C. Pintos Varela, V. Redondo Fernández,
O. Aguín Casal y J. P. Mansilla Vázquez
Estación Fitopatolóxica Do Areeiro,
Deputación Pontevedra, Subida a la Robleda s/n. 36153 Pontevedra, Spain
ABSTRACT: In November
2010, four grapevine plants of cv. Crimson from a vineyard located in Sevilla
(south Spain) revealed trunk cankers. Several pathogens were isolated,
including Cylindrocarpon liriodendri (2), Phaeoacremonium
aleophilum (2), Pleurostomophora richardsiae, Neofusicoccum parvum,
and Botryosphaeria dothidea (2). Among Botryosphaeriaceae fungi
isolated on potato dextrose agar (PDA) were two types that did not fit the
above mentioned species. Isolates of type 1 produced an abundant, gray mycelium
with a diurnal zonation that gradually became dark olivaceous. Mycelium growth
occurred from 5 to 37°C with an optimum at 28°C. Conidia were hyaline,
fusiform, aseptate, thin walled, but gradually became obscured and septate with
age, and measured (18.4-) 21.4 (-24.3) × (4.2-) 5.5 (-7.2) µm with
a length/width (L/W) ratio of 4.0 ± 0.5 (n = 100). Isolates of type 1
were identified as N. mediterraneum (3). Single-spore cultures of type 2
developed a whitish, dense, aerial mycelium and remained white up to 10 days on
PDA and darkened to gray thereafter. Mycelium growth occurred from 3 to
37°C with an optimum at 29 to 30°C. Conidia were hyaline, aseptate,
thick walled, oblong to cylindrical, sometimes becoming light brown and one or
two septate after discharge, and measured (24.6-) 30.2 (-42.8) × (10.9-)
14.3 (-18.6) µm with a L/W ratio of 2.1 ± 0.2 (n =100). Isolates
of type 2 were identified as Diplodia corticola (1). Nucleotide
sequences of the ribosomal internal transcribed spacer (ITS) region and the
ß-tubulin genes were used to confirm the identifications through BLAST
searches in GenBank. Comparison of the sequences of types 1 and 2 showed 99 to
100% homology with N. mediterraneum (HM443604 (4) and GU251836) and
D. corticola (AY268421 (1) and EU673117), respectively. Representative
sequences of N. mediterraneum (JF949757 and JF949756) and D.
corticola (JF949758 and JF949759) were deposited in GenBank. The
pathogenicity of one representative isolate of each of N. mediterraneum
and D. corticola was confirmed by inoculating 10 detached grapevine
canes (averaging 12 mm in diameter and 30 cm long) per isolate. A shallow wound
was made with a scalpel on the internodes. A colonized 6-mm agar plug, from the
margin of an actively growing colony, was inserted in every wound and sealed
with Parafilm. Ten grapevine canes controls received only sterile PDA agar
plugs. Canes were maintained at 25°C and 70% humidity. After 5 weeks, all
inoculated canes developed cankers and pycnidia around the inoculation site.
Vascular necroses that developed on the inoculated canes were an average of
28.6 mm for N. mediterraneum and 27.7 mm for D. corticola.
One-way analysis of variance and Tukey's test confirmed significant differences
in the extent of vascular necroses. The average necroses length in the
inoculated canes was significantly greater (P < 0.05) than the average
length of discoloration induced by the simulated inoculation process in the
control. Both pathogens were reisolated from all inoculated plants but not from
controls. To our knowledge, this is the first report of N. mediterraneum
and D. corticola as pathogens on grapevine in Spain.
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