Unraveling the nature of suppressiveness to Verticillium wilt of specific olive orchard soils

Abstract

Plants have evolved strategies of stimulating and supporting specific groups of antagonistic microorganisms in the rhizosphere as a defense against diseases caused by soilborne plant pathogens. Disease suppressive soils provide the best examples of this strategy. Suppressive soils have been described worldwide for many different pathogens, however for the fungal pathogen Verticillum dahliae the information is very scarce. Olive (Olea europaea L.) is one of the most important crops in Spain with > 2.4 million ha. During the last two decades the phytosanitary status of olive orchards is being threatened mainly due to Verticillium wilt. In previous studies a collection of rhizosphere soils from 90 olive orchards under different management systems and three rhizosphere soils from wild olive havens in Andalusia were characterized by their level of suppressiveness to Verticillium wilt. Results indicated that at least 25% of soils showed a high level of suppressiveness to Verticillium wilt. The objective of this study was to unravel the biotic and abiotic factors that may be associated with this phenomenon. For that purpose we selected a set of suppressive and conducive soils and have performed some “Classical” approaches to identify the biotic factors (microorganisms) involved in this specific suppression that included: 1) Transferring suppressiveness by adding small amounts of suppressive soil to conducive soil which have confirmed its biological nature. 2) Treating suppressive soil with heat to eliminate specific groups of microorganism which have demonstrated loss of suppressiveness at a certain level. 3) Isolating different microbial groups and correlating their presence with suppressiveness. 4) Screening representatives of microbial groups for in vitro and biocontrol activity against the target pathogen. In a second step using molecular approaches such as bar-coded pyrosequencing we have characterized all bacterial communities associated to the rhizosphere of plants growing in those selected soils. Sequences generated from pyrosequencing of rRNA gene amplicons with the GS Junior system (Roche) were processed using the Quantitative Insights Into Microbial Ecology (QIIME 1.6.0) pipeline. Flowgrams were clustered into OTUs at 97% pairwise identity using the seed-based UCLUST algorithm, and representative sequences from each OTU were aligned to the Greengenes bacteria database using PyNAST. In addition, α diversity and β diversity metrics together with rarefaction plots were also calculated to determine the bacterial population structure in those soils. Different multivariate analyses have allowed identifying some climatic parameters and physicochemical soil characteristics that are differentially associated to the level of suppressiveness of those soils as well as to determine which OTUs are specially enriched in the suppressive soils. Furthermore, specific OTUs have been identified as being transferred from the suppressive into the conducive soils in the ‘transferability experiment’ which might be associated as the OTUs being responsible of this transferable suppressive effect. Finally, experiments are being conducted to determine the influence of the indigenous microbiota on inducing plant defense mechanisms in olive plants grown in those suppressive soils. For that purpose both microarray and metagenomic sequencing of total DNA will be used to unravel genes differentially expressed or present under the suppressive conditions.