Marc Fermaud

The role of saprotrophy and virulence in the population dynamics of Botrytis cinerea in vineyards

ABSTRACT Change in relative frequencies of the three main genetic types of Botrytis cinerea (Group I, Group II vacuma, and Group II transposa) were monitored over time from 1998 to 2000 in three Bordeaux vineyards not treated with fungicides. During 2000, Group I isolates, detected mostly at flowering comprised only 2.5% of the entire population. Within Group II, the complementary frequencies of vacuma and transposa isolates differed significantly depending on grapevine phenological stages and organs. Every year and at all sites, including one noble rot site, transposa isolates dominated at every stage, particularly on overwintering canes and at harvest (greater than 86.7% on berries). The complementary frequency of vacuma isolates reached a maximum on senescing floral caps (between 23.5 and 71.4%) and then decreased significantly until harvest on leaves and berries. In pathogenicity tests on grape berries, transposa isolates were significantly more virulent than were vacuma isolates. Mycelial growth rate was negatively correlated with virulence, notably on leaves in transposa and with double resistance to the fungicides carbendazim and vinclozolin. In vacuma, this double resistance was positively correlated with virulence on leaves. Change in the vacuma and transposa frequencies was most likely caused by differences in saprotrophic and pathogenic fitness. Possible interactions between fungicide resistance profiles and fitness are discussed.

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.

Olfactory and gustatory behaviour by larvae of Lobesia botrana in response to Botrytis cinerea

In vineyards, larvae of Lobesia botrana (Denis and Schiffermüller) (Lepidoptera: Tortricidae) and Botrytis cinerea (Persoon: Fries) often occur together. This study was carried out to establish whether first instar larvae are attracted by the fungus. Olfactory and gustatory responses of larvae to the fungus were investigated on grape berry clusters (Vitis vinifera) and in two types of olfactometer. In clusters infected partly by B. cinerea, 82% of first instar larvae settled where berries had been infected. In the olfactometers, first instar larvae detected and discriminated between two tested foods. They significantly preferred synthetic media or grape berries (cv., ‘Alphonse Lavallée’ or ‘Cabernet Sauvignon‘) infected with B. cinerea to non‐infected controls. The olfactory response led to 62.2% to 72.6% of the first instar larvae attracted by the fungus and the association of olfactory with gustatory responses resulted in similar rates (64.1% to 81.6%). So, the larvae were most attracted by synthetic media or grape berries infected by the fungus as compared to the controls. Olfaction appears to be the main sense involved in this attraction process.

Mutualism between a phytopathogenic fungus (Botrytis cinerea) and a vineyard pest (Lobesia botrana). Positive effects on insect development and oviposition behaviour

Abstract In vineyards, larvae of the grape berry moth ( Lobesia botrana ) favour the development of the grey mould fungus Botrytis cinerea . To study the possibility of a mutualistic relationship between these organisms, we investigated the effects of the fungus on the development and oviposition behaviour of the insect. Larvae were reared on whole plants infected or uninfected with the fungus, or on an artificial diet containing either infected grape berries or the mycelium. The presence of the fungus consistently led to a reduction in the duration of insect development (4–6 d) and mortality (20–67 %). The fecundity increased from 12 to 76 %. Moreover, the fact that the females laid eggs preferentially on fungus-infected grape berries confirms the mutualistic association between these organisms. The fungus alone contributed to the positive effects. We showed the existence of a mutualistic relationship between the two organisms which are both damaging to vineyards.

Population structure and temporal maintenance of the multihost fungal pathogen Botrytis cinerea: causes and implications for disease management

Understanding the causes of population subdivision is of fundamental importance, as studying barriers to gene flow between populations may reveal key aspects of the process of adaptive divergence and, for pathogens, may help forecasting disease emergence and implementing sound management strategies. Here, we investigated population subdivision in the multihost fungus Botrytis cinerea based on comprehensive multiyear sampling on different hosts in three French regions. Analyses revealed a weak association between population structure and geography, but a clear differentiation according to the host plant of origin. This was consistent with adaptation to hosts, but the distribution of inferred genetic clusters and the frequency of admixed individuals indicated a lack of strict host specificity. Differentiation between individuals collected in the greenhouse (on Solanum) and outdoor (on Vitis and Rubus) was stronger than that observed between individuals from the two outdoor hosts, probably reflecting an additional isolating effect associated with the cropping system. Three genetic clusters coexisted on Vitis but did not persist over time. Linkage disequilibrium analysis indicated that outdoor populations were regularly recombining, whereas clonality was predominant in the greenhouse. Our findings open up new perspectives for disease control by managing plant debris in outdoor conditions and reinforcing prophylactic measures indoor.

Screening and modes of action of antagonistic bacteria to control the fungal pathogen Phaeomoniella chlamydospora involved in grapevine trunk diseases

The antagonistic activity of 46 bacterial strains isolated from Bordeaux vineyards were evaluated against Phaeomoniella chlamydospora, a major grapevine pathogen involved in Esca. The reduction of the necrosis length of stem cuttings ranged between 31.4% and 38.7% for the 8 most efficient strains. Two in planta trials allowed the selection of the two best strains, Bacillus pumilus (S32) and Paenibacillus sp. (S19). Their efficacy was not dependent on application method; co-inoculation, prevention in the wood and soil inoculation were tested. The involvement of antibiosis by the secretion of diffusible and/or volatile compounds in the antagonistic capacity of these two strains was assessed in vitro. Volatile compounds secreted by B. pumilus (S32) and Paenibacillus sp. (S19) were identified by gas chromatography/mass spectroscopy (GC/MS). The volatile compounds 1-octen-3-ol and 2,5-dimethyl pyrazine were obtained commercially and tested, and they showed strong antifungal activity against P. chlamydospora, which suggested that these compounds may play an important role in the bacterial antagonistic activity in planta. Furthermore, the expression of 10 major grapevine defense genes was quantified by real-time polymerase chain reaction, which demonstrated that the two strains significantly affected the grapevine transcripts four days after their application on the plants. High expression levels of different genes associated with P. chlamydospora infection in B. pumilus pre-treated plants suggests that this strain induces systemic resistance in grapevine. For the first time, we demonstrated the ability of two bacterial strains, B. pumilus and Paenibacillus sp., isolated from grapevine wood, to control P. chlamydospora via direct and/or indirect mechanisms.

Environmental Conditions Affect Botrytis cinerea Infection of Mature Grape Berries More Than the Strain or Transposon Genotype

Effects of environment, Botrytis cinerea strain, and their interaction on the infection of mature grape berries were investigated. The combined effect of temperature (T) of 15, 20, 25, and 30°C and relative humidity (RH) of 65, 80, 90, and 100% was studied by inoculating berries with mycelium plugs. Regardless of the T, no disease occurred at 65% RH, and both disease incidence and severity increased with increasing RH. The combined effect of T (5 to 30°C) and wetness duration (WD) of 3, 6, 12, 24, and 36 h was studied by inoculating berries with conidia. At WD of 36 h, disease incidence was approximately 75% of affected berries at 20 or 25°C, 50% at 15°C, and 30 to 20% at 30 and 10°C; no infection occurred at 5°C. Under favorable conditions (100% RH or 36 h of WD) and unfavorable conditions (65% RH or 3 h of WD), berry wounding did not significantly affect disease incidence; under moderately favorable conditions (80% RH or 6 to 12 h of WD), disease incidence was approximately 1.5 to 5 times higher in wounded than in intact berries. Our data collectively showed that (i) T and RH or WD were more important than strain for mature berry infection by either mycelium or conidia and (ii) the effect of the environment on the different strains was similar. Two equations were developed describing the combined effect of T and RH, or T and WD, on disease incidence following inoculation by mycelium (R2=0.99) or conidia (R2=0.96), respectively. These equations may be useful in the development of models used to predict and control Botrytis bunch rot during berry ripening.

Bacteria in a wood fungal disease: characterization of bacterial communities in wood tissues of esca-foliar symptomatic and asymptomatic grapevines

Esca is a grapevine trunk disease (GTD) associated with different pathogenic fungi inhabiting the woody tissues. Bacteria can also be found in such tissues and they may interact with these fungal colonizers. Although such types of microbial interactions have been observed for wood diseases in many trees, this has never been studied for grapevine. In this study, the bacterial microflora of different vine status (esca-symptomatic and asymptomatic), different anatomical part (trunk and cordon) and different type of tissues (necrotic or not) have been studied. Based on Single Strand Conformation Polymorphism (SSCP) analyses, data showed that (i) specific complexes of bacterial microflora colonize the wood of both necrotic and non-necrotic tissues of esca-foliar symptomatic and asymptomatic vines, and also that (ii) depending on the anatomical part of the plant, cordon or trunk, differences could be observed between the bacterial communities. Such differences were also revealed through the community-level physiological profiling (CLPP) with Biolog EcoplatesTM. Two hundred seventeen bacterial strains were also isolated from plant samples and then assigned to bacterial species based on the 16S rRNA genes. Although Bacillus sp. and Pantoea agglomerans were the two most commonly isolated species from all kinds of tissues, various other taxa were also isolated. Inoculation of vine cuttings with 14 different bacterial species, and one GTD fungus, Neofusicoccum parvum, showed no impact of these bacteria on the size of the wood necroses caused by N. parvum. This study showed, therefore, that bacterial communities differ according to the anatomical part (trunk or cordon) and/or the type of tissue (necrotic or non-necrotic) of wood of grapevine plants showing external symptoms of esca disease. However, research into bacteria having a role in GTD development needs further studies.

Classification of winegrape cultivars in Chile and France according to their susceptibility to Botrytis cinerea related to fruit maturity

Background and Aims The susceptibility of winegrape cultivars to Botrytis cinerea is a debated topic, and the available classifications are based on professional experience rather than on experimental data. The main aim of this study was to compare and classify the susceptibility of several winegrape cultivars to B. cinerea and its relation to fruit maturity under two contrasting climatic and cropping conditions. Methods and Results Between 2011 and 2015, three field trials were undertaken in Chile and France, including 13 common cultivars. Both the incidence and severity of the disease were evaluated at harvest, and indices of susceptibility and maturity were calculated on a per site basis. The significant difference in incidence and severity observed among cultivars led to a similar susceptibility classification in both countries. Cabernet Sauvignon, Cabernet Franc, Grenache Noir and Petit Verdot were the most resistant cultivars, whereas Gewurztraminer and Sauvignon Blanc were the most susceptible. Moreover, an exponential and positive relationship was established between indexes of susceptibility and maturity. Conclusions The classification of cultivars according to the susceptibility to B. cinerea was similar in both countries, despite the contrasting climatic conditions and cropping practices. Significance of the Study These findings might be of interest for choosing cultivars that are more resistant to B. cinerea to reduce the number of fungicide applications.