J Gen Plant Pathol (2010) 76:358361
DOI 10.1007/s10327-010-0255-0
DISEASE NOTE
Choanephora rot of floral tops of Hyoscyamus muticus caused
by Choanephora cucurbitarum
Fatma F. Abdel-Motaal Magdi A. El-Sayed
Soad A. El-Zayat Mortada S. M. Nassar
Shin-ichi Ito
Received: 11 February 2010 / Accepted: 14 July 2010 / Published online: 17 August 2010
The Phytopathological Society of Japan and Springer 2010
Abstract Floral rot of Egyptian henbane (Hyoscyamus
muticus L.) was found on potted plants in a greenhouse in
Yamaguchi city, Japan, in the late summer of 2008 and
2009. The symptoms were identical to those of rots caused
by Choanephora species. The pathogen was isolated and
identified as C. cucurbitarum (Berkeley and Ravenel)
Thaxter. This new disease was named Choanephora rot
(Kougai-kabi-byo) of Egyptian henbane.
Keywords Choanephora rot
Choanephora
cucurbitarum
Egyptian henbane
Hyoscyamus muticus
Introduction
Egyptian henbane (Hyoscyamus muticus L.) is a solana-
ceous plant that produces several tropane alkaloids among
which hyoscyamine and scopolamine are important as
pharmaceutical agents (Oksman-Caldentey and Hiltunen
1996). Because of the complexity and difficulties involved
in the chemical synthesis of these alkaloids, both com-
pounds are instead extracted from the plant (Hashimoto
et al. 1993). Although a recent study showed that hyo-
scyamine and other non-alkaloidal agents produced by this
plant have antifungal activity against several fungal species
(Abdel-Motaal et al. 2009b), when these plants were grown
F. F. Abdel-Motaal
M. A. El-Sayed
S. Ito (&)
Department of Biological and Environmental Sciences,
Faculty of Agriculture, Yamaguchi University,
1677-1 Yoshida, Yamaguchi 753-8515, Japan
e-mail: shinsan@yamaguchi-u.ac.jp
S. A. El-Zayat
M. S. M. Nassar
Department of Botany, Faculty of Science,
South Valley University, Aswan 81528, Egypt
123
under certain greenhouse conditions, they developed a leaf
spot disease caused by Cladosporium herbarum (Abdel-
Motaal et al. 2009a). During the flowering of Egyptian
henbane grown in greenhouses in the late summer of 2008
and 2009, the flowering tops, including flowers, leaves and
stem, developed a severe rot that killed the entire top. We
isolated and identified the pathogen; when we inoculated
host plants, the rot symptoms appeared, and we then
reisolated the pathogen from the inoculated plant.
Pathogen and symptoms
The flowering tops of H. muticus plants grown in pots in
greenhouses rotted in late summer, when the temperature
was between 20 and 30C and the humidity was high. At
first, the disease developed on aged flower petals, wilting
and rotting them, and then it extended to the calyx and
flower stalk where the stalk joins the stem (Fig. 1a). The
rot then extended up the stem to the entire flower top,
including the leaves and other flower buds. Subsequently,
the entire upper part of the plant withered and died
(Fig. 1b). Fungal hyphae and abundant sporangiophores
with fertile sporangial heads were clearly seen on all the
infected parts of the plant (Fig. 1ce).
Isolation and identification of the pathogen
The diseased flower was detached from the plant and cul-
tured on potato dextrose agar (PDA) medium either by
shaking the spores directly onto the medium in the petri
dish or by placing the flower on it. The cultured petri dishes
were incubated at 25C. Within 2 days, the fungal mycelia
had visibly colonized the PDA, and a single hypha was
J Gen Plant Pathol (2010) 76:358361 359

Fig. 1 Choanephora rot in

Egyptian henbane (Hyoscyamus
muticus L.) and morphology of
the causal agent Choanephora
cucurbitarum. a Natural rot on
Egyptian henbane grown in a
greenhouse. b Symptoms
eventually develop in fruits
of the plant. c Monosporous
sporangiophores and
monosporous sporangiola
(arrows) growing on infected
fruit stalk. Micrographs of
d monosporus sporangiophores
with monosporous sporangiola
on branches, e apex of
sporangiophore with primary
vesicle that arises from a simple
or dichotomously branched
stalk, f monosporous
sporangiola. g Colony of
C. cucurbitarum culture on
PDA. hj Micrographs of
morphology of C. cucurbitarum
cultured for 7 days on PDA:
h few-spored sporangium and
sporangiophores, i fusiform
sporangiospores with
appendages, j chlamydospores
in chains. km Disease severity
gradually increased on
C. cucurbitarum over the 8 days
after inoculation with a conidial
suspension with the isolate
obtained from single-spore
culture: k wilting and rotting
of the flower at 3 days, l rotting
had extended to the calyx at
5 days, m symptoms are more
severe and have extended to the
flower base, stalk (arrow A),
and stem (arrow B) after 8 days

transferred to fresh PDA plates. The fungus grew rapidly
on PDA, and mycelia covered the whole plate within
3 days (Fig. 1g). On the basis of microscopic examination
of the fungus, the morphology was identical to that of
Choanephora cucurbitarum (Berkeley & Ravenel) Thaxter
(Agrios 1997; Farr et al. 1989; Kobayashi et al. 1992;
Kwon et al. 2001). Initially, the fungal colony was color-
less; as it aged, it became white to yellowish brown and
had aseptate hyphae with irregular branching. Monosporous
sporangiophores were long, slender, and branched at the
apex with monosporous sporangiola on each branch
(Fig. 1d, e). The monosporous sporangiola were sub-
spherical to ovate in shape and 13.125.3 9 8.215.0 lm
in size (Fig. 1f). The few-spored sporangia were subglo-
bose in shape and 43.4121.2 lm in size (Fig. 1h). The
dark brown sporangiospores were fusiform or elliptical to
ovoid and 810 9 1216 lm in size with hair-like
appendages on both ends (Fig. 1i).
Four-day-old cultured vegetative cells of two isolates,
HM16-N and HM16-V, were streaked 1 cm apart on a new
PDA plate and incubated at 25C for 1520 days to test
mating. Heterothallic, hemispherical zygospores were
black, 45.385.6 lm in diameter, and formed between the
tips of entwining branches. Chlamydospores were found in
chains in old cultures (Fig. 1j).
C. cucurbitarum causes fruit rots, and flower and leaf
blights on a variety of plants including okra, squash,
pumpkin, pepper, pea, bean, and cucumber (Yu and Ko
1997). Diseases caused by the pathogen in Japan have been
recorded on pea, four oclock, cabbage, cucumber, white

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360 J Gen Plant Pathol (2010) 76:358361

clover, sugar beet, garden petunia, spinach, Mesembryan- Table 1 Growth of Choanephora cucurbitarum for 7 days on potato
themum spp., pumpkin, and eggplant (National Institute of dextrose agar at various temperatures
Agrobiological Sciences 2010). Temp. (C) Mean diameter (mm) Abundance at 7 days
Nuclear DNA from isolate HM16-N was extracted using
2 days 7 days Sporangia Zygospores
a Dr. GenTLE Kit (from yeast) (Takara Bio, Otsu, Japan)
according to the manufacturers instructions. Nucleotide 15 27.5 80.0 ? -
sequences of the D1/D2 region of the 28S ribosomal DNA 20 50.0 80.0 ?? -
(rDNA), the internal transcribed spacer region of the rDNA 25 65.0 80.0 ??? -
(ITS), and the mitochondrial small subunit (mtSSU) 30 0.0 13.5 - ???
rDNA of the fungal isolate were determined directly from 37 0.0 0.0 - -
the products of a polymerase chain reaction (PCR) (White
Temp., temperature; ???, abundant; ??, medium; ?, few; -, none
et al. 1990). Sequence analysis showed that the rDNA-ITS
and 28S rDNA of the isolate had 96% similarity with those
were wilting, and symptoms had begun to appear. The
of choanephoraceae species (EF060399), while the 28S
rotting extended to the remaining parts of the flower within
rDNA of the isolate had maximum similarity (95%) with
8 days and reached the stem and leaves after 10 days;
that of C. cucurbitarum. The sequence database did not
eventually the floral tops wilted and died (Fig. 1km).
contain information about the (mtSSU) rDNA of the cho-
C. cucurbitarum was reisolated from the diseased plants
anephoraceae family. Sequence analysis of 28S rDNA
but not from the control plants.
supported the morphological identification of the isolate.
The nucleotide sequences determined in this study have
been deposited in the DNA Data Bank of Japan (DDBJ) as
Name of the disease
accessions AB536740, AB536741 and AB536742 for the
mtSSU, D1/D2 and ITS regions, respectively.
The morphological and genetic characteristics confirmed that
On the basis of these results, the isolate HM16-N was
the disease in H. muticus was caused by C. cucurbitarum.
identified as C. cucurbitarum (Berkeley & Ravenel)
This is the first report on Choanephora rot of this plant. We
Thaxter and deposited in Genebank, National Institute of
thus propose the name Choanephora rot (Kougai-kabi-byo) of
Agrobiological Sciences as accession MAFF 242429.
Egyptian henbane for this new disease.

Acknowledgments This study was supported in part by the

Growth at various temperatures
Growth of C. cucurbitarm HM16-N at different tempera-
tures (15, 20, 25, 30 and 37C) was tested on PDA media
for up to 7 days. The best vegetative growth was recorded
at 25C followed by 20C, at which the colony diameter
was the largest, and both mycelia and sporangia formation
were abundant. The growth rate was reduced at decreasing
temperatures to 15C. High temperature (37C) completely
inhibited growth (Table 1). Only 7 days were needed to
produce zygospores at 30C, whereas zygospores were not
observed at lower (15, 20 and 25C) or higher (37C)
temperatures after 7 days (Table 1).
Pathogenicity test
Inoculum was prepared from 610-day-old pure cultures of
C. cucurbitarm HM16-N on PDA plates. A monosporous
sporangiola suspension (2 9 104 sporangiola/ml sterilized
water) was applied on newly opened flowers. The control
flowers received the same amount of sterilized water. The
plants were covered with plastic bags to increase the
moisture content. Three days later, the inoculated flowers
Department of Missions (Ministry of Higher Education and Scientific
Research, Egypt).
References
Abdel-Motaal FF, El-Sayed MA, El-Zayat SA, Nassar MSM, Ito S
(2009a) Leaf spot disease of Hyoscyamus muticus (Egyptian
henbane) caused by Cladosporium herbarum. J Gen Plant Pathol
75:437439
Abdel-Motaal FF, Nassar MSM, El-Zayat SA, El-Sayed MA, Ito S
(2009b) Responses of fungi to tropane alkaloids produced by a
medicinal plant Hyoscyamus muticus (Egyptian henbane). Folia
Microbiol 54:207212
Agrios GN (1997) Plant pathology, 4th edn. San Diego, Academic
Press, pp 283286
Farr DF, Bills GF, Chamuris GP, Rossman AY (1989) Fungi on plants
and plant products in the United States. APS Press, St. Paul,
p 1252
Hashimoto T, Yun DJ, Yamada Y (1993) Production of tropane
alkaloids in genetically engineered root cultures. Phytochemistry
32:713718
Kobayashi T, Katsumoto K, Abiko K, Abe Y, Kakishima M (1992)
Illustrated genera of plant pathogenic fungi in Japan (in
Japanese). Zenkoku Noson Kyoiku Kyokai Publishing, Tokyo,
pp 7071
Kwon J, Shen S, Park C (2001) Pod rot of cowpea (Vigna sinensis)
caused by Choanephora cucurbitarum. Plant Pathol J
17:354356

123
J Gen Plant Pathol (2010) 76:358361
National Institute of Agrobiological Sciences (ed) (2010) Database of
plant diseases in Japan, http://www.gene.affrc.go.jp/databases-
micro_pl_diseases_en.php. Accessed 3 April 2010
Oksman-Caldentey KM, Hiltunen R (1996) Transgenic crops for
improved pharmaceutical products. Field Crops Res 45:5769
White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct
sequencing of fungal ribosomal RNA genes for phylogenetics.
361
In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR
protocols: a guide to methods and applications. Academic Press,
San Diego, pp 315322
Yu MQ, Ko WH (1997) Factors affecting germination and the mode
of germination of zygospores of Choanephora cucurbitarum.
J Phytopathol 145:357361
123