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  2. Emerging diseases of vine in the central part of Spain wood   (05/05/2011)

This news article was originally written in Spanish. It has been automatically translated for your convenience. Reasonable efforts have been made to provide an accurate translation, however, no automated translation is perfect nor is it intended to replace a human translator. The original article in Spanish can be viewed at Enfermedades emergentes de la madera de vid de la zona centro de España
Current status and perspectives on their management and control

Emerging diseases of vine in the central part of Spain wood

May 5, 2011

The so-called 'diseases of wood' of the vine due to fungi are responsible for considerable economic losses worldwide. In recent years, there is a growing concern due to the increase observed in the incidence of this type of pathology in almost all international producing areas. The present contribution offers an update on the day of knowledge on epidemiology and characterization of pathologies of wood of Grapevine, offering an overview of their impact on different national wine-growing areas, with particular reference to the community of Madrid. In addition, provides a vision integrating on the different current strategies of management and control of these diseases, both in the production of plant nursery, as in the field. Finally, presents the results obtained in the Imidra in relation to some developments of research on etiology and control of these diseases in our region.
Vicente González and María Luisa Tello, of the Madrid Institute for research and Rural Development, agriculture and food (Imidra)
In Spain, the cultivation of the vine is of great importance, both economic and social levels. Viticulture in Spain is extended to almost all of the autonomous communities, where more than fifty regions and/or regions wine with denominación de origen (D.O.) are currently registered.
The so-called 'diseases of wood' of the vine are responsible for considerable economic losses worldwide. The interest of the plant, grape and wine-producing sectors has increased over the past three decades as a result of two fundamental facts. Firstly, in the last 10 - 20 years has detected a sensitive increase of the mortality of young plants of vine from nursery, in the 1-3 years after planting in field. In addition, the progressive suspension of the use of fungicides of chemical origin as sodium arsenite, single control measure used traditionally in pathologies of wood as la yesca, has led a progressive increase in the incidence, dispersion and losses due to these diseases. Along with these events, it is believed that there are other factors involved, such as changes in cultural practices, less protection in the wounds of pruning or reduction of the health inspection in certified propagation material (Graniti and col.)(, 2000). In addition, in recent years, the situation and etiology of diseases of Grapevine wood has become more complex with the emergence and description of new pathogens, causing agents in some cases of different pathologies, but in many others shared by non-specific symptoms with well-known actors.
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Objectives

The main objective of this work is to give a vision integrating to compile the current knowledge of the pathologies that affect vine wood, from the most classic to the newest syndromes, with special emphasis on the methods of handling and control under development. Provides knowledge about the incidence and distribution of these pathologies in different national production areas and especially in the peninsula Center. Finally, described and set out from results of experiments carried out by the Group of plant pathology of Imidra, on etiology and development of new alternatives for control of diseases of Grapevine wood.

Current status of knowledge: Etiology of diseases of Grapevine wood

The concept of disease of Grapevine wood covers various pathologies of fungal origin who share as a common feature internal alteration of the wood they parasitize, producing phenomena of necrosis or rot. His progression often lead to the death of the individual attacked over a period of indeterminate time, which can vary from 1-30 years. Until recently, so-called yesca and eutipiosis diseases were considered as the most important. The first one is characterized by a spongy part tissue degradation accompanied by other symptoms characteristic in other tissues of the plant, such as chlorosis and necrosis of the spaces internerviales of the leaves (Graniti and col., 2000;) (Mugnai et al., 1999). La yesca is today considered a complex disease in which speakers and acting synergetically a number of species of fungi of origin and diverse systematic position. Among them are ' Phaeomoniella chlamydospora' and various species of 'phaeoacremonium', between the mitospóricos fungi, or the basidiomiceto Fomitiporia mediterranea.
Recently it has been observed in all countries producers (including Spain), the emergence and development of symptoms in wood at very early ages, even in the first year of plantation (Armengol and col., 2001;) Giménez-Jaime and col., 2006; (etc.). This syndrome associated with young plants is the generic name of disease of Petri and is associated with 'pachlamydospora' and several species of the genus 'phaeoacremonium' as aetiologic agents. Petri disease can cause high losses in new plantations. The symptoms associated with the syndrome includes phenomena of progressive decay and stunted growth, along with foliar manifestations (marginal necrosis, chlorosis, etc.) and wilt, and can evolve to the sudden collapse of the entire plant. Internally, the tissue show a characteristic darkening of the vessels xilemáticos forming longitudinal stripes. May also appear tilosas or formation of rubber in the vessels of the xylem (from the Rio et al., 2001), structures usually produced as a defense against the invasion of bacteria or fungi (Scheck and col., 1998;) (Rego et al., 2000).
Recently it has been observed in all countries producers (including Spain), the emergence and development of symptoms in wood at very early ages, even in the first year of planting
Infected young vines with ' Pa chlamydospora' suffer a slow establishment or inappropriate development, even failures can occur in the welding of graft when the pattern is infected before the graft. The infected vineyards which initially grow well, can later accuse a loss of productivity. There are also other biotic and abiotic factors that collaborate decisively in the process of disease. Numerous studies suggest that species involved in the disease of Petri are also present in the processes associated with la yesca. In fact, some authors think that the associated pathogens may be necessary for the further development of the syndrome of yesca in adult plants, where other species such as ' Fomitiporia mediterranea' are capable of producing infection and secondary rot in tissues previously parasitised by species associated with the disease of Petri (Graniti and col.)(, 2000).
Another disease which have recently been major outbreaks is caused by ' Cylindrocarpon spp. ' and known as 'black foot'. These fungi in soil are associated with basal protects, especially in young grape plants (Halleen et al., 2006), although they also affect plants of 2-10 years of age (Gubler et al., 2004) as set out in the vineyard. These species infectarían patterns during the process of rooting in the production of plant grafted nursery (Rego et al., 2001). The eutipiosis for its part, is attributed to the ascomiceto 'eutypa lata' and characterized by producing a delay in the development of the plant with rickets in the budding of the affected branches, which often is accompanied by chlorosis and foliar deformations (Carter)(, 1994).

Along with the previous ones, have been described other syndromes and agents pathogens associated with the vine which, to a greater or lesser extent, also have some relevance in the plant health status of cultivation at the national level. Among these is noteworthy for their special impact in certain areas the so-called 'dead arm' or 'black dead arm' (BDA), caused by a group of fungi belonging to the family Botryosphariaceae, including 'botryosphaeria', 'diplodia', 'fusicoccum', 'neofusicoccum' and 'Lasiodiplodia', of increasing importance in certain areas of cultivation around the world. The symptoms they produce is specific and similar to those already described for other diseases of wood, being able to confuse its manifestations with episodes of yesca and/or eutipiosis. In advanced stages, it is frequent to observe complete leaf desecaciones accompanied by involvement in inflorescences and fruits already formed. On the outside of the stem appear necrotic areas where the crust separates easily (Lecomte et al., 2005).

A sectoral necrosis Brown and 'V'-shaped looms in cross sections. In addition, (Van Niekerk et al., 2006) can be observed pits and necrosis around the spinal cord central cylinder.

In addition, in recent years have been isolated and identified a number of taxa from vines whose pathogenic potential and involvement in the previously described pathologies is yet to decide. Among these, include some species of the genus 'phaeoacremonium' (' p. krajdenii', ' p. parasiticum', ' p. subulatum', ' p. venezuelense' and 'P. wine'), 'phialemonium' (' p. curvatum') or 'cadophora' ('C. luteo-olivacea') (Halleen and col., 2007;) Mostert and col., 2006, etc.). Finally, there is described in the literature other pathologies associated with decay of vines of certain cultivars, such as the so-called 'Syrah Decline', observed since the beginning of the 1990s in many producing areas worldwide, and characterized by swelling and rupture of the area of graft, with the presence of deep furrows, rapid leaf discoloration and death of the plant after a period of 1-4 years (Batanny et al., 2004).

Control methods

Traditionally the control of diseases of Grapevine wood degradation has been basically on two fronts: the cultural practices and fungicide treatments. In Spain, the most ancient method of fight against la yesca of the vine, which was to make a cut in the trunk of the strain and keep it open through the introduction of a stone, was almost completely replaced by the application of sodium arsenitewhich lasted for decades. Sodium arsenite is a highly toxic chemical, mutagenic and carcinogenic. However, it is the only product that could eliminate the external symptoms of the disease, its use and abuse became widespread in the Spanish field. A single treatment after pruning and budding of buds, repeated on a cycle of 2-3 years, sufficient to control the disease (of the Rivero and García Marí, 1984). Due to their extreme danger, both for the environment and for the man, sodium arsenite is banned in Europe in 1991 and Spain, France and Portugal in 2003.

The control currently in phase of implementation or development techniques include the following:

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- Obtaining of material of propagation and planting healthy: the material of propagation is one of the main routes of spread of these diseases. The use of thermotherapy with hot, but with mixed results has been tested under nursery conditions. Some authors (Crous and col., 2001;) (Fourie and Halleen, 2004) point out that dives of the material to 50 ° C could be effective in the Elimination of ' Pa chlamydospora' and ' Phaeoacremonium spp. ' However, other studies have shown that both genders can withstand the application of 51 ° C for 30 minutes (Whiting et al., 2001). In addition, thermotherapy treatments have been ineffective with a series of varidades of grape (e.g. Pinot Noir, Cabernet-Sauvignon, etc.) (Rooney and Gubler, 2001). Currently evaluates the increase of temperature in immersion treatments to enhance the effectiveness of the same thickness and col. (2009a).
- Recognition of natural resistance in the plant host: knowledge of the susceptibility of the plant material to infection by the aetiologic agents is still scarce and limited to a small part of the involved fungi. In the case of the disease of Petri, Eskalen et al. (2001) conducted a study of sensitivity to ' Pa chlamydospora' and 'phaeoacremonium' several species in 20 different rootstocks, finding no resistance, although Yes differences in the degree of sensitivity to pathogens.
- Reducing the burden of inoculum in the field: currently under investigation on the role of soil in the dynamics of these diseases to assess the possibilities of its transmission in it, or the capacity to host reservoirs of inoculum which can remain viable and produce new cycles of infection. In the case of the disease of Petri, previous studies (Scheck and col., 1998;) (Khan et al., 1999) have shown that these taxa can act as pathogens from soil in artificial inoculations, and even some of them (e.g. ' p inflatipes') have been isolated from natural vineyard soils. Despite the above, no known experiences the effects of treatments of disinfection of the soil in these populations.
- Control measures based on cultural practices: proven that pruning is a practice which considerably influences the development of diseases of Grapevine. The wounds produced therein constitute potential routes of entry for some of the pathogens involved. Some authors advise late pruning, double pruning or surgical pruning techniques able to limit the damage by eutipiosis (Herche and Gubler, 2010;) (Sosnowski et al., 2010). The protection of the natural routes of entry by means of chemical substances with thiophanate-methyl or myclobutanyl show promising results (Herche and Gubler, 2010). Some studies point to the desirability that pruning is carried out in dry weather, as in the case of ' p. chlamydospora' has been observed a higher rate of infection when pruning is carried out under conditions of rain. Other authors (Mugnai et al., 1999) claim that one of the measures which may allow the reduction in the level of inoculum in the vineyards, is the removal and destruction of plant remains of dead plants and pruning.
In recent years, as a substitute for sodium arsenite, alternative chemicals, such as dinitrorthocresol, fenarimol, furmentamide, benodamil, fosetyl - are evaluated to the and several triazole
- Chemical control: in recent years, as a substitute for sodium arsenite, alternative chemicals, such as dinitrorthocresol, fenarimol, furmentamide, benodamil, fosetyl - are evaluated to the and several triazole (Mugnai and col., 1999;) (Jaspers, 2001). They have been tested in field, on diseased vines, but have not been fully effective (Di Marco et al., 1997), at least at the levels achieved with arsenite. On the other hand, in experiments carried out in nurseries with young plants, have been achieved acceptable results with foliar applications of fosetil-, not noting later foliar symptoms in treated plants. In the absence of convincing answers, are being applied to the cultivation in excessive form different products (usually phosphorus derivatives and lignatos with aluminium) nutritional or invigorating, with mediocre or negative results. On the other hand, are tested chemical commercial formulations for use as a disinfectant in the processes of handling of seedlings of vine in the nursery, although the works are still in phase of experimentation and are not ready for transfer to the production sector. The methods tested with more promising results include the immersion of the stakes during the phase of moisturizing with fungicides carbendazima and didecil-dimethyl-ammonium (Grammage et al, 2009b).
- Biocontrol: currently there is no any micro-organism at an advanced stage of development (registry or exploitation of patent) as a potential agent of control of diseases of Grapevine. Search and development of antagonists from the microbiological equity of the plant, it is innovative in the context of vine diseases, because knowledge about the possibilities of introducing alternative control methods from microbial antagonists, have been based largely on the use of micro-organisms or substances derived from them, of commercial origin, belong almost entirely to the genus 'trichoderma' (Harvey and Hunt, 2006;) (Di Marco and Osti, 2007). For this reason, current research focus on prospecting, isolation and characterization of species of fungal Endophytes associated with vine, to assess their location and possibilities of development and introduction into the agricultural market of new agents for biological control of fungal diseases of Grapevine.

In summary, the current research for an integrated control system that increases the chances of success at the time that minimize environmental risks. In this sense, in the Imidra our research focuses on three aspects: the etiology of the disease, identification of endophyte vid agencies as potential agents of biocontrol and the effectiveness of fungicides in applications to soil substances.

Methodology

- Etiology of diseases of the wood of Grapevine in the zone Centre: during the last 10 years carry out systematic surveys in 25 commercial vineyards in 12 locations of the three sub-areas of the D.O. wines of Madrid and Toledo. In each plot vines were identified with symptoms of diseases of degradation of wood, both foliar symptoms such as necrosis and pudriciones in outbreaks, arms, and trunk. In selected plants were sampling repeated periodically, at least in three seasons of the year: winter, spring and summer. Prospectados vineyards are a cross-section of Madrid viticulture and the central part of Spain, because they include a wide range of climatic and terrain conditions, age of the plants (from 2 to 100 years), systems of cultivation (low vase and trellis system) and commercial varieties.

Once in the laboratory, samples of wood are seccionaron longitudinal and transversely and informative data on the type and aspect of the present pudriciones, according to a definite scale were registered in them (Mugnai et al. 1999, Redondo 2003). Some subsamples are incubated in moist Chamber to promote the development of fungal structures and necrotic areas of other pieces of tissue of 2 cm3 which was sterilized superficially were extracted using sodium hypochlorite solutions (4-5%) and/or with ethanolfollowed by washing with sterile water. After this, were planted in the synthetic means of cultivation, agar-potato-dextrose (PDA) and agar-malta (MEA), supplemented with antibiotics to promote growth of fungal colonies and limit the bacterial contamination, and they are incubated at 22-25 ° C in darkness. The plates were regularly every 2-3 days to detect the development of fungal colonies. For its isolation and purification, the emerging colonies were transferred to means of maintenance (PDA, MEA, etc.).

The taxonomic identification of fungal species were used to specific identification keys for each group of fungi, based mainly on morphological criteria and morphometric, and in some cases, applied molecular techniques based on the direct sequencing of specific regions of DNA (region ribosomal).

- Search and characterization of new biocontrol agents for their use against pathogens of vine wood: samples of plant material is carried out in vineyards distributed throughout the community of Madrid following the guidelines outlined in the previous section, with the caveat that this study Lakes both asymptomatic and symptomatic plants, and on each floor, trunks, shoots and leaves. After the selection of plants and various tissues and parts of interest extraction, they were tagged in the field and identified (variety, rootstock, age, etc.), and its position fixed by means of a system of global positioning (GPS).

Plant laboratory materials were processed in a manner similar to the one described in the previous point. The preliminary identifications of the isolates obtained and maintained in pure culture, were performed using taxonomic determination of classical techniques, using studies of comparative morphology of the different taxa. To obtain one sufficient number of diagnostic characters in each case, were employed in addition techniques of microscopy and Microbiology (study of growth at different temperatures, different synthetic media, etc.). Taxonomic determinations were made from the available and up-to-date bibliography for each body or groups of organisms (monographs, studies, etc.). The molecular characterisation was carried out for those taxa in which the presence of fruiting (sexual/asexual) was not obvious, or those Endophytes where pure crops did not show sufficient diagnostic characters. The molecular identification based on ITS1 and ITS2 regions nucleotide sequences, obtained and analyzed according to the methods described by Sánchez-Torres et al. (2008). Sequences of the ITS region IDs were by comparison with the 'Fasta' application to public GenBank sequence database.

-Evaluation of fungicidas commercial of application to the floor for the control of 'Phaeomoniella chlamydospora': it tested the efficiency of two active matters with effect fungicida and suitable formulation for his direct application to the floor: himexazol (dicarboximida) and tiram (ditiocarbamato). Both products applied to the concentrations recommended by the manufacturer (1.600 ppm for tiram and 360 ppm for the himexazol), in an alone application, to plants injertadas \1CV\2 Tempranillo/ 140-Ru) of 6 months of age, cultivated in plant pots of 4 l in conditions of invernadero (Tª half= 30±2 °C). Like sustrato used a mix of natural floor and perlita (5:1 v/v), that previously checked was free of the responsible pathogens of the illnesses of Petri and black foot. Two days after the treatment fungicida, the floor inoculated artificially with a suspension of conidios of 5 isolated of 'Pa. chlamydospora' To two concentrations (106 and 102 with/mL). To the cape of 2 months the plants extracted of the plant pots, seccionaron longitudinalmente and measured in them the length of the necrosis vascular ascendants from the base of the estaquilla. The variables analysed estadísticamente were the incidence (number of plants sintomáticas) and the gravity of the illness (length of the necrosis vascular).

Results and discussion

-Etiology of diseases of the wood of Grapevine in the zone Center: fungal agents related to necrosis or vine wood protects isolated from the plant material collected in the sampling of the center of Spain pointsas well as their frequency of isolation, they are shown in Figure 1. She follows the high presence of the genres that encompass the main pathogenic species involved in diseases of wood of Grapevine, such as yesca, Petri, blackroot or dead arm disease. So, are representatives of the genera 'phaeoacremonium', 'phaeomoniella', 'fomitiporia' and 'Curranii', along with those of the Group of the Botryosphaeriaceae more regularly. The first two genera have been widely cited and isolated in the whole of Spanish wine-growing areas, including producing nurseries, and both of young plants as adult vineyards and in almost all the varieties and forms of cultivation prospectadas (Tello and col.)2010; (Martin and Cobos, 2007). Our results are consistent with similar research carried out in other Spanish and international wine-growing regions (Rego et al, 2000, Armengol et al, 2001, Gubler et al. 2004;) Hallen et al. 2006; Van Niekerk et al. 2006; (Martin and Cobos, 2007), with the exception of that in our case we find Fomitiporia mediterranea with a high frequency, as opposed to what seems to occur in Castilla y León (Martín Cobos, 2007).
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Figure 1: Frequency of isolation of the main fungal species associated with the wood of vines in the area pudriciones Center of Spain during 1999-2009.
Also noted the low representation of the basidiomiceto ' Stereum hirsutum', traditionally associated with la yesca of vine, who was preferably isolated in the trunk of the strain, and whose involvement in the process of the disease is currently in question. However, Sánchez-Torres et al. (2008) noted that, in the Valencian Community, ' Stereum hirsutum' showed a frequency of isolation high in affected vineyards of yesca, even beyond the observed for 'fomitiporia'. In addition, Gonzalez et al. (2008) reported that under certain conditions of cultivation in the Mediterranean coastal zone d. mediterranea was replaced by another lignícola species of the genus 'inonotus' (' i. hispid ').

On the other hand, emphasizes the absence of representatives of the genus 'eutypa'. This result corresponds with the obtained recently by other authors, who points out that 'eutypa' seems to be a predominant pathogen in the wine-growing regions more humid and cold, while the representatives of the Group of the Botryosphaeriaceae prefer warm and dry climates (Martin and Cobos)2007; (Muruamendiaraz et al., 2009). Thus, the North of Spain (Rioja alavesa), South and center of France and North of Italy are areas in which the eutipiosis is common, while in Catalonia, Valencian Community and area Spain Center, dominated by the pudriciones produced by Botryosphaera and related genera.

The isolates were selectively depending on the type of rot that featured wood (Redondo, 2003). These studies follows a certain specialization, so that the clear and soft pudriciones would correspond with the massive isolation of the basidiomiceto ' Fomitiporia mediterranea'. The necrotic pits in vessels are related to the fungus ' Phaeomoniella chlamydospora'. The dark and harsh necrosis would preferably correspond to the 'Phaeoacremonium' and the Botryosphaerias, while the pink and dark that appeared with preference in the basal part of the plant would correspond to 'Curranii'. ' Phaeoacremonium aleophilum' was isolated with high frequency mainly associated with brown or pardo-rosáceas necrosis. These results are consistent with the other authors (Mugnai et al., 1999). The younger vines predominate pathogens ' Pa. chlamydospora', ' WP. aleophilum' and ' Cylindrocarpon spp. ', while the basidiomycete fungi ' Fomitiporia mediterranea' and ' Stereum hirsutum' are associated to the older strains.
- Search and characterization of new biocontrol agents for their use against pathogens of vine wood: some preliminary results of this line of research for the control of diseases during the period 2008-2009, have shown a high degree of fungal diversity in the interior of the tissues of vinesexpressed both in terms of the number of isolated and in the composition of species of fungi. As well as for some of the few studies on micoflora vid endofítica made in other countries (Halleen and col., 2007;) Casieri and col., 2009, etc.), our work shows that among ascomycetous fungi and fungus mitospóricos represent the main contingent of endófitas species associated with the crop (table 1). In General, the abundance and species composition does not differ significantly between the different types of wine varieties. Finally, and as expected, some of the isolated genres are referred usually in literature as biocontrol agents, which makes them potential candidates for studies on microbial antagonism. These include strains belonging to the genera 'acremonium' and 'phoma' (' p. glomerata') or 'chaetomium' (' ch. globosum'), who have shown potential activity in previous studies in the laboratory.
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Table 1: endófitas fungal species isolated from plants of vine from six of the community of Madrid-producing areas included in the appellation 'Wines of Madrid'.
- Evaluation of commercial fungicides of implementation on the ground for the control of ' Phaeomoniella chlamydospora': plants in greenhouse conditions tests show that the himexazol was not effective to the dose recommended by the manufacturer. On the contrary, the tiram managed to significantly reduce both the incidence and the severity of the disease to the dose recommended by the manufacturer and only an application of the fungicide to the ground. The inoculated fungus was reaislado of the knitted vascular necrotic. Our results suggest that preventative tiram applications to the soil or substrate can reduce primary inoculum of the pathogen, delay effects and, in combination with other measures, to contribute to their control.

Conclusions

The situation in Spain is similar to the one described in other producing countries, exist in the past 15 years a progressive increase in the extent and severity of diseases of wood of Grapevine of fungal origin. Worldwide research groups addressing his study, mainly its incidence, extent and etiology. Such studies have enabled remarkable progress in the knowledge of these pathologies in a short time. Currently, no method of control is totally effective to control degradation of the vine diseases, so that research in this field continues to be essential.
The situation in Spain is similar to the one described in other producing countries, exist in the past 15 years a progressive increase in the extent and severity of diseases of vine of fungal origin wood
The Group of plant pathology of Imidra carried out trials to check the effectiveness of some substances of chemical origin, as an alternative to the use of sodium arsenite, for application both the soil and plants nursery. At the same time he studied characterization of the biodiversity of fungal Endophytes associated with different varieties and types of cultivation, with the aim of identifying new antagonist agents for the control. With these lines of research, in which the group is a pioneer at the national level, is developing alternative or complementary methods of control in wine, in order to reduce the economic losses caused by these pathogens in the productive sectorusing techniques consistent with the preservation of the natural environment and the regulations of the European Union.
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