Endophytic and rhizosphere actinobacteria isolated from the root system of 1-year-old grafted Vitis vinifera plants were evaluated for their activities against fungi that cause grapevine trunk diseases. A total of 58 endophytic and 94 rhizosphere isolates were tested. Based on an in vitro bioassay, 15.5% of the endophytic isolates and 30.8% of the rhizosphere isolates exhibited antifungal activity against the fungal pathogen Diplodia seriata, whereas 13.8% of the endophytic isolates and 16.0% of the rhizosphere isolates showed antifungal activity against Dactylonectria macrodidyma (formerly Ilyonectria macrodidyma). The strains which showed the greatest in vitro efficacy against both pathogens were further analyzed for their ability to inhibit the growth of Phaeomoniella chlamydospora and Phaeoacremonium minimum (formerly Phaeoacremonium aleophilum). Based on their antifungal activity, three rhizosphere isolates and three endophytic isolates were applied on grafts in an open-root field nursery in a 3-year trial. The field trial led to the identification of one endophytic strain, Streptomyces sp. VV/E1, and two rhizosphere isolates, Streptomyces sp. VV/R1 and Streptomyces sp. VV/R4, which significantly reduced the infection rates produced by the fungal pathogens Dactylonectria sp., Ilyonectria sp., P. chlamydospora, and P. minimum, all of which cause young grapevine decline. The VV/R1 and VV/R4 isolates also significantly reduced the mortality level of grafted plants in the nursery. This study shows that certain actinobacteria could represent a promising new tool for controlling fungal trunk pathogens that infect grapevine plants through the root system in nurseries. IMPORTANCE Grapevine trunk diseases are a major threat to the wine and grape industry worldwide. They cause a significant reduction in yields as well as in grape quality, and they can even cause plant death. Trunk diseases are caused by fungal pathogens that enter through pruning wounds and/or the root system. Although different strategies have recently been developed to protect pruning wounds using antifungal compounds (natural or synthetic) or biocontrol agents, no tools are yet available for controlling soil pathogens that infect plants through their root system. This study shows that different actinobacterial isolates, when applied to grafts in a nursery, can significantly reduce the infection rate caused by fungal pathogens that enter through the root system. This is a new, promising, and green alternative for preventing the decline of young grapevines in nurseries and vineyards.

Grapevine trunk fungal pathogens, such as Diplodia seriata and Phaeomoniella chlamydospora, can infect plants through pruning wounds. They cause grapevine trunk diseases and are involved in grapevine decline. Accordingly, the protection of pruning wounds is crucial for the management of grapevine trunk diseases. The efficacy of different natural antifungals in inhibiting the growth of several fungi causing grapevine trunk diseases was evaluated in vitro. The fungi showing greater in vitro efficacy were tested on autoclaved grape wood assays against D. seriata and P. chlamydospora. Based on results from these assays, chitosan oligosaccharide, vanillin, and garlic extract were selected for further evaluation on pruning wounds inoculated with D. seriata and P. chlamydospora in field trials. A significant decrease in plant mortality was observed after 2 years of growth in the plants treated with the different natural antifungals compared to the mortality rate observed in infected plants that were not treated with antifungals. Also, the infection rate for the inoculated pathogens was significantly reduced in plants treated with the selected natural antifungals. Therefore, natural antifungals represent a promising alternative for disease control and could provide significant economic benefits for the grape-growing industry. Grapevine trunk diseases are a major threat to the wine and grape industry. They have been reported in most grapevineproducing regions worldwide, causing serious economic losses due to a significant reduction of both yields and the quality of the grapes (1-3). The diseases include Botryosphaeria dieback, esca, Eutypa dieback, Petri disease, black foot, and Phomopsis dieback as the most relevant pathologies.Planting material produced in nurseries is frequently infected with fungal pathogens, especially those involved in Petri and black foot diseases (4, 5). In mature vineyards, pathogens such as Ilyonectria sp. mainly penetrate through the root system (6). However, it is widely accepted that for many pathogens, the annually produced pruning wounds are the main route of entry into young and adult plants (7-9). Consequently, numerous studies have been conducted to assess the susceptibility of pruning wounds to fungal pathogens causing trunk diseases. Susceptibility to Botryosphaeriaceae species has been studied by several authors (8-10). Eutypa lata infection has been extensively analyzed by authors like Munkvold and Marois (11) (8) also studied susceptibility to Phomopsis viticola infection. All of these studies support the hypothesis that pruning wounds are the main route of entry of these pathogens in the plant, and, therefore, the development of measures to protect pruning wounds would be an essential tool in the management of vineyards with better sanitation. Accordingly, numerous tests treating pruning wounds with chemical fungicides and/or biological control agents (BCA) have been carried out. Munkvold and Marois (16) checked the efficacy of natural epiphytes and colonizers of pruning wounds fo...

The elderly account for a disproportionate share of all tuberculosis cases, and the population ageing may not fully explain this phenomenon. We have performed in vitro infection experiments to investigate whether there is an immunological basis for the apparent susceptibility of elders to tuberculosis. In our infection model, Mycobacterium tuberculosis induces a higher production of interleukin (IL)-6 and reactive oxygen species in macrophages from elders than from younger adults. This response did not prevent, however, an increased multiplication of M. tuberculosis in macrophages from elders as compared with the growth observed within cells from adults. By performing a factorial experiment, we have found that IFN-γ, but not IL-1β, IL-6 or TNF-α, stimulate the macrophages to restrict the multiplication of the bacterium in macrophages from elders. Although monocytes from elders seem to be in a higher level of activation, we present evidences that protein tyrosine phosphorylation response induced by M. tuberculosis is stronger in monocytes from adults than from elders. Using a protein array that detects 71 tyrosine phosphorylated kinases, we identified Pyk2 as the only kinase that displayed a difference of intensity larger than 50 % in adults than in elders. Furthermore, monocytes from elders that were incubated in the presence of tyrosine kinase inhibitors (genistein and PP2) allowed a higher level of bacterial multiplication. These observations may help to explain the susceptibility of elders to tuberculosis. An unexpected result was that both genistein and its negative control, daidzein, abundant soy isoflavones, promoted intracellular mycobacterial growth.

Many phytopathogenic fungi produce necrosis and ethylene inducing peptide 1 (Nep1-like proteins or NLP) that trigger leaf necrosis and the activation of defense mechanisms. These proteins have been widely studied in plant pathogens as Moniliophthora perniciosa or Botrytis cinerea between others, but little is known about their biological roles in grapevine trunk pathogens. Advances in the sequencing of genomes of several fungi involved in grapevine trunk diseases have revealed that these proteins are present in several copies in their genomes. The aim of this project was to analyze the presence of genes encoding NLP proteins in the Diplodia seriata genome and to characterize their putative role as virulence factors associated to grapevine trunk diseases. In this study, we characterized four NLPs from Diplodia seriata. All proteins showed highly similar amino acid sequences and contained the characteristic peptide motifs of NLPs. DserNEPs slightly reduced the viability of Vitis vinifera L. cell cultures. The cytolytic activity from DserNEP1 was stronger than that from DserNEP2, even at low concentrations. Purified DserNEPs also produced necrosis in leaves when they were inoculated into micropropagules of V. vinifera L. This is the first record of Nep1-like proteins from a fungus associated with grapevine trunk diseases and also from a member of the Botryosphaeriaceae family.

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