Epiphytic and Endophytic Bacteria on Olive Tree Phyllosphere: Exploring Tissue and Cultivar Effect.

Variation on bacterial communities living in the phyllosphere as epiphytes and endophytes has been attributed to plant host effects. However, there is contradictory or inconclusive evidence regarding the effect of plant genetics (below the species' level) and of plant tissue type on phyllosphere bacterial community assembly, in particular when epiphytes and endophytes are considered simultaneously. Here, both surface and internal bacterial communities of two olive (Olea europaea) cultivars were evaluated in twigs and leaves by molecular identification of cultivable isolates, with an attempt to answer these questions. Overall, Proteobacteria, Actinobacteria and Firmicutes were the dominant phyla, being epiphytes more diverse and abundant than endophytes. Host genotype (at cultivar level) had a structuring effect on the composition of bacterial communities and, in a similar way, for both epiphytes and endophytes. Plant organ (leaf vs. twig) control of the bacterial communities was less evident when compared with plant genotype and with a greater influence on epiphytic than on endophytic community structure. Each olive genotype/plant organ was apparently selective towards specific bacterial operational taxonomic units (OTUs), which may lead to specific feedbacks on fitness of plant genotypes. Bacterial recruitment was observed to happen mainly within epiphytes than in endophytes and in leaves as compared with twigs. Such host specificity suggested that the benefits derived from the plant-bacteria interaction should be considered at genetic levels below the species.

Large and variable genome size unrelated to serpentine adaptation but supportive of cryptic sexuality in Cenococcum geophilum.

Estimations of genome size and its variation can provide valuable information regarding the genetic diversity of organisms and their adaptation potential to heterogeneous environments. We used flow cytometry to characterize the variation in genome size among 40 isolates of Cenococcum geophilum, an ectomycorrhizal fungus with a wide ecological and geographical distribution, obtained from two serpentine and two non-serpentine sites in Portugal. Besides determining the genome size and its intraspecies variation, we wanted to assess whether a relationship exists between genome size and the edaphic background of the C. geophilum isolates. Our results reveal C. geophilum to have one of the largest genome sizes so far measured in the Ascomycota, with a mean haploid genome size estimate of 0.208 pg (203 Mbp). However, no relationship was found between genome size and the edaphic background of the sampled isolates, indicating genetic and demographic processes to be more important for shaping the genome size variation in this species than environmental selection. The detection of variation in ploidy level among our isolates, including a single individual with both presumed haploid and diploid nuclei, provides supportive evidence for a possible cryptic sexual or parasexual cycle in C. geophilum (although other mechanisms may have caused this variation). The existence of such a cycle would have wide significance, explaining the high levels of genetic diversity and likelihood of recombination previously reported in this species, and adds to the increasing number of studies suggesting sexual cycles in previously assumed asexual fungi.

Endophytic fungal community structure in olive orchards with high and low incidence of olive anthracnose.

Fungal endophytes have been increasingly recognized to promote host plant protection to pathogens, but knowledge of the multiple effects that they could have in crop diseases is still scarce. This work attempts to understand the role of fungal endophytes in crop diseases, specifically in reducing disease development and interfering on lifestyle transition of the pathogen. To accomplish this, the endophytic fungal community of reproductive organs of olive tree from two orchards showing different levels of anthracnose incidence, a major disease of olive fruits, was characterized and compared between them. The two orchards showed distinct endophytic communities, differing in species richness, abundance and composition, with highest isolation rates and richness of endophytes in the orchard with low anthracnose incidence. These differences among orchards were greater on fruits than on flowers, suggesting that these changes in endophytic fungal composition may influence the lifestyle shifts in pathogen (from latent to pathogen). A number of fungal taxa were found to be positively associated to one of the two orchards. The fungal endophytes best correlated with high incidence of anthracnose are pathogens, while endophytes-associated to low anthracnose incidence are described to protect plants. Altogether, the results suggest varying pathogen-endophyte interactions among the two orchards.

Screening the Olive Tree Phyllosphere: Search and Find Potential Antagonists Against Pseudomonas savastanoi pv. savastanoi.

Olive knot (OK) is a widespread bacterial disease, caused by Pseudomonas savastanoi pv. savastanoi (Pss), which currently has not effective control methods. The use of naturally occurring microbial antagonists, such as bacteria, as biocontrol agents could be a strategy to manage this disease. The objective of this work was to select bacteria from olive tree phyllosphere able to antagonize Pss using in vitro and in planta experiments. The elucidation of their modes of action and the potential relationship between antagonism and bacteria origin has been investigated, as well. To this end, 60 bacterial isolates obtained from the surface and inner tissues of different organs (leaves, twigs, and knots), from two olive cultivars of varying susceptibilities to OK, were screened for their in vitro antagonistic effect against Pss. A total of 27 bacterial strains were able to significantly inhibit Pss growth, being this effect linked to bacteria origin. Strains from OK-susceptible cultivar and colonizing the surface of plant tissues showed the strongest antagonistic potential. The antagonistic activity was potentially due to the production of volatile compounds, siderophores and lytic enzymes. Bacillus amyloliquefaciens P41 was the most effective antagonistic strain and their capacity to control OK disease was subsequently assayed using in planta experiments. This strain significantly reduces OK disease severity (43.7%), knots weight (55.4%) and population size of Pss (26.8%), while increasing the shoot dry weight (55.0%) and root water content (39.6%) of Pss-infected olive plantlets. Bacterial isolates characterized in this study, in particular B. amyloliquefaciens P41, may be considered as promising biocontrol candidates for controlling OK disease.

Impact of plant genotype and plant habitat in shaping bacterial pathobiome: a comparative study in olive tree.

Plant-inhabiting microorganisms interact directly with each other affecting disease progression. However, the role of host plant and plant habitat in shaping pathobiome composition and their implication for host susceptibility/resistance to a particular disease are currently unknown. For the elucidation of these questions, both epiphytic and endophytic bacterial communities, present in asymptomatic and symptomatic twigs from olive cultivars displaying different susceptibilities to olive knot (OK) disease, were investigated using culturing methods. OK disease was the main driver of the bacterial community, causing changes on their diversity, abundance and composition. OK disease effect was most notorious on OK-susceptible cultivar and when considering the endophytic communities. Plant habitat (epiphytes vs. endophytes) also contributed to the bacterial community assembling, in particular on symptomatic twigs (knots) of OK-susceptible cultivar. In contrast, host cultivar had little effect on the bacterial community composition, but OK-symptomatic twigs (knots) revealed to be more affected by this driver. Overall, the pathobiome seems to result from an intricate interaction between the pathogen, the resident bacteria, and the plant host. Specific bacterial genera were associated to the presence or absence of OK disease in each cultivar. Their ability to trigger and/or suppress disease should be studied in the future.