Endophytic and rhizosphere actinobacteria isolated from the root system of 1-year-old graftedVitis viniferaplants 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 anin vitrobioassay, 15.5% of the endophytic isolates and 30.8% of the rhizosphere isolates exhibited antifungal activity against the fungal pathogenDiplodia seriata, whereas 13.8% of the endophytic isolates and 16.0% of the rhizosphere isolates showed antifungal activity againstDactylonectria macrodidyma(formerlyIlyonectria macrodidyma). The strains which showed the greatestin vitroefficacy against both pathogens were further analyzed for their ability to inhibit the growth ofPhaeomoniella chlamydosporaandPhaeoacremonium minimum(formerlyPhaeoacremonium 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,Streptomycessp. VV/E1, and two rhizosphere isolates,Streptomycessp. VV/R1 andStreptomycessp. VV/R4, which significantly reduced the infection rates produced by the fungal pathogensDactylonectriasp.,Ilyonectriasp.,P. chlamydospora, andP. 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.IMPORTANCEGrapevine 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 diseases (GTDs) are one of the most devastating pathologies that threaten the survival and profitability of vineyards around the world. Progressive banning of chemical pesticides and their withdrawal from the market has increased interest in the development of effective biocontrol agents (BCAs) for GTD treatment. In recent years, considerable progress has been made regarding the characterization of the grapevine microbiome, including the aerial part microbiome (flowers, berries and leaves), the wood microbiome, the root environment and vineyard soil microbiomes. In this work, we review these advances especially in relation to the etiology and the understanding of the composition of microbial populations in plants affected by GTDs. We also discuss how the grapevine microbiome is becoming a source for the isolation and characterization of new, more promising BCAs that, in the near future, could become effective tools for controlling these pathologies.

On average less than 1% of the total phosphorous present in soils is available to plants, making phosphorous one of the most limiting macronutrients for crop productivity worldwide. The aim of this work was to isolate and select phosphate solubilizing bacteria (PSB) from the barley rhizosphere, which has other growth promoting traits and can increase crop productivity. A total of 104 different bacterial isolates were extracted from the barley plant rhizosphere. In this case, 64 strains were able to solubilize phosphate in agar plates. The 24 strains exhibiting the highest solubilizing index belonged to 16 different species, of which 7 isolates were discarded since they were identified as putative phytopathogens. The remaining nine strains were tested for their ability to solubilize phosphate in liquid medium and in pot trials performed in a greenhouse. Several of the isolated strains (Advenella mimigardefordensis, Bacillus cereus, Bacillus megaterium and Burkholderia fungorum) were able to significantly improve levels of assimilated phosphate, dry weight of ears and total starch accumulated on ears compared to non-inoculated plants. Since these strains were able to increase the growth and productivity of barley crops, they could be potentially used as microbial inoculants (biofertilizers).

Grapevine trunk fungal pathogens, such asDiplodia seriataandPhaeomoniella 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 evaluatedin vitro. The fungi showing greaterin vitroefficacy were tested on autoclaved grape wood assays againstD. seriataandP. chlamydospora. Based on results from these assays, chitosan oligosaccharide, vanillin, and garlic extract were selected for further evaluation on pruning wounds inoculated withD. seriataandP. chlamydosporain 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.

Background: The phytopathogenic fungus Diplodia seriata, whose genome remains unsequenced, produces severe infections in fruit trees (fruit blight) and grapevines. In this crop is recognized as one of the most prominent pathogens involved in grapevine trunk disease (or grapevine decline). This pathology can result in the death of adult plants and therefore it produces severe economical losses all around the world. To date no genes or proteins have been characterized in D. seriata that are involved in the pathogenicity process. In an effort to help identify potential gene products associated with pathogenicity and to gain a better understanding of the biology of D. seriata, we initiated a proteome-level study of the fungal mycelia and secretome. Results: Intracellular and secreted proteins from D. seriata collected from liquid cultures were separated using two-dimensional gel electrophoresis. About 550 cytoplasmic proteins were reproducibly present in 3 independent extractions, being 53 identified by peptide mass fingerprinting and tandem mass spectrometry. The secretome analysis showed 75 secreted proteins reproducibly present in 3 biological replicates, being 16 identified. Several of the proteins had been previously identified as virulence factors in other fungal strains, although their contribution to pathogenicity in D. seriata remained to be analyzed. When D. seriata was grown in a medium supplemented with carboxymethylcellulose, 3 proteins were up-regulated and 30 down-regulated. Within the up-regulated proteins, two were identified as alcohol dehydrogenase and mitochondrial peroxyrredoxin-1, suggesting that they could play a significant role in the pathogenicity process. As for the 30 down-regulated proteins, 9 were identified being several of them involved in carbohydrate metabolism. Conclusions: This study is the first report on proteomics on D. seriata. The proteomic data obtained will be important to understand the pathogenicity process. In fact, several of the identified proteins have been reported as pathogenicity factors in other phytopathogenic fungi. Moreover, this proteomic analysis supposes a useful basis for deepening into D. seriata knowledge and will contribute to the development of the molecular biology of this fungal strain as it has been demonstrated by cloning the gene Prx 1 encoding mitochondrial peroxiredoxin-1 of D. seriata (the first gene to be cloned in this microorganism; data not shown).

Phaeomoniella chlamydospora and Phaeoacremonium aleophilum are the two main fungal causal agents of Petri disease and esca. Both diseases cause significant economic losses to viticulturalists. Since no curative control measures are known, proactive defensive measures must be taken. An important aspect of current research is the development of sensitive and time-saving protocols for the detection and identification of these pathogens. Real-time PCR based on the amplification of specific sequences is now being used for the identification and quantification of many infective agents. The present work reports real-time PCR protocols for identification of P. chlamydospora and P. aleophilum . Specificity was demonstrated against purified DNA from 60 P. chlamydospora isolates or 61 P. aleophilum isolates, and no amplification was obtained with 54 nontarget DNAs. The limits of detection (i.e., DNA detectable in 95% of reactions) were around 100 fg for P. chlamydospora and 50 fg for P. aleophilum . Detection was specific and sensitive for P. chlamydospora and P. aleophilum . Spores of P. chlamydospora and P. aleophilum were detected without the need for DNA purification. The established protocols detected these fungi in wood samples after DNA purification. P. chlamydospora was detectable without DNA purification and isolation in 67% of reactions. The detection of these pathogens in wood samples has great potential for use in pathogen-free certification schemes.