First report of shoot blight of persimmon caused byDiaporthe
neotheicolain Australia
H. Golzar &Y. P. Ta n &R. G. Shivas &C. Wang
Received: 27 March 2012 / Accepted: 24 May 2012 / Published online: 23 June 2012
#
Australasian Plant Pathology Society Inc. 2012
AbstractShoot blight symptom was found on persimmon
(Diospyros kaki) in southern Western Australia in December
2010. The pathogen was isolated and identified asDia-
porthe neotheicolaon the basis of morphology, sequence
analysis of the internal transcribed spacer (ITS) and the
translation elongation factor 1-α(TEF). A pathogenicity test
was conducted and Koch’s postulates were fulfilled by re-
isolation of the fungus from diseased tissues. This is the first
report ofD.neotheicolacausing shoot blight on persimmon
in Australia and worldwide.
KeywordsDiospyros kaki.
Phomopis.
Shoot blight.
Teleomorph
The genusDiaporthe(includingPhomopsis)containsa
large number of endophytes, saprobes and plant pathogens
that cause blights, cankers, die-backs and wilts on a range of
plants of economic importance (Uecker1988; Mostert et al.
2001; Udayanga et al.2011).Diaporthe neotheicola(syno-
nymPhomopsis theicola) has been reported as a weak
pathogen from several hosts, includingAspalathus linearis
(van Rensburg et al.2006),Camellia sinsensis(Uecker
1988),Foeniculum vulgare(Santos and Phillips2009),Pru-
nus dulcis(Diogo et al.2010)andProtea,Pyrus,Vi t i svinifera(Mostert et al.2001; van Niekerk et al.2005).
Phomopsis diospyriwas reported causing shoot blight and
dieback on persimmon in Greece (Thomidis and Navrozidis
2009). In ItalyP. maliwas reported as the cause of severe
dieback of persimmon (Rosciglione1983). In both cases the
identifications of the fungi were not confirmed by molecular
methods and the species names utilized should be treated
with caution. There has been no report, in the scientific
literature, of the occurrence ofD.neotheicolaon persimmon
in Australia or elsewhere in the world, although the Austra-
lian Plant Disease Database (APDD) includes records of
Phomopsissp. on persimmon from NSW, QLD, VIC and
WA. Persimmon is a deciduous tree that has been cultivated
in Australia for over 100 years. Japanese cultivars of non-
astringent persimmons were introduced into Australia in the
1980s. Persimmon production is spread across the states and
Western Australia produces 5 % percent of total production
in Australia (PAI.2009).
In December 2010, persimmon plants with shoot blight,
twig girdling and wilt symptoms (Fig.1a) from southern
Western Australia were sent to the Department of Agricul-
ture and Food, Plant Diseases Diagnostic lab at South Perth.
Severe decline of the persimmon trees has occurred due to
loss of blighted shoots and it has become prevalent in the
persimmon production areas in southern Western Australia.
The disease developed during spring on the current season’s
shoots, extended rapidly, disrupted vascular tissue, and
caused shoots to wilt in the late spring and early summer.
Tissue was excised from the leading edge of stem lesions
and surface-sterilised by immersion in a 1.25 % aqueous
solution of sodium hypochlorite for 2 min, rinsed in sterile
water and dried in a laminar flow cabinet. The pieces were
then either (a) placed on potato dextrose agar (PDA) and
incubated at 22 ± 3 °C for 7 days, fungal colonies subse-
quently sub-cultured onto PDA and then single-spored to
H. Golzar (*) :C. Wang
Department of Agriculture and Food Western Australia,
3 Baron-Hay Court South Perth,
South Perth, WA 6151, Australia
e-mail: hossein.golzar@agric.wa.gov.au
Y. P. T a n
:R. G. Shivas
Department of Employment, Economic Development
and Innovation, Plant Pathology Herbarium (BRIP),
Plant Biosecurity Science,
GPO Box 267, Brisbane, QLD 4001, Australia Australasian Plant Dis. Notes (2012) 7:115–117
DOI 10.1007/s13314-012-0061-y
obtain pure cultures; or (b) placed in trays on moist filter
paper and incubated at 25 °C with a 12-h dark and light
cycle for 2 weeks. Fungal colonies with similar morpholog-
ical characteristics were consistently isolated both from agar
plates and incubated plant specimens. After 14 d the fungal
colonies were cottony white, pale-grey on the reverse side of
the agar plates. Conidiomata were globose to subglobose,
360–700μm in diam. Pycnidia were dark brown, scattered
or in groups. Conidiophores cylindrical hyaline 6.5–25 ×
1.5–3μm. Alpha conidia unicellular, guttulate, fusoid with
obtuse ends 7–10 × 2.5–4μm. Beta conidia hyaline, asep-
tate, filiform curved with rounded ends 23–30 × 1.2–1.5
(Fig.2). Some pycnidia produced only one type of conidia.
Pycnidia were embedded in the stem tissues, sub-epidermal
scattered with a short neck protruding to the surface
(Fig.1b). Both cultural and morphological characteristics
of the fungal isolates were similar to those described forD.
neotheicola(Santos and Phillips2009). Representative iso-
lates were deposited in the Western Australia Plant Pathogen
Collection (WAC13427) and the Queensland Plant Pathol-
ogy Herbarium (BRIP 54603).
Two representative isolates identified asD. neotheicola
were grown on PDA for 2 weeks at 25 °C. DNA wasextracted from fungal mycelium with the Gentra Puregene
kit (Qiagen, Melbourne, Vic., Australia) according to the
manufacturer’s instructions. PCR amplification was con-
ducted using the Phusion High-Fidelity PCR Master Mix
(Finnzymes, Espoo, Finland), which consisted of 12.5μLof
2 x Master Mix with HF Buffer, 0.5μL each of 10 mM of
forward and reverse primers, and 1μL of DNA template.
The internal transcribed spacer (ITS) region was amplified
with primers ITS1 and ITS4 (White et al.1990), and part of
the translation elongation factor 1-α(TEF) was amplified
with primers EF1-728 F (Carbone & Kohn1999) and EF2
(O’Donnell et al.1998). PCR products were amplified in a
Bio-Rad C1000 thermal cycler (Bio-Rad Laboratories, Her-
cules, California, USA) using the following conditions: 98 °C
for 30 s, 30 cycles at 98 °C for 10s, 55 °C for 30 s, 72 °C for
30 s, followed by a 5 min final extension at 72 °C. The
products were sequenced by Macrogen Incorporated (Seoul,
Korea) using the AB 3730xl DNA Analyser (Applied
Biosystems, Forster City, California, USA). The ITS and
TEF sequences of BRIP 54603 (GenBank accession
JQ809272 and JQ809273, respectively) are identical to
the ex-type culture ofD. neotheicolaCBS 123208
(EU814480 and GQ250315).
acb
Fig. 2Diaporthe neotheicola.
(a) conidiophores andα
conidia; (b) conidiophores and
βconidia; (c)αandβconidia.
Bars010μm
b a
Fig. 1Diaporthe neotheicola.
(a) shoot blight symptoms on
persimmon; (b) pycnidia on the
infected shoots. Bars05 and
0.5 cm respectively
116H. Golzar et al.
A pathogenicity test was carried out on 3 year old per-
simmon plants in the glasshouse. Two representative iso-
lates ofD. neotheicola(14-d-old cultures) were used for
inoculation of wounded and non-wounded shoots. The
shoots were trimmed to a uniform length of 30 cm. A single
wound was made in each of the shoots by pin-prinking them
with a syringe needle. One mycelial plug (5 mm in diam.)
was placed on each wound. Mycelial plugs were fixed onto
the wound sites with parafilm to prevent contamination and
desiccation, and then removed after 7 day. Agar plugs
without fungal colonies were placed on wounded and non-
wounded branches of plants, serving as controls. Inoculated
plants were placed in the glasshouse at 25 °C and a 12-
h dark and light cycle. After 3 weeks disease symptoms
were identical to those seen on naturally infected plants. The
symptom development was accelerated on wound-
inoculated compared with non-wound inoculated shoots.
Controls remained asymptomatic. Koch’s postulates were
fulfilled by re-isolation ofD. neotheicola. This fungus was
hence shown to be pathogenic to persimmon causing shoot
blight. The identity ofD. neotheicolawas confirmed by
morphology and DNA sequence data. To our knowledge,
this is the first report ofD. neotheicolacausing shoot blight
on persimmon in Australia and worldwide.
AcknowledgmentsThe authors would like to thank Ms Nuccia
Eyres and Ms Christine Wood for providing information and technical
assistance.
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