Emilie Bruez

Analyses of the Temporal Dynamics of Fungal Communities Colonizing the Healthy Wood Tissues of Esca Leaf-Symptomatic and Asymptomatic Vines

Esca, a Grapevine Trunk Disease (GTD), is of major concern for viticulture worldwide. Our study compares the fungal communities that inhabit the wood tissues of vines that expressed or not foliar esca-symptoms. The trunk and rootstock tissues were apparently healthy, whether the 10 year-old plants were symptomatic or not. The only difference was in the cordon, which contained white rot, a typical form of esca, in 79% of symptomatic plants. Observations over a period of one year using a fingerprint method, Single Strand Conformation Polymorphism (SSCP), and the ITS-DNA sequencing of cultivable fungi, showed that shifts occurred in the fungal communities colonizing the healthy wood tissues. However, whatever the sampling time, spring, summer, autumn or winter, the fungi colonizing the healthy tissues of asymptomatic or symptomatic plants were not significantly different. Forty-eight genera were isolated, with species of Hypocreaceae and Botryosphaeriaceae being the most abundant species. Diverse fungal assemblages, made up of potentially plant-pathogenic and -protective fungi, colonized these non-necrotic tissues. Some fungi, possibly involved in GTD, inhabited the non-necrotic wood of young plants, but no increase in necrosis areas was observed over the one-year period.

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.

Biocontrol of Rhizoctonia Root Rot in Tomato and Enhancement of PlantGrowth using Rhizobacteria Naturally associated to Tomato

In the present study, 25 rhizobacterial isolates, obtained from rhizosphere of healthy tomato plants collected from various tomato-growing sites in Tunisia, were tested in vitro and in vivo against Rhizoctonia solani. This bacterial collection, composed of isolates belonging to Bacillus spp., Enterobacter cloacae, Chryseobacterium jejuense, and Klebsiella pneumoniae, was assessed for its antifungal potential against R. solani the causative agent of Rhizoctonia Root Rot disease in various crops including tomato. Antifungal activity of diffusible and volatile metabolites derived from these isolates was tested against target pathogen using dual and distance culture bioassays, respectively. Growth inhibition rates, recorded after 5 days of incubation at 25°C, depended significantly upon tested bacterial isolates and screening methods and reached 34-59% and 18-45% for diffusible and volatile metabolites, respectively. The screening of disease-suppressive and plant growth-promoting abilities of these tomato-associated rhizobacteria showed 47-100% decrease in disease severity and significant increments in plant height by 62-76%, roots fresh weight by 53-86%, and aerial parts fresh weight by 34-67% compared to pathogen-inoculated and untreated control. B. thuringiensis B2 (KU158884), B. subtilis B10 (KT921327) and E. cloacae B16 (KT921429) were found to be the most efficient isolates in decreasing R. solani radial growth, suppressing disease severity, and enhancing plant growth.

Characterization of Tomato-associated Rhizobacteria Recovered fromVarious Tomato-growing Sites in Tunisia

In the present study, a total of 200 rhizobacterial isolates were obtained from rhizosphere of healthy tomato plants grown in fields with a history of severe soilborne diseases and mainly crown and root rots. Screened their capacity to suppress in vitro growth of Sclerotinia sclerotiorum and Rhizoctonia solani, 69 and 57 isolates out of the 200 tested were shown able to inhibit significantly the mycelial growth of target pathogens by 11-62% relative to control. The 25 most effective isolates, leading to suppression of both fungi by more than 45% over control, were selected and subjected to morphological, biochemical, molecular, and metabolic characterizations. This collection of tomato-associated rhizobacteria exhibited a great morphological and biochemical diversity. Sequencing of 16S rRNA and rpoB genes led to the identification of four genera namely Bacillus, Chryseobacterium, Enterobacter, and Klebsiella. The most frequent species were B. amyloliquefaciens, B. thuringiensis, B. megaterium, B. subtilis, E. cloacae, C. jejuense, and K. pneumoniae. Screening for their plant growth-promoting properties, 20 isolates were shown able to produce siderophore, 18 had solubilized phosphate, and 19 were capable to synthesize indole-3- acetic acid (IAA). PCR amplification of lipopeptide biosynthetic genes revealed the presence of genes encoding fengycin A and bacillomycin D biosynthesis in 18 and 16 isolates, respectively. Metabolic characterization performed using Biolog™ Ecoplates indicated that tomato-associated rhizobacteria displayed a large metabolic activity and they were able to use a wide range of carbon sources with the increase of the incubation duration. Based on their metabolic profiles, these rhizobacterial isolates were grouped into eight major clusters generated at the different sampling times (24, 48 and 120 h of incubation). Average well-color development (AWCD) values were found to be positively correlated with the Shannon diversity index.