Identification of phytotoxins from Botryosphaeria obtusa , a pathogen of black dead arm disease of grapevine

A bioassay-guided fractionation of a culture filtrate of Botryosphaeria obtusa led to the isolation of four dihydroisocoumarins, named mellein 1, 4-hydroxymellein 2, 7-hydroxymellein 3 and the new 4,7-dihydroxymellein 4. LC-UV-DAD-MS analysis of vine wood infected by B. obtusa revealed the presence of mellein (1). Botryosphaeria obtusa was also able to oxidise wood δ -resveratrol into the dimer delta-viniferin. The structures of isolated phytotoxins were established on the basis of IR, MS, 1D and 2D NM

Preliminary Studies on the Biology of «Phaeoacremonium»

Comparative microflora studies of one-year-old pruned and unpruned canes carried out during dormancy period over three consecutive years showed that Phaeoacremonium chlamydosporum was more frequently isolated from pruned than unpruned canes, while Phaeoacremonium aleophilum was found as much in pruned as in unpruned canes. Inoculations of pruning wounds made in two consecutive winters by suspensions of conidia of P. chlamydosporum and P. aleophilum showed that both fungi were able to invade through wounds during winter. Infections varied with pruning date and age of pruning wound, but were more serious and of longer duration with early pruning (December, January). Trapping studies showed that spores of P. chlamydosporum were captured throughout the whole year, while spores of P. aleophilum were mostly trapped during the vegetative period. Microscopic examination of adhesive tape applied to without bark zones of the canes showed the presence of conidia of P. aleophilum. From these studies, it emerged that only P. chlamydosporum is able to contaminate pruning wounds during winter. P. chlamydosporum and P. aleophilum may be propagated by infected canes in the nursery. They can be considered airborne fungi during a period of their biological cycle

Observation on Black Dead Arm in French Vineyards

Black dead arm was first identified in French vineyards in 1999. It produces symptoms in the vine wood, causing decline and eventually death. Previously, this disease was confused with esca because of the similarity of the foliar symptoms. Two forms of black dead arm, a mild and a severe form, were observed in French vineyards on Cabernet Sauvignon and Sauvignon. Isolations from 23 vines collected from four vineyards located in three viticultural districts of Bordeaux showed that the main fungi associated with the brown streaks were Botryosphaeria obtusa, B. dothidea and Trichoderma spp. Pathogenicity tests showed that B. obtusa and B. dothidea caused dark lesions on 1- year-old canes. Therefore, these two fungi merit further study to determine their role in causing black dead arm

Characterization of Neopestalotiopsis, Pestalotiopsis and Truncatella species associated with grapevine trunk diseases in France

Pestalotioid fungi associated with grapevine wood diseases in France are regularly found in vine growing regions, and research was conducted to identify these fungi. Many of these taxa are morphologically indistinguishable, but sequence data can resolve the cryptic species in the group. Thirty pestalotioid fungi were isolated from infected grapevines from seven field sites and seven diseased grapevine varieties in France. Analysis of internal transcribed spacer (ITS), partial β-tubulin (TUB) and partial translation elongation factor 1-alpha (TEF) sequence data revealed several species of Neopestalotiopsis, Pestalotiopsis and Truncatella associated with the symptoms. Three Neopestalotiopsis spp. and one Pestalotiopsis sp. are reported for the first time associated with wood diseases in grapevine in France and worldwide, and include Neopestalotiopsis asiatica, N. javaensis, Neopestalotiopsis sp. and Pestalotiopsis biciliata. The sequence data indicate that Truncatella angustata was also associated with wood grapevine diseases in France; this species was previously reported on grapevine in Iran.  The importance of controlling Pestalotioid fungi associated with wood grapevine diseases is discussed, which was previously considered of minor importance. These fungi are isolated from plants in nurseries before marketing

Extracellular compounds produced by fungi associated with Botryosphaeria dieback induce differential defence gene expression patterns and necrosis in Vitis vinifera cv. Chardonnay cells

Three major grapevine trunk diseases, esca, botryosphaeria dieback and eutypa dieback, pose important economic problems for vineyards worldwide, and currently, no efficient treatment is available to control these diseases. The different fungi associated with grapevine trunk diseases can be isolated in the necrotic wood, but not in the symptomatic leaves. Other factors seem to be responsible for the foliar symptoms and may represent the link between wood and foliar symptoms. One hypothesis is that the extracellular compounds produced by the fungi associated with grapevine trunk diseases are responsible for pathogenicity. In the present work, we used Vitis vinifera cv. Chardonnay cells to test the aggressiveness of total extracellular compounds produced by Diplodia seriata and Neofusicoccum parvum, two causal agents associated with botryosphaeria dieback. Additionally, the toxicity of purified mellein, a characteristic toxin present in the extracellular compounds of Botryosphaeriaceae, was assessed. Our results show that the total extracellular compounds produced by N. parvum induce more necrosis on Chardonnay calli and induce a different defence gene expression pattern than those of D. seriata. Mellein was produced by both fungi in amounts proportional to its aggressiveness. However, when purified mellein was added to the culture medium of calli, only a delayed necrosis and a lower-level expression of defence genes were observed. Extracellular compounds seem to be involved in the pathogenicity of the fungi associated with botryosphaeria dieback. However, the doses of mellein used in this study are 100 times higher than those found in the liquid fungal cultures: therefore, the possible function of this toxin is discussed

Phytotoxic metabolites from Neofusicoccum parvum, a pathogen of Botryosphaeria dieback of grapevine

Liquid chromatography-diode array screening of the organic extract of the cultures of 13 isolates of the fungus Neofusicoccum parvum, the main causal agent of botryosphaeria dieback of grapevine, showed similar metabolites. One strain was selected for further chemical studies and led to the isolation and characterisation of 13 metabolites. Structures were elucidated through spectroscopic analyses, including one- and two-dimensional NMR and mass spectrometry, and through comparison to literature data. The isolated compounds belong to four different chemical families: five metabolites, namely, ( )-terremutin (1), (+)-terremutin hydrate (2), (+)-epi-sphaeropsidone (3) ( )-4-chloro-terremutin hydrate (4) and(+)-4- hydroxysuccinate-terremutin hydrate (5), belong to the family of dihydrotoluquinones; two metabolites, namely, (6S,7R) asperlin (6) and (6R,7S)-dia-asperlin (7), belong to the family of epoxylactones; four metabolites, namely, (R)-( )-mellein (8), (3R,4R)-4-hydroxymellein (9), (3R,4S)-4-hydroxymellein (10) (R)( )-3-hydroxymellein (11), belong to the family of dihydroisocoumarins; and two of the metabolites, namely, 6-methyl-salicylic acid (12) and 2-hydroxypropyl salicylic acid (13), belong to the family of hydroxybenzoic acids. We determined the phytotoxic activity of the isolated metabolites through a leaf disc assay and the expression of defence-related genes in Vitis vinifera cells cv. Chardonnay cultured with ( )-terremutin (1), the most abundant metabolite. Finally, analysis of the brown stripes of grapevine wood from plants showing botryosphaeria dieback symptoms revealed the presence of two of the isolated phytotoxinsinfo:eu-repo/semantics/publishedVersio

Management of grapevine trunk diseases: knowledge transfer, current strategies and innovative strategies adopted in Europe

Since the early 1990s, grapevine trunk diseases (GTDs) have posed threats for viticulture. Esca complex, Eutypa- and Botryosphaeria- diebacks, mostly detected in adult vineyards, are currently responsible for considerable economic losses in the main vine-growing areas of the world. Other GTDs, such as Petri- (Esca complex) and Black-foot diseases, are emerging problems in grapevine nurseries (resulting in grafting failures and/or loss of saleable plants) and in young vineyards. The impacts of GTDs in modern viticulture depend on several factors, some related to their complexity, and others linked to host plant characteristics, changes in vineyard management and to the scarcity of simple tools for their control. For these reasons control of GTDs remains difficult, also depending on knowledge transfer from research to field and vice versa. This paper outlines the main preventive and curative techniques currently applied, scientifically tested or not that have resulted from the outcomes of “Winetwork”, a European Union funded project with special emphasis on the promising and innovative approaches.