Table 1.
Isolates of Epicoccum layuense and related species used for phylogenetic analyses.
Ex-type isolates are shown in bold type.
Fig 1.
Schematic representation of the inoculation and re-isolation areas in grapevine rooted cuttings.
(A) E24 inoculation via mycelium plug; (B) pathogen inoculation via spore suspension; (C) re-isolation area 15 mm below the pathogen inoculation; (D) re-isolation area 45 mm below the pathogen inoculation.
Fig 2.
Phylogenetic tree inferred from a Maximum likelihood analysis based on a concatenated alignment of ITS, rpb2 and tub2 sequences of 25 isolates of Epicoccum and one isolate of Didymella.
The RAxML bootstrap support values (MLBS) and Bayesian posterior probabilities (BPP) are given at the nodes (BPP/MLBS). The tree was rooted to Didymella exigua (CBS 183.55). Ex-type cultures are emphasised in bold type. The scale bar indicates 0.1 expected changes per site.
Fig 3.
In vitro inhibition of esca-related fungi by Epicoccum spp. isolates.
Growth was measured 14 days post-inoculation on PDA medium in dual-culture with (A) Pch, Phaeomoniella chlamydspora; (B) Pmin, Phaeoacremonium minimum and (C) Fmed, Fomitiporia mediterranea. ‘Control s’ and ‘Control d’ indicate the colony area of the pathogen in single culture or dual culture, respectively, and ‘E’ indicates the Epicoccum isolate. Error bars represent the standard deviation from the mean; different letters indicate statistically significant differences (Tukey post hoc test, p ≤ 0.05).
Fig 4.
Interaction pathogen—Antagonist or pathogen—Pathogen after 14 d of incubation on PDA.
(i) Phaeomoniella chlamydospora (a), Phaeoacremonium minimum (b) and Fomitiporia mediterranea (c) inoculated on the left side, against Epicoccum layuense strain E24 inoculated on the right; (ii) the same pathogens against E. mezzettii strain E17, (iii) dual cultures of each pathogen.
Table 2.
Percent growth inhibition of esca-associated fungi by Epicoccum spp.
Fig 5.
Morphological changes of vegetative structures of esca-associated fungi upon interaction with Epicoccum layuense isolate E24 in dual culture plates.
Phaeomoniella chlamydospora isolate CBS 161.90 (left side) and E. layuense (right side) at interaction area at day eight (A); hyphae of P. chlamydospora with swollen chlamydospores (arrow) and an increasing of sporulation in the contact line with E. layuense (B-D) when compared to P. chlamydospora growing alone (E) at day ten. Phaeoacremonium minimum isolate CBS 110713 (left side) and E. layuense (right side) at the interaction zone at day eight (F); intermingled hyphae of both fungi at day ten (G) after the hyphae of P. minimum have tried to change their growth direction (left arrow) to avoid the contact with the antagonist (right arrow) at day six (H); agglomerates of P. minimum hyphae intermingled with the antagonist hyphae (I), conidia of P. minimum from the interaction zone (J) when compared to conidia from the P. minimum single culture (K), and hyphae of P. minimum growing alone (L), all at day ten. Fomitiporia mediterranea (left side) and E. layuense (right side) at the interaction zone at day eight (M); agglomerates and strands of F. mediterranea hyphae in an attempt to block the advance of E. layuense (N); hyphae of F. mediterranea denoting plasmolysis (arrow) (O) and clamp connections (arrow) (P); mycelium of F. mediterranea growing alone (Q). Scale bars represent A, F, M = 1 mm; B, G, H, L, N = 50 μm; C, E, I, O-Q = 20 μm; D, J, K = 10 μm.
Fig 6.
Effects of Epicoccum layuense E24 on grapevines.
Measurements of the shoot length (A) and brown wood streaking length (B), in cv. Touriga Nacional, four months after inoculation. Error bars represent the standard deviation from the means.
Table 3.
Shoot length (cm) of grapevine potted plants inoculated with water (control) or with Epicoccum layuense strain E24, Phaeoacremonium minimum (Pmin), Phaeomoniella chlamydospora (Pch) and the combination of E24 with Pmin (E24 x Pmin) or Pch (E24 x Pch).
Fig 7.
Brown wood streaking length in inoculated grapevine potted plants.
Grapevines were inoculated with water (control) or with Epicoccum layuense strain E24, Phaeoacremonium minimum (Pmin) and Phaeomoniella chlamydospora (Pch) alone, or combined with E24 (E24 x Pmin and E24 x Pch). Bars followed by the same letter do not differ statistically according to the Tukey’s test (p = 0.05).
Fig 8.
Frequency of Epicoccum layuense re-isolation from two grapevine cultivars and two areas of re-isolation (C 15 mm and D 45 mm).
E. layuense was inoculated alone (E24) or in combination with Phaeoacremonium minimum (E24 x Pmin) or Phaeomoniella chlamydospora (E24 x Pch). Different letters on the top of the bars represent statistical differences according to the Tukey’s test (p = 0.05).
Fig 9.
Frequency of re-isolation of inoculated pathogenic fungi.
Phaeoacremonium minimum (Pmin) and Phaeomoniella chlamydospora (Pch) were either inoculated alone, or in combination with Epicoccum layuense E24 (E24 x Pmin, E24 x Pch). Re-isolations occurred at 15 mm (C) and 45 mm (D) below the pathogens’ inoculation point in cultivars Cabernet Sauvignon and Touriga Nacional. Different letters indicate statistically significant differences after a Tukey’s post hoc test (p = 0.05).