Verticillium dahliae Inoculation and in vitro Propagation Modify the Xylem Microbiome and Disease Reaction to Verticillium Wilt in a Wild Olive Genotype

Abstract

Host resistance is the most practical, long-term, and economically efficient disease control measure for Verticillium wilt in olive caused by the xylem-invading fungus Verticillium dahliae (Vd), and it is at the core of the integrated disease management. Plant's microbiome at the site of infection may have an influence on the host reaction to pathogens; however, the role of xylem microbial communities in the olive resistance to Vd has been overlooked and remains unexplored to date. This research was focused on elucidating whether in vitro olive propagation may alter the diversity and composition of the xylem-inhabiting microbiome and if those changes may modify the resistance response that a wild olive clone shows to the highly virulent defoliating (D) pathotype of Vd. Results indicated that although there were differences in microbial communities among the different propagation methodologies, most substantial changes occurred when plants were inoculated with Vd, regardless of whether the infection process took place, with a significant increase in the diversity of bacterial communities when the pathogen was present in the soil. Furthermore, it was noticeable that olive plants multiplied under in vitro conditions developed a susceptible reaction to D Vd, characterized by severe wilting symptoms and 100% vascular infection. Moreover, those in vitro propagated plants showed an altered xylem microbiome with a decrease in total OTU numbers as compared to that of plants multiplied under non-aseptic conditions. Overall, 10 keystone bacterial genera were detected in olive xylem regardless of infection by Vd and the propagation procedure of plants (in vitro vs nursery), with Cutibacterium (36.85%), Pseudomonas (20.93%), Anoxybacillus (6.28%), Staphylococcus (4.95%), Methylobacterium-Methylorubrum (3.91%), and Bradyrhizobium (3.54%) being the most abundant. Pseudomonas spp. appeared as the most predominant bacterial group in micropropagated plants and Anoxybacillus appeared as a keystone bacterium in Vd-inoculated plants irrespective of their propagation process. Our results are the first to show a breakdown of resistance to Vd in a wild olive that potentially may be related to a modification of its xylem microbiome and will help to expand our knowledge of the role of indigenous xylem microbiome on host resistance, which can be of use to fight against main vascular diseases of olive.

Keywords: Verticillium dahliae; host resistance; microbiome; micropropagation; olive; xylem.

FIGURE 1

Verticillium wilt disease progression in “Ac-18” in vitro (standard and adapted) and nursery propagated olive plants inoculated with the defoliating pathotype of Verticillium dahliae. “Picual” and “Ac-15” olive genotypes were used as positive controls to determine the inoculation success and development of the disease. Each point represents the mean disease severity (0–4 scale: 0 = healthy, 4 = dead plant) of data and error bars show the standard error from six plants per treatment.

FIGURE 2

Boxplots of Richness, Shannon, and Evenness alpha diversity indices at OTU taxonomic level in olive xylem from Verticillium dahliae (Vd)-inoculated and non-inoculated “Ac-18” plants following in vitro (standard and adapted) and nursery propagation methods. Boxes represent the interquartile range, while the horizontal line inside the box defines the median and whiskers represent the lowest and highest values of six values for each treatment combination. For all three indexes and propagation methods, values on Vd-inoculated plants were significantly higher compared to that on non-inoculated treatments according to the Scheirer–Ray–Hare test at P < 0.05.

FIGURE 3

Principal coordinates plot of weighted UniFrac distances of bacterial communities at OTU taxonomic level in olive xylem from Verticillium dahliae (Vd)-inoculated and non-inoculated “Ac-18” plants following in vitro (standard and adapted) and nursery propagation methods. Points are colored by plant inoculation treatment and shaped by propagation methods.

FIGURE 4

Prevalence Venn diagram showing the unique and shared bacterial genera in olive xylem from the core microbiome (A) or by each propagation approach (B) obtained from Verticillium dahliae (Vd)-inoculated and non-inoculated (NI) “Ac-18” plants following in vitro (standard and adapted) and nursery propagation methods.

FIGURE 5

Bar plots showing the relative bacterial abundance taxa at phylum (A) and genera (B) level present in olive xylem from Verticillium dahliae (Vd)-inoculated and non-inoculated “Ac-18” plants following in vitro (standard and adapted) and nursery propagation methods.

FIGURE 6

Cladogram representation from LEfSe analysis showing the taxonomic ranks from the innermost phylum ring to the outermost genera ring. Each point is a member within each taxonomic rank. Significant taxa (P < 0.05) appearing as dominant for each treatment when comparing each propagation method by inoculation treatment (A,B), or each inoculation treatment by propagation method (C–E) are shown in different colors (red, green, or blue) associated to the legend of each individual cladogram.