledge of the mechanisms of interactions of this pathogenic bacteria with host is largely unknown, the plant-pathogen system of BN is commonly used as a model system for studying grapevine yellows diseases. We applied here a conceptual model of general plant pathology – a disease triangle for describing interactions among the host plant, the pathogen and the environment. We generated a proof-of-concept statistical model for disease triangle using original experimental data and different statistical and data mining approaches for a selected system of ‘Ca. P. solani’ infection of cv. ‘Chardonnay’ grapevine plants. We monitored individual plants from a single vineyard over a period of six years. Phytoplasma content, the expression of 21 selected grapevine genes and environmental conditions were recorded and related to disease severity. Our model predicts that in described conditions BN is a function of the expression of grapevine gene VvDMR6, summer rainfall and abundance of ‘Ca. P. solani’. The greatest impact among elements of the disease triangle is attributed to the pathogen, and is independent of the pathogen titer. We showed that this first de facto representation of the disease triangle is useful for showing disease dynamics over several years and could be applied to other plant-pathogen systems. The overall results of this study will contribute to understanding of ‘Ca. P. solani’ biology and its interactions with grapevine host.     

Spatial distribution of the dagger nematode Xiphinema index and its associated Grapevine fanleaf virus in French vineyard

The nematode Xiphinema index is, economically, the major virus vector in viticulture, transmitting specifically the Grapevine fanleaf virus (GFLV), the most severe grapevine virus disease worldwide. Increased knowledge of the spatial distribution of this nematode, both horizontally and vertically, and of correlative GFLV plant infections, is essential to efficiently control the disease. In two infested blocks of the Bordeaux vineyard, vertical distribution data showed that the highest numbers of individuals occurred at 40 to 110 cm depth, corresponding to the two layers where the highest densities of fine roots were observed. Horizontal distribution based on a 10 x 15 m grid sampling procedure revealed a significant aggregative pattern but no significant neighborhood structure of nematode densities. At a finer scale ( approximately 2 x 2 m), nematode sampling performed in a third block confirmed a significant aggregative pattern, with patches of 6 to 8 m diameter, together with a significant neighborhood structure of nematode densities, thus identifying the relevant sampling scale to describe the nematode distribution. Nematode patches correlate significantly with those of GFLV-infected grapevine plants. Finally, nematode and virus spread were shown to extend preferentially parallel to vine rows, probably due to tillage during mechanical weeding.     

Virulence affected by assay parameters during grapevine pathogenicity studies with Botryosphaeriaceae nursery isolates

This study assessed the symptoms that developed when 114 Botryosphaeriaceae isolates from grapevine nursery plant materials were used to inoculate excised green shoots and 1‐year‐old rooted canes of Sauvignon blanc. The experiments showed that all isolates and species were able to produce lesions. Overall, the Neofusicoccum species were shown to be highly pathogenic in both tissue types while the Diplodia species were highly pathogenic on canes but not on green shoots. Isolates of the most prevalent species, N. luteum and N. parvum, showed varying lesion lengths on excised green shoots and canes. An evaluation of the factors associated with lengths of lesions showed that they were significantly affected by experimental batch which reflected inherent host and environmental factors over time. Reisolation from inoculated canes also indicated that most isolates of all species except D. seriata were able to spread internally beyond the lesions. Genetic variability analysis using UP‐PCR showed that N. luteum isolates were genetically diverse but no association was observed between the phylogenetic group and degree of pathogenicity caused by the isolates. This study demonstrated that all Botryosphaeriaceae species from grapevine nurseries were pathogenic to grapevines and that the lesion lengths varied between species, among isolates within a species and among nursery sources, and was affected by the test method.     

Molecular tracing of the transmission routes of bois noir in Mediterranean vineyards of Montenegro and experimental evidence for the epidemiological role of Vitex agnus‐castus (Lamiaceae) and associated Hyalesthes obsoletus (Cixiidae)

Epidemiological aspects and transmission routes of bois noir (BN), a grapevine yellows disease induced by ‘Candidatus Phytoplasma solani’, have been exhaustively studied in the affected vineyards of continental Europe but not in the Mediterranean coastal zone. Because ‘Ca. Phytoplasma solani’ and its principal vector Hyalesthes obsoletus presumably originate from the Mediterranean, gaining knowledge of the epidemiological peculiarities of the disease in this area is essential for understanding its global spread and diversification, as well as for designing local management strategies. In this study, molecular epidemiology was applied to trace transmission pathways of ‘Ca. Phytoplasma solani’ in the Mediterranean vineyards of Montenegro, using multilocus sequence typing of tuf, vmp1 and stamp genes of the isolates associated with various hosts. Thus, ‘Ca. Phytoplasma solani’ was tracked from a tentative reservoir plant (inoculum source) through an associated vector population to the infected grapevine. Three pathways of transmission were documented, originating from Urtica dioica, Convolvulus arvensis and Vitex agnus‐castus; however, only the route originating from U. dioica was direct, whereas the latter two were overlapping and could be intermixed. Vitex agnus‐castus is a natural source of ‘Ca. Phytoplasma solani’, representing an important link in disease epidemiology in the Mediterranean and a possible origin of several genotypes occurring in central Europe. Experimental confirmation of the role of Vitex‐associated H. obsoletus in BN transmission in Montenegrin vineyards indicates its tentative role as a vector in the wide area of the Mediterranean, where some of the major wine‐producing regions are located.     

Aetiology of anthracnose on grapevine shoots in Brazil

Anthracnose is an important disease in vineyards in south and southeast Brazil, the main grape‐producing regions in the country. This study aimed to identify the causal agents of grapevine anthracnose in Brazil through multilocus phylogenetic analyses, morphological characterization and pathogenicity tests. Thirty‐nine Elsinoë ampelina and 13 Colletotrichum spp. isolates were obtained from leaves, stems and berries with anthracnose symptoms collected in 38 vineyards in southern and southeastern Brazil. For E. ampelina isolates, the internal transcribed spacer (ITS), histone H3 (HIS3) and elongation factor 1‐α (TEF) sequences were analysed. HIS3 was the most informative region with 55 polymorphic sites including deletions and substitutions of bases, enabling the grouping of isolates into five haplotypes. Colonies of E. ampelina showed slow growth, variable colouration and a wrinkled texture on potato dextrose agar. Conidia were cylindrical to oblong with rounded ends, hyaline, aseptate, (3.57–) 5.64 (?6.95) μm long and (2.03–) 2.65 (?3.40) μm wide. Seven species of Colletotrichum were identified: C. siamense, C. gloeosporioides, C. fructicola, C. viniferum, C. nymphaeae, C. truncatum and C. cliviae, with a wide variation in colony and conidium morphology. Only E. ampelina caused anthracnose symptoms on leaves, tendrils and stems of Vitis vinifera and V. labrusca. High disease severity and a negative correlation between disease severity and shoot dry weight were observed only when relative humidity was above 95%. In this study, only E. ampelina caused anthracnose symptoms on grapevine shoots in Brazil.     

A Survey of Trunk Disease Pathogens within Rootstocks of Grapevines in Spain

Grapevine trunk disease pathogens, and specifically Petri disease pathogens, can be spread by planting infected plants. Due to the increasing incidence of Petri disease and other young grapevine declines reported lately in Spain, a sampling of plants used before for new vineyards were carried out in 2002 and 2004. A total number of 208 plants (grafted and non grafted) were collected, of which 94 plants (45.2%) were infected with at least one of the following pathogens: Phaeomoniella chlamydospora, and species of Phaeoacremonium, Botryosphaeria, Cylindrocarpon, and Phomopsis. Species of the genera Phaeoacremonium and Botryosphaeria isolated in 2004 were identified using morphological and molecular characters. Species of Phaeoacremonium identified were P. aleophilum and P. parasiticum; and those of Botryosphaeria were B. obtusa, B. dothidea and B. parva. This is the first report of P. parasiticum and B. parva occurring on grapevines in Spain. Distribution of pathogens within the plants was studied in 2004. Phaeomoniella chlamydospora was not detected in the graft union of any plant; however, species of Botryosphaeria and Phomopsis were detected along the plant, but mainly in the graft union; Phaeoacremonium aleophilum was detected along the grafted plants, but not in rooted rootstocks. The results suggest that infected plants used for new plantings in Spain are an important source of primary inoculum of the pathogens associated with grapevine trunk diseases in the field.     

Identification and pathogenicity of Fusarium species associated with pokkah boeng of sugarcane in Brazil

Brazil is the world’s biggest producer of sugarcane (Saccharum spp. hybrids). Pokkah boeng is an important fungal disease in this crop caused by members of the Fusarium fujikuroi species complex (FFSC) and characterized by deformation of the aerial part of the plant and stem rot. While the occurrence of symptoms has been reported in plantations in Brazil, no official reports of the disease exist. In this study, species of the FFSC were identified that are associated with sugarcane plants with symptoms of pokkah boeng in Brazil. This was achieved using two-loci molecular phylogeny, sexual compatibility and analysis of morphological markers. The ability of strains to cause disease in plants of sugarcane, maize, sorghum and millet was also evaluated. The 39 isolates studied were identified as F. sacchari, F. proliferatum and another, still unknown, phylogenetic lineage that is sister to F. andiyazi. Crossing field isolates of F. sacchari and F. proliferatum with their respective tester strains produced fertile perithecia and viable ascospores. All three species induced symptoms of pokkah boeng on inoculated sugarcane plants and caused stem rot in maize, sorghum and millet. Symptoms on sugarcane are chlorosis and necrosis of leaves, punctured lesions, twisted leaves, reduction of the total leaf area, death of the top of the plant and stalk rot. The findings confirmed the aetiology of the disease in Brazil, generated basic knowledge for the development of strategies for diagnosis and monitoring of the disease and support breeding programmes for selecting resistant germplasm.     

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.     

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.     

Genetic diversity and aggressiveness of Calonectria pteridis in Eucalyptus spp.

Calonectria leaf blight, caused by Calonectria pteridis, is currently one of the main foliar diseases in eucalypt plantations in Brazil. In warm and high rainfall regions, the disease can be a limiting factor for eucalypt production when planting susceptible genotypes. The most effective method for controlling this disease in the field is the use of resistant genotypes, which requires knowledge of the genetic variability and aggressiveness of the pathogen population for effective deployment of plant resistance. This work evaluated the genetic diversity and aggressiveness of C. pteridis populations obtained from infected eucalypt plants in Monte Dourado (Pará state) and Imperatriz (Maranhão state), Brazil. To study the genetic diversity, 16 ISSR primers were tested, five of which amplified polymorphic, reproducible and informative bands. Thirty-one closely related genotypes were identified from 84 isolates studied, indicating that the population has a low genetic diversity. The aggressiveness of seven isolates, selected according to geographic origin and their clustering in the ISSR-based dendogram, was determined by inoculation of a hybrid Eucalyptus grandis × E. urophylla clone under controlled conditions. Disease severity was assessed by both measuring the percentage of plant defoliation and assigning a score according to a diagrammatic scale of symptoms. A high correlation between the two evaluation methods was observed, which revealed significant differences in aggressiveness among the isolates. The diagrammatic scale is recommended for disease evaluation because results are obtained much faster, before the occurrence of severe defoliation. No correlation between clustering in the ISSR-based phylogenetic analysis and aggressiveness was observed.     

Soybean cultivar performance in the presence of soybean Asian rust,in relation to chemical control programs

Soybean rust is caused by an obligate parasite (Phakopsora pachyrhizi) which has spread in Brazil in each new season since 2001 and, despite the efforts to control the disease, losses have occurred every year. Its control demands several tactics amongst which chemical control with fungicides is the main method and remains indispensable. Control strategies such as the use of cultivars with partial resistance are desirable, but are not yet commercially available. The present study analyzed the existing differences in the reactions of short, medium and long cycle soybean cultivars against Asian rust and their responses to fungicide sprays. The experiment was conducted at Uberlandia-MG, Brazil, under field conditions from December 2007 to May 2008, in the Syngenta Seeds Experimental Station. The high pressure of the disease in the experiment simulated the natural pressure that the disease often reaches in Brazil. The studied variables were: visual severity (percentage of infected leaf area), percentage defoliation and productivity (kg ha⁻¹). Disease severity was expressed as AUDPC (area under disease progress curve). Variance analysis and comparison of means by the Tukey test (5% significance) were done for all variables studied. Significant differences were observed between cultivar effects and chemical control programs. The results obtained here indicate that the cultivars M-Soy 8199RR and Emgopa 315RR were less susceptible to disease, and that a control program termed “monitoring” (in which the appearance of new pustules of the pathogen were monitored to make the decision at each fungicide spray) was the most effective.     

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.     

Detection of Agrobacterium tumefaciens in grapevine cuttings1

Crown gall is the most important bacterial disease of grapevine and is primarily associated with Agrobacterium tumefaciens biovar 3. The systemic distribution of the agrobacteria in grapevine requires the checking of nursery propagation material to reveal their presence in the xylem vessels. The methods adopted to demonstrate systemic survival are quite lengthy and, thus, this study aimed to develop a serological sampling procedure to rapidly detect A. tumefaciens in xylem extracts, obtained by vacuum extraction with suitable washing fluid, from asymptomatic one year-old dormant grape cuttings. The suspension was centrifuged and the final concentrate used for immunofluorescence staining. The sensitivity threshold of the vacuum washing system and the effectiveness of the detection method were experimentally evaluated. The antiserum specificity was tested in a comparative study with a collection of A. tumefaciens strains from grapevine and other common grapevine contaminants. The vacuum washing system is discussed as an indexing tool in a much hoped for certification programme.     

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.     

New Mealybug Species Vectoring Grapevine Leafroll-Associated Viruses-1 and -3 (GLRaV-1 and -3)

Many grape viruses, such as filamentous Grapevine leafroll-associated viruses in the Closteroviridae family, are spread primarily through infected propagating material. However, there is increasing evidence that leafroll disease are spread in the field by insect vectors, namely mealybugs and other scale insects. This study was carried out in the northern wine-growing regions of France where Grapevine leafroll-associated virus-1 and -3 (GLRaV-1 and -3) are the most widespread grape Ampelovirus species. The vineyards were inspected for presence of mealybug and scale insects and grapes infected by GLRaV-1 and -3. Mealybugs, Heliococcus bohemicus, Phenacoccus aceris (Pseudococcidae) and the soft scale insect Parthenolecanium corni (Coccidae), were capable of a transmission efficiency of 14%, 23% and 29% respectively. GLRaV-1 and -3 infections that resulted from virus transmission were confirmed with DAS-ELISA using polyclonal antibodies. This is the first report of GLRaV-1 and -3 transmission by mealybug and coccid species in France, and the first report of the ability of H. bohemicus and Phenacoccus aceris to transmit these viruses to grapevines. The relevance of these findings with regards to maintenance of virus-free grapevine stocks and to control leafroll spread in commercial vineyards is discussed.     

<Emphasis Type="Italic">Grapevine virus A</Emphasis> transmission by larvae of <Emphasis Type="Italic">Parthenolecanium corni</Emphasis>

Grapevine virus A (GVA, Vitivirus) was transmitted experimentally by first and second instars of the scale insect Parthenolecanium corni from grapevine to grapevine and to the herbaceous host Nicotiana benthamiana. This is the first report of GVA transmission by P. corni. Grapevine leafroll-associated virus-1 (Ampelovirus) was always present in the donor grapevines and, in every case, GVA was transmitted simultaneously with this ampelovirus from grapevine to grapevine, suggesting possible interactions between the two viruses for transmission.