Pascal Lecomte

Phenotypic Differences Between vacuma and transposa subpopulations of Botrytis cinerea

One hundred and twenty-one single-spore strains of Botrytis cinerea isolated from Bordeaux vineyards were molecularly characterized as either transposa or vacuma, two subpopulations of B. cinerea distinguished by the presence of transposable elements. Forty-three vacuma and 68 transposa strains were distributed into two main classes (mycelial or sclerotial) by morphological phenotype according to the organ of origin. Strains isolated from overwintering sclerotia produced exclusively sclerotial colonies. The mycelial growth rate of 21 transposa and 13 vacuma strains was significantly influenced by agar-medium and temperature. The mycelial growth rate was significantly strain-dependent at favourable temperatures (15, 20 and 25 °C), but not at limiting ones (5 and 28 °C): vacuma strains showed the fastest growth rates. The strains of the two subpopulations were similar in virulence on both host species tested (Vitis vinifera and Nicotiana clevelandii). The grapevine leaves were significantly more susceptible to B. cinerea than those of tobacco. A significant negative correlation was established between virulence and mycelial growth rate. The epidemiological consequences concerning population structure of B. cinerea in vineyards are discussed.

PCR Assays That Identify the Grapevine Dieback Fungus Eutypa lata

ABSTRACT Eutypa lata is the causal fungal agent ofEutypa dieback, a serious grapevine necrotic disease. The erratic and delayed (1 to 2 months) appearance of characteristic conidia on culture media and the presence of numerous microorganisms in decaying wood make it difficult either to identify or to detectE. lata in grapevine wood samples. We designed six pairs of PCR primers for diagnosis of E. lata. Three primer pairs were derived from ribosomal DNA internal transcribed spacer sequences, and three pairs were derived from randomly amplified polymorphic DNA fragments. The six primer pairs could be used to amplify DNAs extracted from all of the E. lata isolates tested. They did not amplify DNAs from fungi and bacteria representing more than 50 different species of microorganisms associated with grapevine. We developed a simple protocol, leading to a rapid release of DNA, that enabled us to identify E. lata from pure or mixed cultures as well as from grapevine wood samples. Identification of E. lata in wood was achieved within a few hours, instead of the several weeks required for classical cultures on agar medium. We believe that the procedure described here can be adapted to detect other microorganisms involved in woody plant diseases.

A transcriptomic study of grapevine (Vitis vinifera cv. Cabernet-Sauvignon) interaction with the vascular ascomycete fungus Eutypa lata

Eutypa dieback is a vascular disease that may severely affect vineyards throughout the world. In the present work, microarrays were made in order (i) to improve our knowledge of grapevine (Vitis vinifera cv. Cabernet-Sauvignon) responses to Eutypa lata, the causal agent of Eutypa dieback; and (ii) to identify genes that may prevent symptom development. Qiagen/Operon grapevine microarrays comprising 14 500 probes were used to compare, under three experimental conditions (in vitro, in the greenhouse, and in the vineyard), foliar material of infected symptomatic plants (S+R+), infected asymptomatic plants (S–R+), and healthy plants (S–R–). These plants were characterized by symptom notation after natural (vineyard) or experimental (in vitro and greenhouse) infection, re-isolation of the fungus located in the lignified parts, and the formal identification of E. lata mycelium by PCR. Semi-quantitative real-time PCR experiments were run to confirm the expression of some genes of interest in response to E. lata. Their expression profiles were also studied in response to other grapevine pathogens (Erysiphe necator, Plasmopara viticola, and Botrytis cinerea). (i) Five functional categories of genes, that is those involved in metabolism, defence reactions, interaction with the environment, transport, and transcription, were up-regulated in S+R+ plants compared with S–R– plants. These genes, which cannot prevent infection and symptom development, are not specific since they were also up-regulated after infection by powdery mildew, downy mildew, and black rot. (ii) Most of the genes that may prevent symptom development are associated with the light phase of photosynthesis. This finding is discussed in the context of previous data on the mode of action of eutypin and the polypeptide fraction secreted by Eutypa.

Identification by PCR analysis on plasmid pEA29 of isolates of Erwinia amylovora responsible of an outbreak in Central Europe

A collection of 127 strains of Erwinia amylovora, the causative agent of fire blight, was tested by PCR amplification of a fragment of the plasmid pEA29. A variability in the length of the DNA fragment obtained was observed after digestion by MspI and Sau3A restriction enzymes. Strains were distributed into three groups according to the length of the DNA product. Most of the strains analysed were placed into two groups. Thirteen strains were clustered into a third group which was linked with the geographical origin of strains: they were all isolates from recently reported outbreaks of fire blight in Austria and in southern Bavaria in Germany. The variation in the length of the amplified fragment is probably due to an insertion into this fragment.

Evaluation of a trunk injection technique to control grapevine wood diseases

Vineyard experiments were conducted over five years in the Bordeaux area to evaluate the effectiveness of trunk injections in controlling Eutypa dieback (4 trials) and esca (1 trial). Single treatments were applied in winter 2001 or 2002 using the tree injector StemJect®. Three compounds were tested: two triazole-derived fungicides, propiconazole and difenoconazole, and one elicitor, 2-hydroxybenzoic acid. Symptomatic vines of two susceptible cultivars, Cabernet Sauvignon and Cabernet Franc, had first been identified in summer in the year before the treatments were started. A disease scale was used to rate the severity of the foliar symptoms. After treatment, disease development was recorded on the same vines in the following years, from 2002 to 2005. Analyses were based on the evolution of foliar symptoms and on the development of wood symptoms (% area of dead wood). This novel procedure made it possible to determine the sanitary status of each vine in terms of three classes of disease severity: remission of symptoms, stability or worsening. No treatment had a significantly durable effect on disease expression irrespective of the site, the compound or the disease studied. Some phytotoxic effects with the triazole fungicides were noticed. Prospects for trunk injections as a means to solve these insidious problems in viticulture are discussed.

Fire blight risk assessment during bloom in an experimental orchard using BIS (Billing's Integrated System)

The value of BIS for blossom blight risk assessment was studied from data collected in an experimental orchard in south-west France. Trees observed included mature commercial pear and apple trees and some young trees in experimental plots. There was a weather station in the orchard and beehives were present. Field records included flowering times of the pear and apple cultivars studied (mostly Passe-Crassane and Beurré Hardy, Royal Gala and Golden Delicious) and dates when blossom blight was first seen on each cultivar. Between 1980 and 1991, records of blight were available for 25 cases. In most cases, one or more infection risk (IR) days, as defined for BIS, could be found during bloom. DD13 mean sums (sums of degree days above a mean temperature of 13 °C) gave good guidance on times when early signs of blossom blight were present in 14 cases. There was only a slight divergence from BIS guides in a further five cases. Possible reasons for divergence and for non-fit in the remaining six cases are discussed. It is concluded from this study that BIS should give useful guidance on optimal times for protective spray applications and for timing of searches for signs of early blossom blight in south-west of France. Graphical presentations of data provide additional information.