Linking belowground microbial network changes to different tolerance level towards Verticillium wilt of olive.
ABSTRACT: BACKGROUND:Verticillium wilt of olive (VWO) is caused by the soilborne fungal pathogen Verticillium dahliae. One of the best VWO management measures is the use of tolerant/resistant olive cultivars. Knowledge on the olive-associated microbiome and its potential relationship with tolerance to biotic constraints is almost null. The aims of this work are (1) to describe the structure, functionality, and co-occurrence interactions of the belowground (root endosphere and rhizosphere) microbial communities of two olive cultivars qualified as tolerant (Frantoio) and susceptible (Picual) to VWO, and (2) to assess whether these communities contribute to their differential disease susceptibility level. RESULTS:Minor differences in alpha and beta diversities of root-associated microbiota were detected between olive cultivars regardless of whether they were inoculated or not with the defoliating pathotype of V. dahliae. Nevertheless, significant differences were found in taxonomic composition of non-inoculated plants' communities, "Frantoio" showing a higher abundance of beneficial genera in contrast to "Picual" that exhibited major abundance of potential deleterious genera. Upon inoculation with V. dahliae, significant changes at taxonomic level were found mostly in Picual plants. Relevant topological alterations were observed in microbial communities' co-occurrence interactions after inoculation, both at structural and functional level, and in the positive/negative edges ratio. In the root endosphere, Frantoio communities switched to highly connected and low modularized networks, while Picual communities showed a sharply different behavior. In the rhizosphere, V. dahliae only irrupted in the microbial networks of Picual plants. CONCLUSIONS:The belowground microbial communities of the two olive cultivars are very similar and pathogen introduction did not provoke significant alterations in their structure and functionality. However, notable differences were found in their networks in response to the inoculation. This phenomenon was more evident in the root endosphere communities. Thus, a correlation between modifications in the microbial networks of this microhabitat and susceptibility/tolerance to a soilborne pathogen was found. Moreover, V. dahliae irruption in the Picual microbial networks suggests a stronger impact on the belowground microbial communities of this cultivar upon inoculation. Our results suggest that changes in the co-occurrence interactions may explain, at least partially, the differential VWO susceptibility of the tested olive cultivars. Video abstract.
Project description:Among biotic constraints affecting olive trees cultivation worldwide, the soil-borne fungus Verticillium dahliae is considered one of the most serious threats. Olive cultivars display differential susceptibility to the disease, but our knowledge on the pathogen's responses when infecting varieties differing in susceptibility is scarce. A comparative transcriptomic analysis (RNA-seq) was conducted in olive cultivars Picual (susceptible) and Frantoio (tolerant). RNA samples originated from roots during the first two weeks after inoculation with V. dahliae defoliating (D) pathotype. Verticillium dahliae mRNA amount was overwhelmingly higher in roots of the susceptible cultivar, indicating that proliferation of pathogen biomass is favored in 'Picual'. A significant larger number of V. dahliae unigenes (11 fold) were only induced in this cultivar. Seven clusters of differentially expressed genes (DEG) were identified according to time-course expression patterns. Unigenes potentially coding for niche-adaptation, pathogenicity, virulence and microsclerotia development were induced in 'Picual', while in 'Frantoio' expression remained negligible or null. Verticillium dahliae D pathotype transcriptome responses are qualitatively and quantitatively different, and depend on cultivar susceptibility level. The much larger V. dahliae biomass found in 'Picual' roots is a consequence of both host and pathogen DEG explaining, to a large extent, the higher aggressiveness exerted over this cultivar.
Project description:Verticillium wilt of olive (VWO) is caused by the vascular pathogen Verticillium dahliae. One of the best VWO management measures is the use of tolerant cultivars; however, our knowledge on VWO tolerance/resistance genetics is very limited. A transcriptomic analysis was conducted to (i) identify systemic defense responses induced/repressed in aerial tissues of the tolerant cultivar Frantoio upon root colonization by V. dahliae, and (ii) determine the expression pattern of selected defense genes in olive cultivars showing differential susceptibility to VWO. Two suppression subtractive hybridization cDNA libraries, enriched in up-regulated (FU) and down-regulated (FD) genes respectively, were generated from "Frantoio" aerial tissues. Results showed that broad systemic transcriptomic changes are taking place during V. dahliae-"Frantoio" interaction. A total of 585 FU and 381 FD unigenes were identified, many of them involved in defense response to (a)biotic stresses. Selected genes were then used to validate libraries and evaluate their temporal expression pattern in "Frantoio." Four defense genes were analyzed in cultivars Changlot Real (tolerant) and Picual (susceptible). An association between GRAS1 and DRR2 gene expression patterns and susceptibility to VWO was observed, suggesting that these transcripts could be further evaluated as markers of the tolerance level of olive cultivars to V. dahliae.
Project description:Verticillium wilt, caused by Verticillium dahliae, challenges olive cultivation and an Integrated Disease Management (IDM) approach is the best-suited tool to combat it. Since 1998, an IDM strategy in an orchard (called Granon, Spain) of the susceptible cv. Picual was conducted by increasing planting density with moderately resistant cv. Frantoio, chemical weed control, and replanting of dead olives with cv. Frantoio following soil solarization. The Verticillium wilt epidemic in Granon orchard was compared to the epidemic in a non-IDM orchard (called Ancla, Spain) with plowed soil and dead Picual olives replanted with the same cultivar. Field evaluations (2012-2013) showed an incidence and severity of the disease as Picual-Ancla > Picual-Granon > Frantoio-Granon. The spatiotemporal dynamics of the Verticillium epidemics from 1998 to 2010 were monitored with digital images using SIG. The annual tree mortalities were 5.6% for Picual olives in Ancla orchard, and 3.1 and 0.7% for Picual and Frantoio olives in Granon orchard, respectively. There was a negative relationship between the mortality of olive trees (%) by the pathogen and the height (m) above sea level. The annual mortality of cv. Picual olives was positively correlated with spring rainfalls. The Index of Dispersion and beta-binomial distribution showed aggregation of Verticillium-dead olives. In conclusion, this IDM strategy considerably reduced the disease in comparison with traditional agronomic practices.
Project description:It is well-known that different plant species, and even plant varieties, promote different assemblages of the microbial communities associated with them. Here, we investigate how microbial communities (bacteria and fungi) undergo changes within the influence of woody plants (two olive cultivars, one tolerant and another susceptible to the soilborne fungal pathogen Verticillium dahliae, plus wild Holm oak) grown in the same soil but with different management (agricultural versus native). By the use of metabarcoding sequencing we determined that the native Holm oak trees rhizosphere bacterial communities were different from its bulk soil, with differences in some genera like Gp4, Gp6 and Solirubrobacter. Moreover, the agricultural management used in the olive orchard led to belowground microbiota differences with respect to the natural conditions both in bulk soils and rhizospheres. Indeed, Gemmatimonas and Fusarium were more abundant in olive orchard soils. However, agricultural management removed the differences in the microbial communities between the two olive cultivars, and these differences were minor respect to the olive bulk soil. According to our results, and at least under the agronomical conditions here examined, the composition and structure of the rhizospheric microbial communities do not seem to play a major role in olive tolerance to V. dahliae.
Project description:BACKGROUND: Gene expression analysis using quantitative reverse transcription PCR (qRT-PCR) is a robust method wherein the expression levels of target genes are normalised using internal control genes, known as reference genes, to derive changes in gene expression levels. Although reference genes have recently been suggested for olive tissues, combined/independent analysis on different cultivars has not yet been tested. Therefore, an assessment of reference genes was required to validate the recent findings and select stably expressed genes across different olive cultivars. RESULTS: A total of eight candidate reference genes [glyceraldehyde 3-phosphate dehydrogenase (GAPDH), serine/threonine-protein phosphatase catalytic subunit (PP2A), elongation factor 1 alpha (EF1-alpha), polyubiquitin (OUB2), aquaporin tonoplast intrinsic protein (TIP2), tubulin alpha (TUBA), 60S ribosomal protein L18-3 (60S RBP L18-3) and polypyrimidine tract-binding protein homolog 3 (PTB)] were chosen based on their stability in olive tissues as well as in other plants. Expression stability was examined by qRT-PCR across 12 biological samples, representing mesocarp tissues at various developmental stages in three different olive cultivars, Barnea, Frantoio and Picual, independently and together during the 2009 season with two software programs, GeNorm and BestKeeper. Both software packages identified GAPDH, EF1-alpha and PP2A as the three most stable reference genes across the three cultivars and in the cultivar, Barnea. GAPDH, EF1-alpha and 60S RBP L18-3 were found to be most stable reference genes in the cultivar Frantoio while 60S RBP L18-3, OUB2 and PP2A were found to be most stable reference genes in the cultivar Picual. CONCLUSIONS: The analyses of expression stability of reference genes using qRT-PCR revealed that GAPDH, EF1-alpha, PP2A, 60S RBP L18-3 and OUB2 are suitable reference genes for expression analysis in developing Olea europaea mesocarp tissues, displaying the highest level of expression stability across three different olive cultivars, Barnea, Frantoio and Picual, however the combination of the three most stable reference genes do vary amongst individual cultivars. This study will provide guidance to other researchers to select reference genes for normalization against target genes by qPCR across tissues obtained from the mesocarp region of the olive fruit in the cultivars, Barnea, Frantoio and Picual.
Project description:Verticillium wilt caused by Verticillium dahliae is a common soil-borne disease worldwide, affecting many economically important crop species. Soil microbes can influence plant disease development. We investigated rhizosphere and endosphere microbiomes in relation to cotton cultivars with differential susceptibility to Verticillium wilt. Soil samples from nine cotton cultivars were assessed for the density of V. dahliae microsclerotia; plants were assessed for disease development. We used amplicon sequencing to profile both bacterial and fungal communities. Unlike wilt severity, wilt inoculum density did not differ significantly among resistant and susceptible cultivars. Overall, there were no significant association of alpha diversity indices with wilt susceptibility. In contrast, there were clear differences in the overall rhizosphere and endosphere microbial communities, particularly bacteria, between resistant and susceptible cultivars. Many rhizosphere and endosphere microbial groups differed in their relative abundance between resistant and susceptible cultivars. These operational taxonomic units included several well-known taxonomy groups containing beneficial microbes, such as Bacillales, Pseudomonadales, Rhizobiales, and Trichoderma, which were higher in their relative abundance in resistant cultivars. Greenhouse studies with sterilized soil supported that beneficial microbes in the rhizosphere contribute to reduced wilt development. These findings suggested that specific rhizosphere and endosphere microbes may contribute to cotton resistance to V. dahliae.
Project description:The bacterial and fungal communities from the olive (Olea europaea L.) root systems have not yet been simultaneously studied. We show in this work that microbial communities from the olive root endosphere are less diverse than those from the rhizosphere. But more relevant was to unveil that olive belowground communities are mainly shaped by the genotype of the cultivar when growing under the same environmental, pedological and agronomic conditions. Furthermore, Actinophytocola, Streptomyces and Pseudonocardia are the most abundant bacterial genera in the olive root endosphere, Actinophytocola being the most prevalent genus by far. In contrast, Gp6, Gp4, Rhizobium and Sphingomonas are the main genera in the olive rhizosphere. Canalisporium, Aspergillus, Minimelanolocus and Macrophomina are the main fungal genera present in the olive root system. Interestingly enough, a large number of as yet unclassified fungal sequences (class level) were detected in the rhizosphere. From the belowground microbial profiles here reported, it can be concluded that the genus Actinophytocola may play an important role in olive adaptation to environmental stresses. Moreover, the huge unknown fungal diversity here uncovered suggests that fungi with important ecological function and biotechnological potential are yet to be identified.
Project description:Worldwide olive industry has expanded into new climatic regions outside the Mediterranean basin due to an increase in extra virgin olive oil demand posing new challenges. This is the case of Uruguay, South America, where the olive crop area reached 10,000 hectares in the last 15 years and is intended to the production of EVOO. Uruguay has a temperate humid climate with mean precipitations above 1,100 mm per year but unequally distributed, mild winters, and warm summers, with mean annual temperatures of 17.7°C. Different agroecological conditions require local knowledge to achieve good productivity whereby the objective of this work was to show the feasibility and potential of olive oil production under our climatic conditions. For this the agronomic performance of Arbequina, Barnea, Frantoio, Leccino, Manzanilla de Sevilla, and Picual cultivars was evaluated along 10 years of full production. Phenology behavior, vegetative growth rate, productive efficiency, alternate bearing, and oil yield were determined. Sprouting and flowering processes occur in a wide window within the annual cycle between the months of August to November with great interannual variation. More than 8 t/ha fruit yield and 40% oil yields in dry weight basis were obtained in promising cultivars. However, alternate bearing arose as the main production limiting factor, with ABI values greater than 0.60 for most cultivars. We conclude that olive oil production in humid climate regions is feasible and the most promising cultivars based on productive efficiency are Arbequina and Picual.
Project description:Sustainable management of crop productivity and health necessitates improved understanding of the ways in which rhizosphere microbial populations interact with each other, with plant roots and their abiotic environment. In this study we examined the effects of different soils and cultivars, and the presence of a soil-borne fungal pathogen, Verticillium dahliae, on the fungal microbiome of the rhizosphere soil and roots of strawberry plants, using high-throughput pyrosequencing. Fungal communities of the roots of two cultivars, Honeoye and Florence, were statistically distinct from those in the rhizosphere soil of the same plants, with little overlap. Roots of plants growing in two contrasting field soils had high relative abundance of Leptodontidium sp. C2 BESC 319 g whereas rhizosphere soil was characterised by high relative abundance of Trichosporon dulcitum or Cryptococcus terreus, depending upon the soil type. Differences between different cultivars were not as clear. Inoculation with the pathogen V. dahliae had a significant influence on community structure, generally decreasing the number of rhizosphere soil- and root-inhabiting fungi. Leptodontidium sp. C2 BESC 319 g was the dominant fungus responding positively to inoculation with V. dahliae. The results suggest that 1) plant roots select microorganisms from the wider rhizosphere pool, 2) that both rhizosphere soil and root inhabiting fungal communities are influenced by V. dahliae and 3) that soil type has a stronger influence on both of these communities than cultivar.
Project description:Olive oil phenolic fraction considerably contributes to the sensory quality and nutritional value of this foodstuff. Herein, the phenolic fraction of 203 olive oil samples extracted from fruits of four autochthonous Moroccan cultivars ("Picholine Marocaine", "Dahbia", "Haouzia" and "Menara"), and nine Mediterranean varieties recently introduced in Morocco ("Arbequina", "Arbosana", "Cornicabra", "Frantoio", "Hojiblanca", "Koroneiki", "Manzanilla", "Picholine de Languedoc" and "Picual"), were explored over two consecutive crop seasons (2012/2013 and 2013/2014) by using liquid chromatography-mass spectrometry. A total of 32 phenolic compounds (and quinic acid), belonging to five chemical classes (secoiridoids, simple phenols, flavonoids, lignans and phenolic acids) were identified and quantified. Phenolic profiling revealed that the determined phenolic compounds showed variety-dependent levels, being, at the same time, significantly affected by the crop season. Moreover, based on the obtained phenolic composition and chemometric linear discriminant analysis, statistical models were obtained allowing a very satisfactory classification and prediction of the varietal origin of the studied oils.