Giuseppe Surico

Esca (Black Measles) and Brown Wood-Streaking: Two Old and Elusive Diseases of Grapevines

“Esca” disease of grapevine has long been known wherever grapes are grown. The disease may be as old as vine cultivation itself. References to esca-like symptoms are found in several ancient Greek and Latin works. Greater descriptive accuracy is found in medieval works such as the Kitab al-Felahah by Ibn al-Awam, a Spanish Muslim who lived in Seville at the end of the twelfth century, and the Opus Ruralium Commodorum by Pietro de’ Crescenzi, born around 1233 in Bologna. Esca is a Latin word meaning food, aliment, or, figuratively, bait. The name indirectly refers to the fruiting bodies of certain wood-rotting fungi. For example, Fomes, which in Latin means “tinder,” is the name of a genus of basidiomycetes once used to make a dry, easily ignited material suitable to start fires when using flints. Wood decayed by these fungi, including rotted grapevine wood, burned slowly and was used to keep fires aglow without a flame. Esca is a complex disease that comprises an array of symptoms, some of which have locally given their own names to the disease. For example in California, the dark, tiny spotting of the grapes is called “black measles.” In many grapevine-growing areas of the world, the sudden wilting of esca-affected vines in summer has earned this form of the disease the name “apoplexy.” Research on the etiology of esca, which started at the end of the nineteenth century in France, can be divided into three periods. The first period began in 1898 with Ravaz (61) and ended in 1926 with Viala (79). The overall conclusion of this period was that two basidiomycetous fungi, Stereum hirsutum (Willd.) Pers. and Phellinus (Fomes) igniarius (L.:Fr.) Quél., were the causal agents, although this could not be shown by pathogenicity tests. However, in Italy in 1912, Petri (58) successfully reproduced some early internal esca symptoms with two undetermined species of Cephalosporium and one of Acremonium that had also been associated with the disease. The second period in esca research started in California in 1957 with Hewitt (35) and lasted until 1959, when Chiarappa (9) detected the relationship between internal wood decay and black measles. He also showed how a Cephalosporium sp. reproduced in vivo some of the symptoms observed in the wood of diseased vines, and how P. igniarius caused wood decay in vitro. The third period, initiated by Larignon and Dubos in 1987 (39) and still underway, is directed at understanding the role of the mitosporic (asexually reproducing) fungi that act alone or together with the basidiomycetes to cause esca and related diseases. In the 1990s, studies on esca and its etiology have intensified. This came after a dramatic upsurge in the disease, especially in Germany, Italy, and Greece, where the arsenites, traditionally used to keep esca under control, have been banned and were replaced by less effective fungicides. However, even in France, Portugal, and Spain, where restricted use of sodium arsenite is still permitted, esca is widespread in all vine-growing regions. This paper refers mostly to conditions in the Mediterranean area and focuses on the progress achieved in our understanding of esca over the last few years. Reference is also made to a decline of young grapevines caused by some of the same mitosporic fungi as those commonly associated with esca in adult vines. Finally, the relations between esca and related syndromes or diseases are also discussed.

Fluctuation of bacteria isolated from elm tissues during different seasons and from different plant organs

In this work we isolated a culturable endophytic aerobic heterotrophic bacterial community from the stem and root tissues of elm trees (Ulmus spp.) and analyzed its fluctuations. A total of 724 bacterial isolates were collected at different times (April, June, September and December) from two elm trees, one infected with Elm Yellows phytoplasmas, and one which was healthy-looking. The isolates were grouped into 82 haplotypes, identified by means of amplified ribosomal DNA restriction analysis (ARDRA) using the restriction enzyme AluI, suggesting that the genetic diversity of the bacterial community was very high. The taxonomic position of the isolates belonging to the twelve main haplotypes, representing more than 72% of the total population, was determined by 16S rDNA sequencing. The main genera were Bacillus, Curtobacterium, Pseudomonas, Stenotrophomonas, Sphingomonas, Enterobacter, and Staphylococcus. The fluctuations in the bacterial community, determined by different parameters (seasonal changes, plant organ, presence of phytoplasmas) were studied, revealing that they were influenced both by variations in temperature (warm or cold according to the season) and by the organ examined (roots or stems). The role of the phytopathogenic status in these fluctuations was also discussed.

Phytotoxins Produced by Fungi Associated with Grapevine Trunk Diseases

Up to 60 species of fungi in the Botryosphaeriaceae family, genera Cadophora, Cryptovalsa, Cylindrocarpon, Diatrype, Diatrypella, Eutypa, Eutypella, Fomitiporella, Fomitiporia, Inocutis, Phaeoacremonium and Phaeomoniella have been isolated from decline-affected grapevines all around the World. The main grapevine trunk diseases of mature vines are Eutypa dieback, the esca complex and cankers caused by the Botryospheriaceae, while in young vines the main diseases are Petri and black foot diseases. To understand the mechanism of these decline-associated diseases and the symptoms associated with them, the toxins produced by the pathogens involved in these diseases were isolated and characterised chemically and biologically. So far the toxins of only a small number of these decline fungi have been studied. This paper presents an overview of the toxins produced by the most serious of these vine wood pathogens: Eutypa lata, Phaeomoniella chlamydospora, Phaeoacremonium aleophilum and some taxa in the Botryosphaeriaceae family, and examines how these toxins produce decline symptoms. The chemical structure of these metabolites and in some cases their vivotoxin nature are also discussed.

Older and more recent observations on esca: a critical overview

The main research findings of studies in esca carried out over the last few years in a number of laboratories are discussed. The topics include: the disease model; the relationship between black dead arm and esca; the role of toxins and xylem cavitation in the development of leaf symptoms; the artificial reproduction of leaf symptoms and wood rot; the inoculum sources of the fungi Phaeomoniella chlamydospora, Phaeoacremonium aleophilum and Fomitiporia mediterranea, and the influence of climate on the incidence of esca. The purpose was to sum up the current state of esca research and where possible to present new suggestions and sometimes alternatives to the suggestions of other researchers. The ultimate aim was to draw attention to those questions in esca research that are open to differing and even opposing explanations because the hard data are still lacking.

Sequence analysis of ITS ribosomal DNA in five Phaeoacremonium species and development of a PCR-based assay for the detection of P. chlamydosporum and P. aleophilum in grapevine tissue

Phaeoacremonium aleophilum and P. chlamydosporum are two recently described mitosporic fungi that are involved in the development of symptoms of esca disease and of a decline of young grapevines previously named “black goo”. The Internal Transcribed Spacers (ITS) 1 and 2, plus the interveining 5.8S gene, of ribosomal DNA (rDNA) of representative isolates of the two species and, for comparison, of isolates of the congeneric species P. angustius, P. inflatipes and P. rubrigenum were amplified by Polymerase Chain Reaction (PCR) using the ITS4 and ITS5 universal primers. The size of the entire ITS region (ITS1-5.8S-ITS2), plus the 3’ end of 18S rDNA and the 5’ end of 28S rDNA, was estimated to be about 620 bp, on gel electrophoresis, for all the Phaeacremonium species tested. Eleven restriction enzymes were used singularly in the digestion of the ITS region of 30 isolates of P. chlamydosporum, 16 of P. aleophilum, 2 of P. angustius, 2 of P. inflatipes and 1 of P. rubrigenum. No length-polymorphism could be detected within species (except for P. aleophilum), but there were quite strong differences between species. The PCR products of ITS region of ten representative isolates for the five Phaeoacremonium species were sequenced, and the sequences aligned and compared. Two main groups were clearly distinguishable, one formed by P. chlamydosporum, and the other by P. aleophilum, P. angustius, P. inflatipes and P. rubrigenum, with an homology between the two groups ranging from 64.5% to 66.5%. The sequences of ITS region were used to design two pairs of primers, Pal1N/ Pal2 and Pch1/Pch2, each of which was subsequently shown to be specific for the amplification of predicted-size fragments from genomic DNA of P. aleophilum and P. chlamydosporum, respectively. The identity of the amplified fragments was confirmed by sequencing. The primer pairs were further tested using as template DNA extracted from healthy grapevines and from other fungi commonly isolated from esca-diseased grapevine plants but no amplification was observed. The PCR protocol was shown to be quite sensitive (10 pg of DNA) and able to specifically detect P. chlamydosporum and P. aleophilum in artificially inoculated grapevine plants.

Epidemiology of esca in some vineyards in Tuscany (Italy).

The incidence of esca in 5 vineyards (SCFI, GTFI, CBSI-1, 2, 3) in Tuscany was monitored from 1992 to 1994 in SCFI, from 1993 to 1998 in GTFI, and from 1995 to 1998 in CBSI-1, 2 and 3. Disease incidence varied from vineyard to vineyard and from year to year. SCFI was the vineyard most affected with an incidence of 63% in 1993, 20% in 1994, and 46% in 1992. However, symptom expression in individual plants was characterized by very marked discontinuity from year to year. As a result, only 8% of diseased plants in SCFI had foliar symptoms in all 3 years of the survey period. The percentage of consistently diseased plants went down to less than 1% in GTFI and CBSI-3 and was nil in CBSI-1 and 2. The cumulative disease incidence, calculated by counting all the plants that showed foliar symptoms at least once in the years surveyed, was 82, 50, 19, 12, and 10% in SCFI, GTFI, CBSI-3, 1 and 2 respectively. An examination of rainfall and air temperature parameters in relation to esca incidence did not detect any weather conditions especially conducive to esca. However, it seemed that a fresh, rainy summer is more favorable to the chronic form of esca while a hot, dry summer is more conducive to the acute form.

A cytokinin from the culture filtrate of Pseudomonas syringae pv. savastanoi

Abstract The structure of a new cytokinin, isolated from the culture filtrate of Pseudomonas syringae pv. savastanoi, is assigned on the basis of spectroscopic data including its tetracetyl derivative and comparison with related adenine derivatives. It was identified as 6-(4-hydroxy-1,3-dimethylbut-trans-2-enylamino-9-β- D -ribofuranosyl)purine.

Pseudomonas syringae and related pathogens

Since the early 1990s, bacterial blight has emerged as an important disease of cantaloupe in France, particularly in the south-west but has caused epidemics in all the production regions of this country. The pathogen is a complex composed of strains identified as Pseudomonas syringae pv. aptata (70% of the strains) and the remaining resembling P. viridijlava (2%), P. s. pv. lachrymans (1%) or P. syringae in general but with no correspondence to a known pathovar. To investigate the epidemiology of this disease and to develop disease control strategies, we have attempted to identify sources of inoculum in the field, factors contributing to the explosive development of the disease, resistance of the bacterium to copper pesticides, and sources of disease resistance in Cucumis melo. Based on our observations and on the description of possible outbreaks of this disease elsewhere in the world, we report here a hypothetical scenario of the critical factors triggering disease development and of the potential efficiency of different control strategies.

CHEMICAL STRUCTURE OF TWO PHYTOTOXIC EXOPOLYSACCHARIDES PRODUCED BY PHOMOPSIS FOENICULI

The two main exocellular polysaccharides produced in vitro by Phomopsis foeniculi, a fungal pathogen of fennel, were isolated and characterized by chemical and spectroscopic methods as a galactan with the known structure [-->6)-beta-D-Galf-(1-->5)-beta-D- Galf-(1-->5)-beta-D-Galf-(1-->]n and a mannan. The latter consists of a backbone of alpha-(1-->6)-linked mannopyranose units. Almost all of these are branched at the 2 position with arms containing 2- and 3-linked mannopyranose units. The crude polysaccharide fraction and its components, galactan and mannan, showed phytotoxic effects, i.e. chlorosis, necrosis and/or wilting, on fennel and on two non-host plants, tobacco and tomato.

Phaeomoniella chlamydospora-Grapevine Interaction : Histochemical Reactions to Fungal Infection

Histochemical reactions of one-year-old potted micropropagated vines (rootstock 1103 Paulsen) to inoculation with Phaeomoniella chlamydospora were studied. Microscopic examination of the vine wood showed that the fungus spread through the wood tissue, albeit slowly. Starting from the roots it required nine months to colonise the first 20-25 cm of the grapevine stems. The slow spread of P. chlamydospora is thought to be due to the defence response initiated by the vines: production of tyloses, including accumulation of phenols in the vessels and adjacent tissue, and the deposition of unidentified defence-response substances, probably stilbene-like substances from the cell-wall surrounding the infection