Date Published: January 24, 2019
Publisher: Public Library of Science
Author(s): Sandra González-García, Jose Manuel Álvarez-Pérez, Luis E. Sáenz de Miera, Rebeca Cobos, Ana Ibañez, Alba Díez-Galán, Enrique Garzón-Jimeno, Juan José R. Coque, Zaid Abdo.
The endophytic Streptomyces sp. VV/E1, and rhizosphere Streptomyces sp. VV/R4 strains, isolated from grapevine plants were shown in a previous work to reduce the infection rate of fungal pathogens involved in young grapevine decline. In this study we cloned fragments from randomly amplified polymorphic DNA (RAPD), and developed two stably diagnostic sequence-characterized amplified region (SCAR) markers of 182 and 160 bp for the VV/E1 and VV/R4 strains, respectively. The SCAR markers were not found in another 50 actinobacterial strains isolated from grapevine plants. Quantitative real-time PCR protocols based on the amplification of these SCAR markers were used for the detection and quantification of both strains in plant material. These strains were applied on young potted plants using two methods: perforation of the rootstock followed by injection of the microorganisms or soaking the root system in a bacterial suspension. Both methods were combined with a booster treatment by direct addition of a bacterial suspension to the soil near the root system. Analysis of uprooted plants showed that those inoculated by injection exhibited the highest rate of colonization. In contrast, direct addition of either strain to the soil did not lead to reliable colonization. This study has developed molecular tools for analyzing different methods for inoculating grapevine plants with selected Streptomyces sp. strains which protect them from fungal infections that enter through their root system. These tools are of great applied interest since they could easily be established in nurseries to produce grafted grapevine plants that are protected against fungal pathogens. Finally, this methodology might also be applied to other vascular plants for their colonization with beneficial biological control agents.
Young grapevine decline (YGD) is a major threat to the wine and grape industry, especially since the 1990s when it entered a period of rapid expansion. Many factors are involved in YGD, although it is accepted that the fungal pathogens responsible for grapevine trunk diseases (GTDs) are one of the major culprits . The main GTDs associated with YGD are Petri disease, primarily caused by Phaeomoniella chlamydospora and several species of the genus Phaeoacremonium , and black-foot disease, caused by different species belonging to the genera Campylocarpon, Cylindrocladiella, Dactylonectria and Ilyonectria [2, 3]. Most of these pathogens can penetrate the plant through the root system [1, 4, 5], and therefore, the presence of endogenous pathogens in grafted plants (planting material) produced in nurseries is also highly frequent. This infection is due either to prior infection of the parent plants from which the rootstocks are obtained, or contamination occurring during the propagation process . Accordingly, there has been a growing demand among winemakers for methods to control fungi causing GTDs that infect the plant through the root system, and for reducing the infection rate of planting material produced in nurseries. Unfortunately, there is not much information available about this subject. Some nursery trials have shown that application of different Trichoderma strains to soils or the grapevine root system had a growth-stimulating effect and reduced the infection rate of Cylindrocarpon sp., Phaeoacremonium sp., and P. chlamydospora [5, 6]. Another study involving the application of Trichoderma harzianum at different vine-growth stages in a nursery indicated that its application during the rooting stage showed some efficacy in protecting against P. chlamydospora infections. However, an increase in vine mortality was observed at the end of the growing season .
Bacteria belonging to the Streptomyces genus are well known as important secondary metabolite producers, excreting both antibacterial and antifungal compounds, and for their ability to control plant diseases [19–23]. The recent finding that the grapevine plant root system is home to numerous endophytic and rhizosphere Streptomyces species, and that some of these strains are effective at reducing the infection rate by fungi causing young grapevine decline (YGD)  in grafted grapevine plants, has suggested a putative use for these strains as biocontrol agents in vine nurseries .