Molecular and phenotypic characterisation of novel Phaeoacremonium species isolated from esca diseased grapevines

Abstract

Petri disease and esca are very destructive grapevine decline diseases that occur in most countries where grapevine (Vitis vinifera) is cultivated. Phaeoacremonium species are among the principal hyphomycetes associated with symptoms of the two diseases, producing a range of enzymes and phytotoxic metabolites. The present study compared the phylogeny of a global collection of 118 Phaeoacremonium isolates from grapevines, in order to gain a better understanding of their involvement in Petri disease and esca. Phylogenetic analyses of combined DNA sequence datasets of actin and beta-tubulin genes revealed the presence of 13 species of Phaeoacremonium isolated from esca diseased grapevines. Phaeoacremonium aleophilum was the most frequently isolated species with an incidence up to 80 % of all isolates investigated. Species previously described mainly as human pathogenic species, namely Pm. alvesii, Pm. griseorubrum and Pm. rubrigenum are newly reported on grapevine from Turkey, Italy and Croatia, respectively. Phaeoacremonium viticola and Pm. scotyli represent new records for Italy, as well as Pm. mortoniae for Hungary and Croatia. In addition, four new species of Phaeoacremonium, namely Pm. croatiense, Pm. hungaricum, Pm. sicilianum and Pm. tuscanum are newly described from grapevine based on morphology, cultural characteristics, as well as molecular phylogeny.

Keywords: Phaeoacremonium; actin; esca; morphology; phylogeny; β-tubulin.

Fig. 1

Symptoms associated with esca of grapevine: chlorosis and necrosis on the leaves showing typical ‘tiger-like’ pattern.

Fig. 2

Symptoms associated with esca of grapevine: black measles on the berries.

Fig. 3

Typical wood symptoms in a vine affected by esca: brown-red wood, black streaking, and central white decay.

Fig. 4

Internal symptoms seen when transversal or longitudinal cuts were made in the trunk or cordon of vines used for fungal isolation. a, b. Black spots and dark brown to black streaking of the xylem tissues; c. cross section showing sectorial necrosis; d. longitudinal section showing wood discoloration; e. central brown-red necrosis; f. cross section showing a central white rot surrounded by brown-red wood.

Fig. 5

One of 317 most parsimonious trees obtained from heuristic searches of a combined alignment of the TUB and ACT gene sequences (length = 1304 steps, CI = 0.508, RI = 0.922, RC = 0.468, HI = 0.492). Bootstrap support values above 64 % are shown at the nodes. Pleurostomophora richardsiae was used as outgroup. Accessions numbers of sequences obtained from the GenBank nucleotide database are indicated on the tree in the format TUB/ACT. Ex-type strains are emphasised in bold. Names in blue are novel species described in this study. New species described in this study; Human pathogenic species; Species isolated from grapevine; * Human pathogenic species newly reported from grapevine.

Fig. 5

One of 317 most parsimonious trees obtained from heuristic searches of a combined alignment of the TUB and ACT gene sequences (length = 1304 steps, CI = 0.508, RI = 0.922, RC = 0.468, HI = 0.492). Bootstrap support values above 64 % are shown at the nodes. Pleurostomophora richardsiae was used as outgroup. Accessions numbers of sequences obtained from the GenBank nucleotide database are indicated on the tree in the format TUB/ACT. Ex-type strains are emphasised in bold. Names in blue are novel species described in this study. New species described in this study; Human pathogenic species; Species isolated from grapevine; * Human pathogenic species newly reported from grapevine.

Fig. 6

Phaeoacremonium croatiense. a–c. Sixteen days old colonies on 2 % MEA (a), PDA (b) and OA (c). — d–o. Aerial structures on 2 % MEA; d, e. conidiophores; f–i. type I phialide; j–l. type II phialide; m, n. type III phialide; o. conidia. — p–q. Structures on the surface of and in 2 % MEA: adelophialides with conidia; all from CBS H-20120 (holotype); d–q: DIC. — Scale bars: d–p = 10 μm; scale bar for d applies to e–n and q.

Fig. 7

Phaeoacremonium hungaricum. a–c. Sixteen days old colonies on 2 % MEA (a), PDA (b) and OA (c). — d–p. Aerial structures on 2 % MEA. d–g. conidiophores; h–k. type I phialide; i–o. type II phialide; p. conidia. — q–s. Structures on the surface of and in 2 % MEA; q–r. adelophialides with conidia; s. conidia; all from CBS H-20119 (holotype); d–s: DIC. — Scale bars: d–s = 10 μm; scale bar for d applies to e–k and q–s; bar for l applies to m–o.

Fig. 8

Phaeoacremonium rubrigenum. a–c. Sixteen days old colonies on 2 % MEA (a), PDA (b) and OA (c). — d–q. Aerial structures on 2 % MEA; d. mycelium showing prominent exudate droplets observed as warts; e–h. single conidiophores; i, j. branched conidiophores; k, l. type I phialide; m, n. type II phialide; o, p. type III phialide; q. conidia. — r–t. Structures on the surface of and in 2 % MEA: adelophialides with conidia; all from H-20121 (holotype); d–t: DIC. — Scale bars: d = 10 μm; scale bar for d applies to i–k and k–t.

Fig. 9

Phaeoacremonium sicilianum. a–c. Sixteen days old colonies on 2 % MEA (a), PDA (b) and OA (c). — d–s. Aerial structures on 2 % MEA; d. mycelium showing prominent exudate droplets observed as warts; e–h. single conidiophores; i–l. branched conidiophores; m–p. type I phialide; q. type II phialide; r. type III phialide; s. conidia. — t–u. Structures on the surface of and in 2 % MEA: adelophialides with conidia; all from CBS H-20118 (holotype); d–u: DIC. — Scale bars: d–u = 10 μm; scale bar for d applies to e and i–k; bar for n applies to o–r.

Fig. 10

Phaeoacremonium tuscanum. a–c. Sixteen days old colonies on 2 % MEA (a), PDA (b) and OA (c). — d–q. Aerial structures on 2 % MEA; d. mycelium occurring in bundles of up to nine; e, f. mycelium showing prominent exudate droplets observed as warts; g–j. single conidiophores; k, l. type II phialide; m, n. type I phialide; o, p. type III phialide; q. conidia. — r–v. Structures on the surface of and in 2 % MEA; r, s. coremium-like structures; t–v. adelophialides with conidia; all from CBS H-20118 (holotype); r, s: DM; d–q: DIC. — Scale bars: d–v = 10 μm; scale bar for d applies to e–k and p–s; bar for m applies to n; bar for t applies to u–v.