Quantification of Cadophora luteo-olivacea From Grapevine Nursery Stock and Vineyard Soil Using Droplet Digital PCR.

Cadophora luteo-olivacea is the most prevalent Cadophora species associated with Petri disease and esca of grapevine. Accurate, early, and specific detection and quantification of C. luteo-olivacea are essential to alert growers and nurseries to the presence of the pathogens in soil and to prevent the spread of this pathogen through grapevine planting material. The aim of this study was to develop molecular tools to detect and quantify C. luteo-olivacea inoculum from environmental samples. Species specific primers based on the ß-tubulin gene and a TaqMan probe for droplet digital PCR (ddPCR) and quantitative PCR (qPCR) were first developed to detect and quantify purified DNA of the target fungus. Specificity tests showed that the primers were able to amplify the C. luteo-olivacea DNA (20 isolates) while none of the 29 nontarget fungal species (58 isolates) tested were amplified. The ddPCR was shown to be more sensitive compared with qPCR in the detection and quantification of C. luteo-olivacea at very low concentrations and was further selected to accurately detect and quantify the fungus from environmental samples. Twenty-five of the 94 grafting plants (26.6%) analyzed by ddPCR tested positive to C. luteo-olivacea DNA (>3 copies/µl). C. luteo-olivacea was barely detected from vineyard soils. The procedure employed in this study revealed the presence of the pathogen in symptomless vines, which makes implementation of this technique suitable for certification schemes of C. luteo-olivacea-free grapevine planting material.

Root hairs play a key role in the endophytic colonization of olive roots by Pseudomonas spp. with biocontrol activity.

The use of indigenous bacterial root endophytes with biocontrol activity against soil-borne phytopathogens is an environmentally-friendly and ecologically-efficient action within an integrated disease management framework. The earliest steps of olive root colonization by Pseudomonas fluorescens PICF7 and Pseudomonas putida PICP2, effective biocontrol agents (BCAs) against Verticillium wilt of olive (Olea europaea L.) caused by the fungus Verticillium dahliae Kleb., are here described. A gnotobiotic study system using in vitro propagated olive plants, differential fluorescent-protein tagging of bacteria, and confocal laser scanning microscopy analysis have been successfully used to examine olive roots-Pseudomonas spp. interactions at the single-cell level. In vivo simultaneous visualization of PICF7 and PICP2 cells on/in root tissues enabled to discard competition between the two bacterial strains during root colonization. Results demonstrated that both BCAs are able to endophytically colonized olive root tissues. Moreover, results suggest a pivotal role of root hairs in root colonization by both biocontrol Pseudomonas spp. However, colonization of root hairs appeared to be a highly specific event, and only a very low number of root hairs were effectively colonized by introduced bacteria. Strains PICF7 and PICP2 can simultaneously colonize the same root hair, demonstrating that early colonization of a given root hair by one strain did not hinder subsequent attachment and penetration by the other. Since many environmental factors can affect the number, anatomy, development, and physiology of root hairs, colonization competence and biocontrol effectiveness of BCAs may be greatly influenced by root hair's fitness. Finally, the in vitro study system here reported has shown to be a suitable tool to investigate colonization processes of woody plant roots by microorganisms with biocontrol potential.

Effect of white mustard cover crop residue, soil chemical fumigation and Trichoderma spp. root treatment on black-foot disease control in grapevine.

BACKGROUND: Black-foot disease is one of the main soilborne fungal diseases affecting grapevine production worldwide. Two field experiments were established to evaluate the effect of white mustard cover crop residue amendment and chemical fumigation with propamocarb + fosetyl-Al combined with Trichoderma spp. root treatment on the viability of black-foot inoculum in soil and fungal infection in grafted plants and grapevine seedlings used as bait plants. RESULTS: A total of 876 black-foot pathogen isolates were collected from grafted plants and grapevine seedlings used as bait plants in both fields. White mustard biofumigation reduced inoculum of Dactylonectria torresensis and the incidence and severity of black-foot of grapevine, but no added benefit was obtained when biofumigation was used with Trichoderma spp. root treatments. The effect of white mustard residues and chemical fumigation on populations of D. torresensis propagules in soil was inconsistent, possibly because of varying pretreatment inoculum levels. CONCLUSION: Biofumigation with white mustard plants has potential for improving control of black-foot disease in grapevines. This control strategy can reduce soil inoculum levels and protect young plants from infection, providing grape growers and nursery propagators with more tools for developing integrated and sustainable control systems. © 2018 Society of Chemical Industry.