This is the first report of Neofusicoccum australe associated with grapevine dieback in Italy. Fungal isolates obtained from symptomatic tissues were identified on the basis of morphological and cultural characteristics as well as ITS sequence data. Pathogenicity was verified by inoculation of excised green grapevine shoots from cv. Cannonau under controlled laboratory condition

LINALDEDDU, Benedetto T.

SCANU, Bruno

SCHIAFFINO, Angela

SERRA, Salvatorica

Firenze University Press: E-Journals

417
Phytopathol. Mediterr. (2010) 49, 417−420
Corresponding author: B.T. Linaldeddu
Fax: +39 079 229316
E-mail: ben@uniss.it
Grapevine trunk diseases are a serious eco-
nomic problem in Sardinian vineyards (Serra et 
al., 2008; Serra et al., 2010). These diseases are 
usually linked to infections by Eutypa lata and 
other fungal pathogens belonging to the genera 
Fomitiporia, Phaeomoniella and Phaeoacremo-
nium (Mugnai et al., 1999, Mostert et al., 2006, So-
snowski et al., 2007). Moreover, several species of 
Botryosphaeriaceae have recently been recognized 
as important pathogens of grapevines worldwide 
(Phillips, 2002, Van Niekerk et al., 2006; Úrbez-
Torres and Gubler, 2009, Pitt et al., 2010). Since 
only limited information about the occurrence and 
identity of the Botryosphaeriaceae associated with 
dieback and trunk diseases of grapevines in Sardi-
nia is available, a field survey has been in progress 
since 2009.
Grapevines showing poor growth, defoliation, 
cankers and dieback (Figure 1a), were collected 
from six vineyards (local varieties Cannonau and 
Vermentino) in northern Sardinia. Common inter-
nal wood symptoms were wedge-shaped necrotic 
sectors and/or brown streaks below the bark run-
ning from cordon to rootstock, 2–3 cm wide and 
a few millimetres thick (Figure 1b, c). Fragments 
measuring approximately 5 mm2 were aseptically 
cut from the margin of infected tissues and cul-
tured in Petri dishes containing malt extract agar 
(MEA, Oxoid Ltd, Basingstoke, UK). After incuba-
tion at room temperature for 5 days, fungal colo-
nies resembling Botryosphaeriaceae were sub-cul-
tured onto both potato dextrose agar (PDA, Oxoid 
Ltd) and malt extract yeast agar (MEYA, 2% malt 
extract, 0.2% yeast extract, 1.5% agar) and incu-
bated at room temperature until differentiation 
of the reproductive structures. To induce sporu-
lation, the fungal isolates were sub-cultured on 
PDA with sterile pine needles. Microscope slides 
of conidia and ascospores were prepared in lacto-
phenol for morphological examination. Spore di-
mensions were recorded using an Optika™ Vision 
Pro version 2.7 digital camera connected to a Leitz 
Diaplan (Ernest Leitz, Wetzlar, Germany) micro-
scope. The colony appearance of cultures growing 
Key words: Botryosphaeriaceae, grapevine diseases, Sardinia.
Summary. This is the first report of Neofusicoccum australe associated with grapevine dieback in Italy. 
Fungal isolates obtained from symptomatic tissues were identified on the basis of morphological and cultural 
characteristics as well as ITS sequence data. Pathogenicity was verified by inoculation of excised green gra-
pevine shoots from cv. Cannonau under controlled laboratory conditions.
Benedetto T. LINALDEDDU, Bruno SCANU, AngelA SCHIAFFINO and SAlvAtoricA SERRA
Dipartimento di Protezione delle Piante – sezione di Patologia vegetale, Università degli Studi di Sassari,
Via E. De Nicola 9, 07100 Sassari, Italy
First report of Neofusicoccum australe associated with grapevine
cordon dieback in Italy
Phytopathologia Mediterranea
B. Linaldeddu et al.
418
on PDA at 25°C in the dark for 1 week was also 
recorded. 
Fungal genomic DNA was extracted from 20 
mg of mycelium following the CTAB method of 
Doyle and Doyle (1987). The entire Internal Tran-
scribed Spacer region (ITS) of the rDNA, inclu-
ding the 5.8S gene, was amplified by polymerase 
chain reaction (PCR) using the primers ITS1 and 
ITS4 (White et al., 1990). The amplifications were 
performed using a Hybaid PCR Express and the 
following protocol: 95°C for 3 min initial denatura-
tion; 35 cycles of 95°C for 15 s, 55°C for 20 s, 72°C 
for 1 min; and 72°C for 5 min final extension. The 
PCR products were purified using the Eurogold 
extraction kit (EuroClone S.p.A., Milano, Italy) 
following the manufacturer’s instructions. Both 
strands were sequenced by the BMR Genomics 
DNA sequencing service (www.bmr-genomics.it). 
The nucleotide sequences were read and edited 
with FinchTV 1.4.0 (Geospiza, Inc.; http://www.
geospiza.com/finchtv), aligned using ClustalW 
(www.ebi.ac.uk/clustalw/) and compared with se-
quences deposited in GenBank using the BLAST 
software (http://blast.ncbi.nlm.nih.gov). 
A total of 113 Botryosphaeriaceous isolates 
were obtained from 15 of the plants sampled. 
Some isolates (44%) sporulated within one or two 
months. The conidial morphology and cultural fe-
Figure 1. Grapevine disease symptoms associated with Neofusicoccum australe: cordon dieback (a); cordon wedge-
shaped necrotic sector (b); trunk brown stripes under the bark (c).
a
b c
419Vol. 49, No. 3 December, 2010
B. Linaldeddu et al. First report of Neofusicoccum australe on grapevine in Italy
atures of six isolates from one vineyard (cv. Can-
nonau) were in close agreement with the morpho-
logical description of both Neofusicoccum australe 
and N. luteum (Phillips et al., 2002, Slippers et al., 
2004). On PDA at 25°C, these isolates developed 
a moderately dense mycelium, with a yellow pig-
ment diffusing into the medium that became dark 
grey after 4 to 5 days. The conidia were hyaline, 
aseptate, fusiform with a truncate base and mea-
sured 18.7±2×6.8±0.9 μm, with a length/width ra-
tio of 2.8±0.4 (mean±SD, n=100). Average conidial 
size was less than the 24.7×5.1 μm previously re-
ported for N. australe by Slippers et al. (2004), but 
similar to the size reported for N. australe by Laz-
zizera et al. (2008). Conidial size was also similar 
to the size reported for N. luteum by Phillips et al. 
(2002). Only one isolate produced ascomata and co-
nidiomata in the same Petri dish on PDA within 2 
months. Ascospores were hyaline, aseptate, fusoid 
to ovoid, and measured 20.2±1.2×8.5±0.7 μm, with 
a length/width ratio of 2.4±0.2 (mean±SD, n=50).
The identity of this Botryosphaeriaceae species 
was defined by analysis of the ITS sequence of a re-
presentative isolate. BLAST searches in GenBank 
revealed that the sequence of this isolate differed 
by only 1 bp from the sequence of the ex-type iso-
late of N. australe (AY339262) and other isolates 
(e.g. EU856059, FJ157192). However, it had 100% 
similarity to sequences of other isolates identified 
as N. australe including isolates from olives in 
southern Italy (EF638770–EF638778) and pista-
chio in Spain (EU375516). Furthermore, the se-
quence of our isolate differed by three bp from the 
ex-type isolate (AY259091) and other isolates of 
N. luteum. Thus we consider that the isolate from 
Sardinian grapevines is N. australe.
The representative sequence of N. australe 
strain BOT48 obtained in this study was deposited 
in GenBank (accession number HQ011406). The 
strain BOT48 was stored in the culture collection 
of the Department of Plant Protection at the Uni-
versity of Sassari. Its pathogenicity was verified 
by inoculating it on five excised green grapevine 
shoots (30 cm in length) from cv. Cannonau. A 
mycelial plug (3 to 4 mm2) taken from the margin 
of an actively growing colony on PDA was placed 
in a shallow wound (~3 mm) made by a scalpel on 
the middle of each shoot. The inoculation point 
was covered with cotton wool soaked in sterile 
water and wrapped with Parafilm®. The inocula-
ted shoots were placed in a beaker containing 200 
mL of sterile distilled water and maintained in an 
Figure 2. Grapevine shoot showing dark-brown lesion 12 days after inoculation with Neofusicoccum australe (top). 
Asymptomatic control shoot (bottom).
Phytopathologia Mediterranea
B. Linaldeddu et al.
420
enclosed transparent plastic bag at room tempe-
rature (18–30°C). Twelve days after inoculation, 
the shoots displayed dark-brown to black disco-
loration, measuring 7.3±3.5 cm (mean±SD) of the 
bark and vascular tissues (Figure 2). The patho-
gen was successfully reisolated from the margin of 
symptomatic tissues, thus fulfilling Koch’s postu-
lates. Five control shoots inoculated with sterile 
PDA plugs remained symptomless.
The plurivorous pathogen N. australe was 
originally thought to be native to the southern 
hemisphere (Slippers et al. 2004; Burgess et al., 
2006). However, it has recently been reported on 
a variety of hosts, including grapevine, worldwi-
de (Armengol et al., 2008, Amponsah et al., 2009, 
Espinoza et al., 2009). Until now, in Italy N. au-
strale was only found associated with drupe rot of 
olives (Lazzizera et al., 2008). Interestingly, our 
isolates conform morphologically and phylogene-
tically more closely to the isolates of Lazzizera et 
al. (2008) than to typical isolates of N. australe. 
To our knowledge, this is the first report of N. 
australe on grapevine in Italy. Further investiga-
tions are currently underway to study the spread 
of this pathogen in Sardinian vineyards and its 
role in grapevine dieback.
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Accepted for publication: November 10, 2010


First report of Neofusicoccum australe associated with grapevine cordon dieback in Italy

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First report of Neofusicoccum australe associated with grapevine cordon dieback in Italy

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