J Gen Plant Pathol (2011) 77:132135
DOI 10.1007/s10327-011-0295-0
DISEASE NOTE
Fusarium root rot of prairie gentian caused by a species belonging
to the Fusarium solani species complex
Keisuke Tomioka Yuuri Hirooka
Akane Takezaki Takayuki Aoki Toyozo Sato
Received: 27 September 2010 / Accepted: 16 December 2010 / Published online: 14 January 2011
The Phytopathological Society of Japan and Springer 2011
Abstract Rotting of roots and stem bases and wilting of
entire plants were found on a gentianaceous flowering
plant, prairie gentian (Eustoma grandiflorum), grown in
Kagawa Prefecture in the southwest region of Japan in
April 2001. A mitosporic fungus, isolated repeatedly from
the diseased plants, was identified as a species belonging to
the clade 3 of Fusarium solani species complex based on
the morphology and the sequence of the translation elon-
gation factor gene. It was demonstrated to cause the disease
by inoculating potted plants and reisolating the fungus
from the diseased plants. We propose the name Fusarium
root rot of prairie gentian for this disease.
Keywords Fusarium root rot
Prairie gentian

Fusarium solani species complex
Introduction
Prairie gentian [Russell prairie gentian, Eustoma grandi-
florum (Rafinesque) Shinners], belonging to the family
Part of this paper was presented elsewhere by Tomioka et al. (2002,
2009a, b).
K. Tomioka (&)
T. Aoki
T. Sato
National Institute of Agrobiological Sciences,
Tsukuba 305-8602, Japan
e-mail: tomioka@affrc.go.jp
Y. Hirooka
United States Department of Agriculture,
Agricultural Research Service, Beltsville, MD 20705, USA
A. Takezaki
National Agricultural Research Center,
Tsukuba 305-8666, Japan
123
Gentianaceae, is a popular and important market flower in
Japan (Tomioka 2004). As its production has developed
nationwide, the plant has been affected by several diseases
including those caused by soilborne fungal species of
Fusarium, Pythium, Rhizoctonia, Sclerotium (Anonymous
2000). Rotting of roots and stem bases and wilting of entire
plants of prairie gentian, asymptomatic of a soilborne
Fusarium species, were found on potted plants of cv.
Fukushihai that were grown in glasshouses at 2228C for
studies on cut flower productivity in Zentsuji City, Kagawa
Prefecture in the southwest region of Japan in April 2001.
We isolated and identified the causal pathogen and inocu-
lated the host plants with the isolates to confirm pathoge-
nicity and presence of the causal agent.
Symptoms and pathogen
Lower leaves initially wilted and turned yellow to
brownish (Fig. 1a, b). Roots and stem bases of the diseased
plants had rotted (Fig. 1c), and soon after, the whole plant
wilted. A white mold, consisting of mycelia and conidia of
the pathogen, grew on the lesions under moist conditions
(Fig. 1d). When a representative isolate, EF2, of the fun-
gus, obtained by single-conidium isolation from a conidial
mass on a lesion of a diseased plant, was cultured on
potato dextrose agar (PDA) at 25C in the dark, it formed
white to pale greenish white colonies (Fig. 1e) bearing
many conidiophores, microconidia and macroconidia.
Similar conidiophores and conidia were also produced on
synthetic low nutrient agar (SNA) at 25C in the dark.
Conidiophores were hyaline, 60100 lm long, 12 lm
wide, 24 septate and sometimes formed a side branch
(Fig. 1f, g). Microconidia formed at the tips of
monophialides in false heads were hyaline, ellipsoid to
J Gen Plant Pathol (2011) 77:132135 133

Fig. 1 Symptoms and pathogen

morphology of Fusarium root
rot of prairie gentian caused by
isolate EF2 of the Fusarium
solani species complex.
ae Natural symptoms, signs
and colonies of the pathogen.
a Wilt and blight of the whole
plant, b wilt of the whole plant
at a flowering stage, c plant with
severe root rot, d mycelia and
conidial masses on rotten roots
and stem base, e colonies of
isolate EF2 on potato dextrose
agar grown at 25C in the dark
for 10 days (upper surface side;
lower reverse side).
fj Morphology of the pathogen
(isolate EF2) cultured on
synthetic low nutrient agar at
25C in the dark for 47 days.
f Conidiophores with conidia
(bar 20 lm), g conidiophore
with a side branch (bar 20 lm),
h microconidia (bar 10 lm),
i macroconidia (bar 10 lm),
j chlamydospores (bar 10 lm).
k, l Reproduction of the natural
symptoms after inoculation with
isolate EF2. k Wilted plants
2 weeks after inoculation (left
control), l root rot on wilted
plant (left control)

oblong-ellipsoid, 01 septate and 616 9 24 lm growth of 5.8 mm/day at 30C. Neither sclerotium nor
(Fig. 1h). Macroconidia were falcate with a foot-like cell, teleomorph was found with the isolate. The morphological
13(5) septate and 1238(66) 9 34(5) lm (Fig. 1i). and cultural characters of the mitosporic isolate agreed
Chlamydospores with a single septum were often produced well with traditional descriptions of Fusarium solani
at the tips of hyphae in old cultures (Fig. 1j). The isolate (Martius) Saccardo (Domsch et al. 1980; Gerlach and
grew on PDA plates in the dark at 1038C with maximum Nirenberg 1982; von Arx 1987).

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134 J Gen Plant Pathol (2011) 77:132135

91 Neocosmosporaornamentata (NRRL 22468, AF178349)

100 Neocosmosporaafricana (NRRL 22436, AF178348)
Neocosmosporavasinfecta (NRRL 22166, AF178350)
0.01 substitutions/site 100 Fusariumsolani f. sp. xanthoxyli (MP-IV, NRRL 22163 = MAFF 238540, AF178328)
65 Fusariumsolani f. sp. xanthoxyli (MP-IV, NRRL 22277 = MAFF 238541, AF178336)
The present isolate (EF2 = MAFF 238881)
92 97 Fusariumsolani f. sp. batatas (MP-II, (NRRL 22400, AF178343)
58 Fusariumsolani f. sp. batatas (MP-II, NRRL 22402, AF178344)
Fusariumstriatum (NRRL22101, AF178333) Clade3
Fusariumsolani f. sp. cucurbitae Race-1(MP-I, NRRL 22098, AF178327)
Fusariumsolani f. sp. cucurbitae Race-1(MP-I, NRRL 22153, AF178346)
Fusariumambrosum (NRRL20438, AF178332)
52
100 Fusariumsolani f. sp. cucurbitae Race-2(MP-V, NRRL 22142, AF178347)
Fusariumsolani f. sp. cucurbitae Race-2 (MP-V, NRRL 22141, AF178329)
100 83 Fusarium solani f. sp. robiniae (MP-VII, NRRL 22586, AF178353)
Fusarium solani f. sp. robiniae (MP-VII, NRRL 22161 = MAFF 238542, AF178330)
69 Fusarium solani f. sp. pisi (MP-VI, NRRL 22278, AF178337)
Fusarium solani f. sp. pisi (MP-VI, NRRL 22820, AF178355)
90
Fusarium solani f. sp. mori (MP-III, NRRL 22230 = MAFF 840046, AF178358)
79 Fusarium solani f. sp. mori (MP-III, NRRL 22157 = MAFF 238538, AF178359)
93 Fusariumcuneirostrum (NRRL 22275 = MAFF 239036, AF178335)
100 Fusariumcuneirostrum (NRRL 22158, AF178331)
Fusariumvirguliforme (NRRL 22823, AF178356)
82
100 Fusariumvirguliforme (NRRL 22825, AF178357)
84 Clade2
Fusarium sp. (NRRL 22574, AF178345)
Fusarium sp. (NRRL 22387, AF178339)
59 100 Fusarium sp. (NRRL 22395, AF178341)
Fusarium sp. (NRRL 22412, AF178351)
63 Fusarium sp. (NRRL 22396, AF178342)
100 Fusarium sp.(NRRL 22632, AF178354) Clade1
Fusariumilludens (NRRL 22090, AF178326 )
Fusariumstaphyleae (NRRL 22316, AF178361)

Fig. 2 Phylogenic tree for isolate EF2 and the Fusarium solani
species complex constructed using neighbor-joining analysis of the
DNA sequence of the translation elongation factor 1a gene-coding
region. Sequence data were retrieved from the DDBJ/EMBL/
GenBank databases (accession numbers begin with AF; numbers
starting with NRRL are the accession numbers of the respective
isolates in the Agricultural Research Service Culture Collection at the
National Center for Agricultural Utilization Research, USA). The
sequence for isolate NRRL22316, classified as Nectria atrofusca
(Schweinitz) Ellis & Everhart (AF178361), was used as the outgroup.
Numbers above nodes represent bootstrap values C50% from 1000
replications

Based on extensive phylogenetic analyses of typical
strains of F. solani and related isolates, the Fusarium
solani species complex comprises at least 26 phylogeneti-
cally distinct species (ODonnell 2000). Simple use of the
scientific name F. solani as a taxon without specifying
its detailed phylogenetic position, however, may cause
serious taxonomic confusion regarding the fungal group.
Three large clades, clades 13, have been identified within
the F. solani species complex through molecular phylo-
genic analyses using DNA sequence data of the translation
elongation factor 1a gene-coding region (TEF) (ODonnell
2000). We therefore tried to determine the phylogenetic
position of the isolated fungus within the complex. On the
basis of results of analyses of TEF amplified with the
primers of EF-1H and EF-2T (ODonnell et al. 1998),
isolate EF2 was classified into clade 3 (Fig. 2) and had
97.0% homology with data in the DDBJ/EMBL/GenBank
databases for Fusarium solani f. sp. xanthoxyli Sakurai &
Matuo (MP-IV; AF178328 and AF178336), which causes
trunk blight of Japanese pepper [Xanthoxylum piperitum
(L.) De Candolle, the Rutaceae] (Sakurai and Matuo 1961).
We do not, however, consider it appropriate to identify
isolate EF2 as the forma specialis because of the large
sequence difference. We need to wait for taxonomic rear-
rangement of the F. solani species complex in order to
assign a specific epithet to isolate EF2. Isolate EF2 was
deposited in the Genebank, National Institute of Agrobio-
logical Sciences as accession MAFF238881, and its DNA
sequence data of TEF was registered in the DNA databases
as accession AB426618.
Pathogenicity
Plants of prairie gentian, cv. Fukushihai, grown to the 8- to
10-leaf stages in pots (9 cm in diameter) with composite
soil of equal parts of vermiculite and field soil in a glass-
house at 2228C for 2 months were inoculated with

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J Gen Plant Pathol (2011) 77:132135
isolate EF2. Conidia were collected from 6-day-old PDA
cultures grown at 25C in the dark, and suspended in
sterilized distilled water at 68 9 105 conidia/mL. Roots
of four healthy plants were soaked in the conidial sus-
pension for 2 days. Roots of healthy plants were soaked in
sterilized distilled water as a control. Subsequently, treated
plants were potted, covered with polyethylene bags and
then kept in a glasshouse at 2228C. Polyethylene bags
were removed 2 days after inoculation. Leaves began
wilting 45 days after inoculation, and part of the leaves
turned yellow to brownish. Entire plants began wilting
10 days after inoculation (Fig. 1k) and were completely
withered by 2 months after inoculation. Roots and stem
bases of the diseased plants turned brown and rotted
(Fig. 1l). Symptoms were reproduced on all the plants
inoculated with the isolate, and control plants had no
symptoms. The isolate was consistently reisolated from
diseased plants, but not from healthy controls, demon-
strating that the isolate was the causal agent of the disease.
Conclusion
Diseases of root rot, crown rot, stem rot, damping-off, wilt
and/or stunt of prairie gentian by F. solani (Martius)
Saccardo have been noted in USA (Farr et al. 1989; Tau-
benhaus and Ezekiel 1934), Japan (Matuo et al. 1980),
Argentina (Wolcan et al. 2001) and South Africa (Truter
and Wehner 2004). But there is no previous report speci-
fying the phylogenetic position of these pathogens in the
F. solani species complex. Nor in Japan have any previous
reports described data essential for identifying the patho-
gen and demonstrating its pathogenicity on prairie gentian.
In this study, we demonstrated that the present disease was
caused by a species belonging to clade 3 of the F. solani
species complex. Its diagnostic symptoms are rotting of
roots and stem bases and wilting of entire plants. The
disease is mainly attributed to rotting of roots. We, there-
fore, propose including the fungus as one of the causal
pathogens of Fusarium diseases on prairie gentian and
naming the present disease Fusarium root rot of the Wolcan S, Lori G, Ronco L (2001) First report of Fusarium solani
plant.
135
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