Fungal trunk pathogens associated with wood decay of almond trees on Mallorca (Spain)

Abstract

Severe decline of almond trees has recently been observed in several orchards on the island of Mallorca (Balearic Islands, western Mediterranean Sea). However, the identity of the causal agents has not yet been investigated. Between August 2008 and June 2010, wood samples from branches of almond trees showing internal necroses and brown to black vascular streaking were collected in the Llevant region on the island of Mallorca. Several fungal species were subsequently isolated from the margin between healthy and symptomatic tissue. Five species of Botryosphaeriaceae (namely Botryosphaeria dothidea, Diplodia olivarum, D. seriata, Neofusicoccum australe and N. parvum), Eutypa lata, Phaeoacremonium iranianum and Phomopsis amygdali were identified based on morphology, culture characteristics and DNA sequence comparisons. Neofusicoccum parvum was the dominant species, followed by E. lata, D. olivarum and N. australe. First reports from almond include D. olivarum and Pm. iranianum. Two species are newly described, namely Collophora hispanica sp. nov. and Phaeoacremonium amygdalinum sp. nov.

Keywords: Botryosphaeriaceae; Collophora; Eutypa lata; Phaeoacremonium; Phomopsis amygdali; Prunus dulcis; almond dieback.

Fig. 1

Disease symptoms on almond trees on the island of Mallorca associated with fungal trunk pathogens. a, b. Dieback and wilting of branches; c–h. internal symptoms visible when transversal and longitudinal cuts were made in branches used for fungal isolation: black spots and dark brown to black streaking of the xylem tissue (d, h), circular (c, g) or sectorial necrosis (f), and wood discoloration (e).

Fig. 2

One of two most parsimonious trees obtained from heuristic searches of ITS, GAPDH and EF-1α gene sequences of Collophora species. Bootstrap support (1 000 replicates) above 70 % are shown at the nodes. Cadophora luteo-olivacea CBS 141.41 was used as outgroup. Ex-type strains for each species are indicated with a ‘T’ after the strain number.

Fig. 3

One of 90 most parsimonious trees obtained from heuristic searches of a combined alignment of the TB and ACT gene sequences. Bootstrap support (1 000 replicates) above 60 % are shown at the nodes. Pleurostomophora richardsiae and Wuestneia molokaiensis were used as outgroup. Ex-type strains for each species are indicated with a ‘T’ after the strain number. Thickened lines indicate branches present on strict consensus tree.

Fig. 4

Collophora hispanica. a. Colony on MEA that is stained red by the pigment exuded by the fungus; b. conidioma on pine needle; c–h. conidiogenous cells and conidia on hyphal cells; i, j. conidiophores formed in conidiomata on pine needles; k, l. microcyclic conidiation (indicated by arrows) in conidia from conidiomata (k) and from hyphal cells (l); m. conidia formed in conidiomata after 4 wk; n, o. endoconidia; p, q. conidia formed on hyphal cells after 4 wk; r. conidia formed on hyphal cells after 2 wk. a, b: DIC, c–r: DM. — Scale bars: a = 1 mm; b = 100 μm; c = 5 μm; scale bar for c applies to c–r.

Fig. 5

Phaeoacremonium amygdalinum. a–c. Sixteen-day-old colonies incubated at 25 °C on MEA (a), PDA (b) and OA (c); d–j. aerial structures on MEA; d–f. conidiophores with polyphialides (indicated by arrows); g, h. type III phialides; i. type III and type II phialides (indicated by arrow); j. type I phialides; k–t. aerial structures by using slide culture technique; k–m. branched conidiophores and type I phialides (indicated by arrows); n–p. type II phialides; q. type I phialides; r–s. Microcyclic conidiation; t. conidia; u–w. structures on the surface of and in MEA; u–v. adelophialides with conidia; w. conidia. — Scale bars: d = 10 μm; scale bar for d applies to d–w.