Disease Notes {continued)
First Report of Grapevine Trunk Disease Caused hy Botryosphaerla
obtusa in China. J.-Y. Yan, Y.-L. Peng, and Y. Xie, Department of
Plant Pathology, China Agricultural University, Beijing 100193, China;
X.-H. Li, Institute of Plant and Environment Protection, Beijing Academy
of Agriculture and Forestry Science, Beijing 100097, China; S.-W. Yao,
Laboratory of Plant Resource Utilization, Hunan Agricultural University,
Changsha, China; M.-L. Tang, Yantai Agricultural Science and Technology
Institute, Yantai, 265500, China; and Z.-Y. Wang, Institute of Plant
Protection, Chinese Academy of Agricultural, Beijing 100193, China. The
research was supported by the earmarked fund for Modern Agro-Industry
Technology Research System (nycytx-30). Plant Dis. 95:616, 2011;
published online as doi;10.1094/PDIS-ll-10-082L Accepted for publica-
tion 27 February 2011.
In September 2010, grapevine {Vitis vinifera) trunk diseases were
observed in several vineyards of Yantai District in Shandong Provinces and
Changli County of Hebei Provinces of China. Characteristic symptoms of
Botryosphaeria canker were apparent, including dark brown discoloration
on the trunk (visible in cross-section), cob base shriveling, drying of fruit
clusters, and berry falling (2). To identify the causal pathogen, culturing of
fungi was attempted from 387 small pieces of tissue from the canker mar-
gins of 43 diseased plants. Samples were surface disinfected by placing
them in 75% ethanol for 1 min and rinsing with sterilized water three
times before culturing on potato dextrose agar (PDA) at 28°C for 7 to 10
days. Fungi isolated were single spored to obtain pure cultures. On the
basis of colony characteristics on PDA, 18 isolates from the 387 tissue
pieces were eventually identified as Botryosphaeria obtusa (1), Most of
the other fungi isolated were B. dothidea. B. obtusa colonies were grayish
white, becoming dark brown with age, and pycnidia were formed after
incubation for approximately 7 days. Conidia measured 8 to 11 x 17 to
26 pm (n = 50). Two isolates were used for rDNA internal transcribed
spacer (ITS) sequence analysis with primers ITSl and ITS4 (3). PCR
products were separated by electrophoresis and bands were purified for
legation with PMD-18T (Takara Company, Dalian, China) vector for
sequencing. BLAST searches of two ITS sequences had 99 to 100%
identity to B. obtusa. EFl-a and ß-tubulin sequence analysis gave similar
results. Koch's postulates were completed in the greenhouse on grape
shoots inoculated with two isolates of B. obtusa originally isolated from
diseased plants in the field. Inoculations were made on green shoots of V.
vinifera cv. Dunkelfelder T. Six shoots were inoculated per isolate by
wounding with a 4-mm cork borer (2 mm deep) and placing a colonized
agar plug from a 5-day-old culture on the wound and wrapping it with
Parafilm. Controls were mock inoculated with an agar plug from sterile
PDA. Inoculated shoots were incubated in the dark under moist conditions
in the laboratory for 8 to 10 days at 25°C. Inoculated shoots had necrotic
cankers after 8 to 10 days and B. obtusa was recovered from each canker
margin. The results indicated that some grapevines in China with
symptoms of Botryosphaeria canker were infected by B. obtusa. To our
knowledge, this is the first report of this pathogen causing trunk disease on
grapevine in China.
References: (1) A. Taylor et al. Australas. Plant Pathol. 34:187. 2005. (2) J. R. Úrbez-
Torres et al. Plant Dis. 92:519. 2008. (3) T J. White et al. Page 315 in: PCR Proto-
cols: A Guide to Methods and Applications. Academic Press. San Diego, 1990.
e-
First Report of Basal Stem Rot and Foliar Blight Caused hy Fythium
sylvaticum on Miscanthus sinensis in Illinois. M. O. Ahonsi and
B. O. Agindotan, Energy Biosciences Institute, University of Illinois,
Urbana 61801; and M. E. Gray and C. A. Bradley, Energy Biosciences
Institute and Department of Crop Sciences, University of Illinois, Urbana
61801. Plant Dis. 95:616, 2011; published online as doi:10.1094/PDIS-08-
10-0592. Accepted for publication 14 February 2011.
Miscanthus sinensis Anderss., a perennial grass, is native to eastern
Asia. It has been widely grown as an ornamental in temperate regions of
the world, including the United States, and recently has become an impor-
tant component of public and private sector bioenergy feedstock Miscan-
thus selection programs. In August 2008, stem rot and blight was observed
on M. sinensis plants in two irregular patches, -2 to 2.5 x 1 to 1.5 m each
in a trial plot that was preceded by corn, at the University of Illinois
Energy Farm near Urbana, IL. At the time of the observation, most plants
were dead and the wilted tillers had black, soft rotted basal stems. A few
616 Plant Disease/Vol. 95 No. 5
plants were stunted and the crowns of the tillers had black-to-brown soft
rot. Some tillers' leaves were dead and others had turned light brown.
Sample tissue fragments were surface disinfested in 0.5% NaOCl and
plated on 1% water agar (WA). After 3 days of incubation in the dark at
23°C, colonies were transferred to com meal agar (CMA), potato dextrose
agar (PDA), or 10% V8 juice agar and incubated at 23°C under continuous
white light for up to 2 weeks. Morphological charactedstics of the isolates
correspond to those originally described for Pythium sylvaticum W.A.
Campb. & J.W. Hendrix (1). The mycelia grew and covered the
10-cm-diameter plates within 5 days. On PDA, the culture was a creamy
white mycelial mat of coenocytic hyphae. The isolates produced only
globose, terminal or intercalary hyphal swellings ranging from 28 to
48 |im in diameter, but no oogonia were produced on any of the three
growth media. No zoospores were produced when agar blocks bearing
mycelium were flooded with distilled water or 1% soil water. Sequence
analysis was performed with the internal transcribed spacer (ITS) region
of the rDNA amplified with primer pair ITS1/ITS4 (3) and the
mitochondrially encoded cytochrome c oxydase subunit II (cox II) gene
using primers FM58/FM66 (2). The resulting 871-bp ITS nucleotide
sequence (Accession No. HM991706) was identical among all three
isolates analyzed and 99% identical (100% coverage) to ITS sequences of
multiple isolates of P. sylvaticum in GenBank. Likewise, the 544-bp cox II
sequence (Accession No. HQ454429) was 99% identical (97% coverage)
to cox II sequences of multiple isolates of P. sylvaticum. Six pots of M.
sinensis seedlings were inoculated by placing two CMA plugs of a
2-week-old culture of isolate F71 at the crown. The control pots were
mock inoculated with sterile CMA plugs. The plants were incubated at
-90% relative humidity (RH) and 25°C day and 22°C night for 3 days, and
thereafter left on the greenhouse bench at -65% RH with alternating 9 h of
darkness and 15 h of light. Three weeks after inoculation, two of the
inoculated seedlings wilted, others were stunted with leaves wilting from
the tip downwards and the stems rotting from the crown upward. A thick
mat of mycelia was seen on the rotted basal stems. No symptoms were
observed in the control. P. sylvaticum was reisolated from both the rotted
basal stems and the wilted foliage. To our knowledge, this is the first
report of P. sylvaticum on M. sinensis. Infestation of farm soils with P.
sylvaticum could limit M. sinensis biomass production significantly by
limiting seedling establishment.
References: (1) W. A. Campbell and F. F Hendrix. Myeologia 59:274. 1967. (2) .F
M. Martin. Mycologia 92:711, 2000. (3) T. J. White et al. Page 38 in: PCR Protocols:
A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press. San
Diego. 1990.
* The e-Xtra logo stands for "electronic extra" and indicates this Disease Note online
contains supplemental material not included in the print edition.
First Report of Flyspeck Caused by Zygophiala wisconsinensis on
Sweet Persimmon Fruit in Korea. J. Kim and O. Choi, Department of
Applied Biology and Institute of Agriculture and Life Science,
Gyeongsang National University, Jinju 660-701, Korea; and J.-H. Kwon,
Gyeongsangnam-do Agricultural Research and Extension Services, Jinju
660-360, Korea. Plant Dis. 95:616, 2011; published online as
doi: 10.1094/PDIS-12-10-0917. Accepted for publication 14 February 2011.
Sweet persimmon (Diospyros kaki L.), a fruit tree in the Ebenaceae, is
cultivated widely in Korea and Japan, the leading producers worldwide
(2). Sweet persimmon fruit with flyspeck symptoms were collected from
orchards in the Jinju area of Korea in November 2010. The fruit had fun-
gal clusters of black, round to ovoid, sclerotium-like fungal bodies with no
visible evidence of a mycelial mat. Orchard inspections revealed that
disease incidence ranged from 10 to 20% in the surveyed area (approxi-
mately 10 ha) in 2010. Flyspeck symptoms were observed on immature
and mature fruit. Sweet persimmon fruit peels with flyspeck symptoms
were removed, dried, and individual speck lesions transferred to potato
dextrose agar (PDA) and cultured at 22°C in the dark. Fungal isolates were
obtained from flyspeck colonies on 10 sweet persimmon fruit harvested
from each of three orchards. Fungal isolates that grew from the lesions
were identified based on a previous description (1). To confirm identity of
the causal fungus, the complete intemal transcribed spacer (ITS) rDNA
sequence of a representative isolate was amplified and sequenced using
primers ITSl and ITS4 (4). The resulting 552-bp sequence was deposited
in GenBank (Accession No. HQ698923). Comparison with ITS rDNA
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