Metabarcoding Analysis of Fungal Diversity in the Phyllosphere and Carposphere of Olive (Olea europaea)

Abstract

The fungal diversity associated with leaves, flowers and fruits of olive (Olea europaea) was investigated in different phenological stages (May, June, October and December) using an implemented metabarcoding approach. It consisted of the 454 pyrosequencing of the fungal ITS2 region and the subsequent phylogenetic analysis of relevant genera along with validated reference sequences. Most sequences were identified up to the species level or were associated with a restricted number of related taxa enabling supported speculations regarding their biological role. Analyses revealed a rich fungal community with 195 different OTUs. Ascomycota was the dominating phyla representing 93.6% of the total number of detected sequences followed by unidentified fungi (3.6%) and Basidiomycota (2.8%). A higher level of diversity was revealed for leaves compared to flowers and fruits. Among plant pathogens the genus Colletotrichum represented by three species (C. godetiae syn. C. clavatum, C. acutatum s.s and C. karstii) was the most abundant on ripe fruits but it was also detected in other organs. Pseudocercospora cladosporioides was detected with a high frequency in all leaf samples and to a less extent in ripe fruits. A much lower relative frequency was revealed for Spilocaea oleagina and for other putative pathogens including Fusarium spp., Neofusicoccum spp., and Alternaria spp. Among non-pathogen taxa, Aureobasidium pullulans, the species complex of Cladosporium cladosporioides and Devriesia spp. were the most represented. This study highlights the existence of a complex fungal consortium including both phytopathogenic and potentially antagonistic microorganisms that can have a significant impact on olive productions.

Fig 1. Rarefaction curves at a genetic distance of 3%.

Determined for May leaves (ML), June leaves (JL), October leaves (OL), December leaves (DL), May flowers (MFl), June fertilized fruitlets (JFr), December asymptomatic fruits (DAFr) and December symptomatic fruits (DSFr).

Fig 2. Venn Diagrams reporting the number of OTUs shared among investigated olive sample types in different possible combinations.

Analyzed samples included May leaves (ML), June leaves (JL), October leaves (OL), December leaves (DL), May flowers (MFl), June fertilized fruitlets (JFr), December asymptomatic fruits (DAFr) and December symptomatic fruits (DSFr).

Fig 3. Unweighted UniFrac distance PCoA (A) and Jackknife dendrogram (B) of fungal communities.

Associated with May leaves (ML), June leaves (JL), October leaves (OL), December leaves (DL), May flowers (MFl), June fertilized fruitlets (JFr), December asymptomatic fruits (DAFr) and December symptomatic fruits (DSFr). In B red and yellow colors of nodes indicate 75–100% and 50–75% of bootstrap support, respectively.

Fig 4. Relative abundance of different fungal phyla (top) and classes (bottom).

Detected in May leaves (ML), June leaves (JL), October leaves (OL), December leaves (DL), May flowers (MFl), June fertilized fruitlets (JFr), December asymptomatic fruits (DAFr) and December symptomatic fruits (DSFr).

Fig 5. Relative abundance of fungal genera.

Detected in May leaves (ML), June leaves (JL), October leaves (OL), December leaves (DL), May flowers (MFl), June fertilized fruitlets (JFr), December asymptomatic fruits (DAFr) and December symptomatic fruits (DSFr). Fungal genera representing less than 0.2% of the total population in each sample, were considered as a single taxa and labeled as "Other fungi". In each column fungal genera are listed according to their abundance.

Fig 6. Phylogenetic trees built using unique sequences representative of sequence types (STs) of the most relevant fungal genera in terms of both abundance and/or relevance as olive fungal pathogens, along with validated reference sequences.

Reference sequences were from Pseudocercospora spp. (Crous et al. 2013) (A), Cladosporium spp. (Bensch et al., 2013) (C), Aureobasidium spp. (Zalar et al., 2008) (D), Colletotrichum acutatum s.l. (Damm et al., 2012a) (E), Colletotrichum boninense s.l. 2012 (Damm et al. 2012b) (F), and Devriesia spp. (Li et al. 2013) (B). Sequences of species closely related to Spilocaea oleagina were sourced in GenBank (B). Grey highlighted boxes contain STs identified in the present study (♦) and genetically close reference species with which they were associated. Numbers in parentheses along with STs indicate the number of identical sequences, represented by each ST. Numbers on nodes represent the posterior probabilities for the maximum likelihood method.