Search

Abstract Background The complex and co-evolved interplay between plants and their microbiota is crucial for the health and fitness of the plant holobiont. However, the microbiota of the seeds is still relatively unexplored and no studies have been conducted with olive trees so far. In this study, we aimed to characterize the bacterial, fungal and archaeal communities present in seeds of ten olive genotypes growing in the same orchard through amplicon sequencing to test whether the olive genotype is a major driver in shaping the seed microbial community, and to identify the origin of the latter. Therefore, we have developed a methodology for obtaining samples from the olive seed’s endosphere under sterile conditions. Results A diverse microbiota was uncovered in olive seeds, the plant genotype being an important factor influencing the structure and composition of the microbial communities. The most abundant bacterial phylum was Actinobacteria, accounting for an average relative abundance of 41%. At genus level, Streptomyces stood out because of its potential influence on community structure. Within the fungal community, Basidiomycota and Ascomycota were the most abundant phyla, including the genera Malassezia, Cladosporium, and Mycosphaerella. The shared microbiome was composed of four bacterial (Stenotrophomonas, Streptomyces, Promicromonospora and Acidipropionibacterium) and three fungal (Malassezia, Cladosporium and Mycosphaerella) genera. Furthermore, a comparison between findings obtained here and earlier results from the root endosphere of the same trees indicated that genera such as Streptomyces and Malassezia were present in both olive compartments. Conclusions This study provides the first insights into the composition of the olive seed microbiota. The highly abundant fungal genus Malassezia and the bacterial genus Streptomyces reflect a unique signature of the olive seed microbiota. The genotype clearly shaped the composition of the seed’s microbial community, although a shared microbiome was found. We identified genera that may translocate from the roots to the seeds, as they were present in both organs of the same trees. These findings set the stage for future research into potential vertical transmission of olive endophytes and the role of specific microbial taxa in seed germination, development, and seedling survival.

The market for bacteria as agricultural biofertilizers is growing rapidly, offering plant-growth stimulants; biofungicides; and, more recently, protectors against extreme environmental factors, such as drought. This abundance makes it challenging for the end user to decide on the product to use. In this work, we describe the isolation of a strain of Bacillus velezensis (belonging to the operational group Bacillus amyloliquefaciens) for use as a plant-growth-promoting rhizobacterium, a biofungicide, and a protector against drought. To compare its effectiveness with other commercial strains of the same operational group, Bacillus amyloliquefaciens, we analyzed its ability to promote the growth of pepper plants and protect them against drought, as well as its fungicidal activity through antibiosis and antagonism tests, its ability to solubilize potassium and phosphates, and its ability to produce siderophores. Finally, we used a probit function, a type of regression analysis used to model the outcomes of analyses, to quantify the biostimulatory effectiveness of the different plant-growth-promoting rhizobacteria, developing what we have called the Agricultural Protection Against Stress Index, which allowed us to numerically compare the four commercial strains of the operational group Bacillus amyloliquefaciens, based on a Delphi method—a type of regression analysis that can be used to model a cumulative normal distribution—and integrate the results from our panel of tests into a single value.

Abstract Background Verticillium wilt of olive (VWO), caused by Verticillium dahliae Kleb, is one of the most threatening diseases affecting olive cultivation. An integrated disease management strategy is recommended for the effective control of VWO. Within this framework, the use of biological control agents (BCAs) is a sustainable and environmentally friendly approach. No studies are available on the impact that the introduction of BCAs has on the resident microbiota of olive roots. Pseudomonas simiae PICF7 and Paenibacillus polymyxa PIC73 are two BCAs effective against VWO. We examined the effects of the introduction of these BCAs on the structure, composition and co-occurrence networks of the olive (cv. Picual) root-associated microbial communities. The consequences of the subsequent inoculation with V. dahliae on BCA-treated plants were also assessed. Results Inoculation with any of the BCAs did not produce significant changes in the structure or the taxonomic composition of the ‘Picual’ root-associated microbiota. However, significant and distinctive alterations were observed in the topologies of the co-occurrence networks. The introduction of PIC73 provoked a diminution of positive interactions within the ‘Picual’ microbial community; instead, PICF7 inoculation increased the microbiota’s compartmentalization. Upon pathogen inoculation, the network of PIC73-treated plants decreased the number of interactions and showed a switch of keystone species, including taxa belonging to minor abundant phyla (Chloroflexi and Planctomycetes). Conversely, the inoculation of V. dahliae in PICF7-treated plants significantly increased the complexity of the network and the number of links among their modules, suggestive of a more stable network. No changes in their keystone taxa were detected. Conclusion The absence of significant modifications on the structure and composition of the ‘Picual’ belowground microbiota due to the introduction of the tested BCAs underlines the low/null environmental impact of these rhizobacteria. These findings may have important practical consequences regarding future field applications of these BCAs. Furthermore, each BCA altered the interactions among the components of the olive belowground microbiota in idiosyncratic ways (i.e. PIC73 strongly modified the number of positive relations in the ‘Picual’ microbiota whereas PICF7 mostly affected the network stability). These modifications may provide clues on the biocontrol strategies used by these BCAs.

Soil health and root-associated microbiome are interconnected factors involved in plant health. The use of manure amendment on agricultural fields exerts a direct benefit on soil nutrient content and water retention, among others. However, little is known about the impact of manure amendment on the root-associated microbiome, particularly in woody species. In this study, we aimed to evaluate the effects of ovine manure on the microbial communities of the olive rhizosphere and root endosphere. Two adjacent orchards subjected to conventional (CM) and organic (OM) management were selected. We used metabarcoding sequencing to assess the bacterial and fungal communities. Our results point out a clear effect of manure amendment on the microbial community. Fungal richness and diversity were increased in the rhizosphere. The fungal biomass in the rhizosphere was more than doubled, ranging from 1.72 × 106 ± 1.62 × 105 (CM) to 4.54 × 106 ± 8.07 × 105 (OM) copies of the 18 S rRNA gene g-1 soil. Soil nutrient content was also enhanced in the OM orchard. Specifically, oxidable organic matter, total nitrogen, nitrate, phosphorous, potassium and sulfate concentrations were significantly increased in the OM orchard. Moreover, we predicted a higher abundance of bacteria in OM with metabolic functions involved in pollutant degradation and defence against pathogens. Lastly, microbial co-occurrence network showed more positive interactions, complexity and shorter geodesic distance in the OM orchard. According to our results, manure amendment on olive orchards represents a promising tool for positively modulating the microbial community in direct contact with the plant.

The Mediterranean basin is drastically affected by intense and frequent droughts, which jeopardize the diversity and survival of its forest, for example, Pinus pinaster forests. The dynamics of the bacterial communities inhabiting the rhizosphere of Pinus pinaster and other plants from a pine dominated forest under contrasting hydric conditions was monitored. The forest was located in Sierra de Oria (southeast Spain), and it was mainly composed by P. pinaster, P. halepensis, woody shrub species and herbaceous plants. 18 trees visually belonging to P. pinaster located along the perimeter and across the forest were selected for the analysis. All the trees were separated at least 50 m each other. Although all of them belonged to P. pinaster morphologically according to visual identification, the genotyping of the roots confirmed that they corresponded to P. pinaster, P. halepensis, and other plant species different from genus Pinus, although in the last case it was not possible to identify the plant species. At a distance less than 50 cm from the trunk, the litter and topsoil were removed, and the soil closely attached to non-suberified roots (rhizosphere soil) was collected (depth of 5-25 cm). Sampling was carried out in two seasons with contrasting temperature and rainfall patterns: on July 18, 2017 (summer) and April 24, 2018 (spring). After rhizosphere soil DNA and RNA extraction (and cDNA synthesis), a metabarcoding approach was followed by sequencing the V3-V4 hypervariable regions of the 16S rRNA gene and its derived transcripts by Illumina MiSeq platform. Sequencing reads were bioinformatically processed; specifically, they were filtered, trimmed, clustered into ASV (Amplicon Sequence Variants), and taxonomically identified. As a result, a total of 1,123,209 and 1,089,359 quality sequences were obtained from DNA and RNA-derived libraries, which resulted in 5,241 and 5,231 ASVs, respectively. Total communities (DNA) were mainly dominated by phyla Proteobacteria, Acidobacteria, Actinobacteria, Verrucomicrobia and Bacteroidetes in summer and spring, while potentially active populations (RNA libraries) were rich in Proteobacteria, Acidobacteria, Candidate division WPS-1, Actinobacteria and Verrucomicrobia both in summer and spring. On the other hand, DNA libraries were mainly dominated by genera Sphingomonas and acidobacterial groups Gp4 and Gp6, while potentially active bacteria (RNA) were rich in acidobacterial Gp3, Gp4, Gp6 and Phenylobacterium, although their relative abundance depended on the considered season.This dataset can provide valuable information about bacterial candidates which could be used as bioindicators of drought conditions. In addition to shifts in the bacterial relative abundance due to seasonal changes, the ratio RNA-based cDNA:DNA could be calculated as proxy of the potential activity of bacterial taxa. Moreover, these data could aid in developing bioformulations based on microorganisms which could be resistant to desiccation and involved in the drought resistance mechanisms of the host plant.

The connection between olive genetic responses to environmental and agro-climatic conditions and the composition, structure and functioning of host-associated, belowground microbiota has never been studied under the holobiont conceptual framework. Two groups of cultivars growing under the same environmental, pedological and agronomic conditions, and showing highest (AH) and lowest (AL) Actinophytocola relative abundances, were earlier identified. We aimed now to: i) compare the root transcriptome profiles of these two groups harboring significantly different relative abundances in the above-mentioned bacterial genus; ii) examine their rhizosphere and root-endosphere microbiota co-occurrence networks; and iii) connect the root host transcriptome pattern to the composition of the root microbial communities by correlation and co-occurrence network analyses. Significant differences in olive gene expression were found between the two groups. Co-occurrence networks of the root endosphere microbiota were clearly different as well. Pearson’s correlation analysis enabled a first portray of the interaction occurring between the root host transcriptome and the endophytic community. To further identify keystone operational taxonomic units (OTUs) and genes, subsequent co-occurrence network analysis showed significant interactions between 32 differentially expressed genes (DEGs) and 19 OTUs. Overall, negative correlation was detected between all upregulated genes in the AH group and all OTUs except of Actinophytocola. While two groups of olive cultivars grown under the same conditions showed significantly different microbial profiles, the most remarkable finding was to unveil a strong correlation between these profiles and the differential gene expression pattern of each group. In conclusion, this study shows a holistic view of the plant-microbiome communication.

The impact of the versatile biocontrol and plant-growth-promoting rhizobacteria Pseudomonas simiae PICF7 on the banana holobiont under controlled conditions was investigated. We examine the fate of this biological control agent (BCA) upon introduction in the soil, the effect on the banana root microbiota, and the influence on specific host genetic defense responses. While the presence of strain PICF7 significantly altered neither the composition nor the structure of the root microbiota, a significant shift in microbial community interactions through co-occurrence network analysis was observed. Despite the fact that PICF7 did not constitute a keystone, the topology of this network was significantly modified—the BCA being identified as a constituent of one of the main network modules in bacterized plants. Gene expression analysis showed the early suppression of several systemic acquired resistance and induced systemic resistance (ISR) markers. This outcome occurred at the time in which the highest relative abundance of PICF7 was detected. The absence of major and permanent changes on the banana holobiont upon PICF7 introduction poses advantages regarding the use of this beneficial rhizobacteria under field conditions. Indeed a BCA able to control the target pathogen while altering as little as possible the natural host-associated microbiome should be a requisite when developing effective bio-inoculants.

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.

The Queretaro semi-desert in central Mexico is the most southern extension of the Chihuahua desert. This semi-arid zone shelters a vast cactus diversity with many endemic species. Currently, two cacti species from this semi-desert namely, Echinocactus platyacanthus and Neobuxbaumia polylopha are under a threat to their survival. So far, there are no reports on the bacterial communities associated with these plants. In this study, we assessed the structure and diversity of the rhizospheric bacterial communities associated with Echinocactus platyacanthus and Neobuxbaumia polylopha growing in wild and cultivated conditions. Samples of E. platyacanthus were also approached with culture-based methods in search of isolates with plant growth promoting abilities. Metagenomic DNA was extracted from rhizospheric samples and used for Illumina sequencing of the 16S rRNA gene. α-diversity and amplicon sequence variant (ASV) richness were higher in both groups of E. platyacanthus samples. All samples accounted for 14 phyla, and the major 6 were common to all treatments. The dominant phyla in all four sample groups were Actinobacteria and Proteobacteria. Analysis at family and genus levels showed association patterns with the cultivated samples from both species grouping together, while the wild samples of each cactus species were grouping apart. High abundance values of Rubrobacteraceae (15.9–18.4%) were a characteristic feature of wild E. platyacanthus samples. In total, 2,227 ASVs were scored in all 12 rhizospheric samples where E. platyacanthus samples showed higher richness with 1,536 ASVs. Regarding the growing conditions, both groups of cultivated samples were also richer accounting for 743 and 615 ASVs for E. platyacanthus and N. polylopha, respectively. The isolates from E. platyacanthus rhizosphere were mainly assigned to Bacilli and Gammaproteobacteria. In total 35 strains were assayed for PGPR traits (IAA and siderophore production, phosphate solubilization, and fungal growth inhibition). Strains obtained from plants growing in the wild displayed better PGPR characteristics, stressing that naturally occurring wild plants are a source of bacteria with diverse metabolic activities, which can be very important players in the adaptation of cacti to their natural environments.

Abstract After a forest wildfire, the microbial communities have a transient alteration in their composition. The role of the soil microbial community in the recovery of an ecosystem following such an event remains poorly understood. Thus, it is necessary to understand the plant-microbe interactions that occur in burned soils. By high-throughput sequencing, we identified the main bacterial taxa of burnt holm-oak rhizosphere, then we obtained an isolate collection of the most abundant genus and its growth promoting activities were characterised. 16S rRNA amplicon sequencing showed that the genus Arthrobacter comprised more than 21% of the total community. 55 Arthrobacter strains were isolated and characterized using RAPDs and sequencing of the almost complete 16S rRNA gene. Our results indicate that isolated Arthrobacter strains present a very high genetic diversity, and they could play an important ecological role in interaction with the host plant by enhancing aerial growth. Most of the selected strains exhibited a great ability to degrade organic polymers in vitro as well as possibly presenting a direct mechanism for plant growth promotion. All the above data suggests that Arthrobacter can be considered as an excellent PGP rhizobacterium that may play an important role in the recovery of burned holm-oak forests.

This study aimed to disentangle the structure, composition, and co-occurrence relationships of the banana (cv. Dwarf Cavendish) root endophytome comparing two phenological plant stages: mother plants and suckers. Moreover, a collection of culturable root endophytes (>1000) was also generated from Canary Islands. In vitro antagonism assays against Fusarium oxysporum f.sp. cubense (Foc) races STR4 and TR4 enabled the identification and characterization of potential biocontrol agents (BCA). Eventually, three of them were selected and evaluated against Fusarium wilt of banana (FWB) together with the well-known BCA Pseudomonas simiae PICF7 under controlled conditions. Culturable and non-culturable (high-throughput sequencing) approaches provided concordant information and showed low microbial diversity within the banana root endosphere. Pseudomonas appeared as the dominant genus and seemed to play an important role in the banana root endophytic microbiome according to co-occurrence networks. Fungal communities were dominated by the genera Ophioceras, Cyphellophora, Plecosphaerella, and Fusarium. Overall, significant differences were found between mother plants and suckers, suggesting that the phenological stage determines the recruitment and organization of the endophytic microbiome. While selected native banana endophytes showed clear antagonism against Foc strains, their biocontrol performance against FWB did not improve the outcome observed for a non-indigenous reference BCA (strain PICF7).

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.