Botanical Studies (2006) 47: 273-277.
*
Corresponding author: E-mail: kowh@dragon.nchu.edu.tw;
Tel: 886-4-23302301-507; Fax: 886-4-23338162.
INTRODUCTION
Phytophthora katsurae Ko and Chang (1979) closely
resembles P. heveae Thompson in morphological charac-
teristics with the exception of its verrucose oogonial wall
(Ho et al., 1995; Stamps et al., 1990). Both species char-
acteristically produce abundant oogonia with a funnel-
shaped base and small amphigynous antheridia in a single
culture (Ho et al., 1995). Sporangia of both species are
papillate with hemispherical apical thickenings, and are
non-deciduous in water (Ho et al., 1995). A high level of
isozyme similarity was observed among the isolates of
P. katsurae and P. heveae (Oudemans and Coffey, 1991).
Cooke et al., (2000) showed that, based on ITS sequences
of genomic rDNA, the 47 Phytophthora taxa examined
are divisible into eight clades. Phytophthora katsurae and
P. heveae are closely related sister taxa in a discrete clade.
Recent phylogenetic analysis of Phytophthora species
based on nuclear gene sequence and the combination of
nuclear and mitochondrial gene sequences also showed a
strong support for the close relationship of P. katsurae and
P. heveae (Kroon et al., 2004).
Geographic distribution and host range of P. katsurae
are limited. Its known hosts include only chestnut (Kat-
sura, 1976), coconut (Uchida et al., 1992), and cocoa (Li-
yanage and Wheeler, 1989). The organism is distributed in
Japan, Taiwan, Hawaii, Ivory Coast, Australia, and Papua
New Guinea (Stamps, 1985). Recently, it was reported
from China for the first time. The organism was isolated
from soil on Hainan Island in southern China (Ho et al,
2005).
Phytophthora heveae has slightly wider geographic
distribution and host range than P. katsurae. The organism
causes diseases on Kauri pine, Brazilian nut, guava, rho-
dodendron, and cocoa (Erwin and Ribeiro, 1996). It has
been reported from Malaysia, New Zealand, New Guinea,
Brazil, Ivory Coast, Australia, India, Guatemala, and the
United States (Erwin and Ribeiro, 1996). The organism
has also been recovered from soil from Taiwan (Ho et al.,
1995) and China (Zhang et al., 1995; Ho et al., 2005).
During our survey of the distribution of P. katsurae and
P. heveae in Taiwan, both organisms were recovered from
a protected natural forest in central Taiwan. The possible
origin of these two species of Phytophthora and their pos-
sible evolutionary relationship were, therefore, investi-
gated.
MICROBIOLOGY
The possible origin and relation of Phytophthora
katsurae and P. heveae, discovered in a protected
natural forest in Taiwan
Wen-Hsiung KO
1,
*, S. Y. WANG
2
, and P. J. ANN
2
1
Department of Plant Pathology, National Chung Hsing University, Taichung, TAIWAN
2
Department of Plant Pathology, Taiwan Agircultural Research Institute, Wufeng, Taichung, TAIWAN
(Received April 22, 2005; Accepted January 18, 2006)
ABSTRACT.
When a total of 531 soil samples collected from 1976 to 2000 from various locations distrib-
uted in every county on the island of Taiwan was assayed, sixteen isolates of Phytophthora katsurae and nine
isolates of P. heveae were recovered from three and four counties, respectively. Phytophthora katsurae and
P. heveae were isolated from a protected natural forest located atop a hill at Lenhuachih without higher land
within the range of vision, suggesting that both species are indigenous to Taiwan. Phytophthora katsurae is
distinguished from the similar P. heveae by its verrucose oogonial wall. The number of protrusions produced
by the Taiwanese isolates of P. katsurae varied greatly ranging from 2 to 23 per oogonium, indicating the
unsteadiness of this taxonomical characteristic. Oogonia produced by isolates of P. katsurae obtained from
Hawaii contained very few protrusions. Moreover, most oogonia produced by two of these isolates did not
have any protrusions and were indistinguishable from those produced by P. heveae. Results suggest the devel-
opment of P. heveae directly from P. katsurae by loss of oogonial protrusions. The high level of ITS sequence
similarity between P. heveae and P. katsurae in comparison with their relationships to other Phytophthora
species tested also support the possibility of the recent development of one species from the other species.
Keywords: Phytophthora heveae; Phytophthora katsurae; Oogonial protrusion; Indigenous.
pg_0002
274
Botanical Studies, Vol. 47, 2006
MATERIALS AND METHODS
Collection of soil samples
Soil samples collected from various locations were tak-
en from a depth of 0 to 10 cm after the surface litter was
cleared. Each location represented a different vegetation,
soil type, or elevation, and the sample about 1 to 2 kg was
a composite of three subsamples taken within a circle of
approximately 1 m diameter. From 1976 to 2000, a total of
531 soil samples was collected intermittently from loca-
tions in every county on the island of Taiwan.
Description of the protected natural forest
The protected natural forest at Lenhuachih of Nan Tow
County in central Taiwan (Figure 1) is under administra-
tion of the Taiwan Forestry Research Institute. It is a broad
leaf forest with diverse tree species, a number of which
belong to Lauraceae. The forest sits atop a hill without any
higher land within the range of vision.
Isolation of organisms
Soils were assayed for the presence of P. katsurae and
P. heveae within one week of collection by baiting with
lupine radical (Chee and Newhook, 1965), citrus leaf discs
(Grimem and Alesander, 1973), or leaf discs of camellia
or azalea (Zhou et al., 1992), depending on availability of
baiting materials at the time of soil collection. About 50 g
of soil from each sample was mixed with 150 ml distilled
water in a 400-ml plastic beaker. Each beaker contained
five lupine radicals or leaf discs as baits. After 3-4 days
at 24XC, baits were blotted dry and placed on a selec-
tive medium for pythiaceous organisms (Ko et al., 1978).
The medium consisted of 5% V-8 juice, 0.02% CaCO
3
,
and 2% agar supplemented with 100 ppm ampicillin, 50
ppm nystatin, and 10 ppm pentachloronitrobenzene after
autoclaving. Plates were incubated on the bench at 24XC.
Two plates were used for each soil sample. Phytophthora
katsurae was identified by its verrucose oogonia with a
funnel-shaped base and a small amphigynous antheridium
(Ko and Chang, 1979), while P. heveae was identified by
its smooth oogonia with a funnel-shaped base and a small
amphigynous antheridium (Ho et al., 1995).
Morphological observation
Since the structure of sporangia of P. katsurae and
P. heveae are simple and similar (Ho et al., 1995), only
sexual structures were included in this study. Isolates of P.
katsurae and P. heveae were grown on a medium consist-
ing of 10% V-8 juice, 0.02% CaCO
3
and 2% agar. After
incubation at 24XC in darkness for 10 days, 20 oogonia
from each isolate were randomly selected for determining
the number of protrusion on each oogonium under a mi-
croscope . The experiments were done twice.
Table 1. Species and isolates of Phytophthora used in sequence similarity analysis and GenBank accession numbers for ITS
sequences
a
.
Species and isolate
Associated habitat
Location
Accession number
P. heveae IMI180616
Hevea brasiliensis
Malaysia
AF266770
P. humicola IMI302303
Citurs orchard soil
Taiwan
AF266792
P. katsurae IMI360596
Cocos nucifera
Ivory Coast
AF266771
P. palmivora UQ1294
Theobroma cacao
Papua New Guinea
AF266780
a
From data published by Cooke et al. (2000).
Figure 1. Distribution of Phytophthora heveae (black dots) and
P. katsurae (open circles) in Taiwan, which is 380 km long from
north to south and with an east-west maximum width of 140 km.
Arrow points to the location of Lenhuachih from where both P.
heveae and P. katsurae were isolated from a protected natural
forest. Each number represents a county: 1, Taipei; 2, Tao Yuan;
3, Hsin Chu; 4, I Lan; 5, Miao Li; 6, Taichung; 7, Hua Len; 8,
Nan Tow; 9, Chang Hua; 10, Yun Lin; 11, Chia Yi; 12, Tainan;
13, Kaohsiung; 14, Taitung; 15, Pintung.
pg_0003
KO et al. X
Phytophthora
in Taiwan
275
Sequence similarity analysis
The ITS sequences of genomic rDNA of Phytophthora
species used in this study were published by Cooke et al.
(2000). Their sequences were retrieved from GenBank for
similarity analysis (Table 1). The nucleotide alignments
were carried out using the optimal alignment method of
the DNAMAN software (Version 4.0, Lynnon BioSoft,
Quebec, Canada).
RESULTS AND DISCUSSION
Nine isolates of P. heveae were recovered from soil
samples collected from farms cultivated with pineapple,
citrus, peach, or longan, and a protected natural forest at
Lenhuachih in Nan Tow County. Phytophthora heveae
was also found in the counties of Taichung, Chang Hua
and Chia Yi (Figure 1). A total of 16 isolates of P. katsurae
was recovered from natural forests located at Sutsi in Tao
Yuan County, Lenhuachih in Nan Tow County, and Tuona
in Kaohsiung County (Figure 1).
One isolate of P. katsurae obtained in 1976 was lost.
The number of protrusions on each oogonium produced
by isolates of P. katsurae varied greatly, ranging from 2
to 23 per oogonium. The average number of protrusions
produced by each isolate also varied from 10 to 17
(Table 2). None of the nine isolates of P. heveae obtained
produced oogonia with protrusions.
Both P. katsurae and P. heveae were isolated from the
protected forest at Lenhuachih in central Taiwan (Figure
1). The forest sits atop a hill, and no higher land is within
the visible range. Consequently, it is not possible for these
organisms to be transported to the forest by rain water or
streams from nearest habitation. It is also very unlikely
that these organisms were carried to the forest on the boots
of humans or the hooves of wild animals (Kliejunas and
Ko, 1976) because of the rarity P. katsurae and P. heveae
in Taiwan. Our study, therefore, suggests that both organ-
isms are indigenous to Taiwan. Failure to isolate Phytoph-
thora cinnamomi Rands from undisturbed localities in the
Americas has been used by Zentmyer (1979) as evidence
that it is not indigenous to the Americas.
Phytophthora heveae, with a wider host range, has been
isolated from cultivated land and also from natural forests
in Taiwan. However, P. katsurae has so far only been iso-
lated from natural forests. Chestnut, coconut, and cacao,
the only known hosts of P. katsurae, have been grown
sporadically in Taiwan, but none of them were present in
the forests surveyed. In fact, plants in the areas where P.
katsurae and P. heveae were recovered in the protected
forest at Lenhuachih all appeared healthy. It is conceivable
that bath organisms may be able to colonize live or dead
roots of certain plant species present in the forest without
causing any visible symptoms. Phytophthora cinnamomi
has been isolated from roots of various healthy-looking
plants belonging to 28 species in 22 families on the island
of Hawaii (Kliejunas and Ko, 1976).
Table 2. Oogonal protrusions of Phytophthora katsurae and P.
haveae.
Species and isolate
No. of protrusions/oogonium
Range
Average
P. katsurae
Pk-1
8-16
12
Pk-2
9-16
12
Pk-3
10-20
13
Pk-4
10-22
13
Pk-5
10-19
13
Pk-6
8-13
11
Pk-7
7-14
11
Pk-8
7-18
12
Pk-9
4-16
11
Pk-10
3-13
10
Pk-11
10-22
16
Pk-12
2-17
10
Pk-13
2-15
10
Pk-14
8-17
11
Pk-15
12-23
17
P. heveae
Ph-1
0
0
Ph-2
0
0
Ph-3
0
0
Ph-4
0
0
Ph-5
0
0
Ph-6
0
0
Ph-7
0
0
Ph-8
0
0
Ph-9
0
0
Table 3. Oogonial protrusions of Phytophthora katsurae from
Hawaii.
Isolate No. of protrusions/oogonium Oogonia without
protrusion (%)
Range
Average
H1024 0-5
2.5
20
H1026 0-2
0.2
85
H1027 0-5
1.9
25
H1028 0-4
0.6
75
H1029 1-5
3.3
0
H1032 0-6
2.6
10
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276
Botanical Studies, Vol. 47, 2006
Species of Phytophthora require special chemicals for
production of
a
hormones for formation of sexual organs
such as oogonia (Ko, 1998; Chern et al., 1999; Jee et al.,
2002; Wu et al., 2003). The verrucose oogonial wall is
the morphological characteristic distinguishing P. katsu-
rae from P. heveae (Ho et al., 1995; Stamps et al., 1990)
However, this taxonomical characteristic appears to be
unstable. Oogonia produced by isolates of P. katsurae
from Taiwan contained protrusions ranging from 2 to 23
per oogonium. It is conceivable that P. heveae may have
developed from P. katsurae through a gradual decrease in
protrusion number. If the hypothesis is correct, it should
be possible to find isolates of P. katsurae with very few
protrusions on their oogonia. Six isolates of P. katsurae
isolated from diseased coconut fruits in Hawaii were
obtained from Dr. J. Y. Uchida (Uchida et al., 1992). All
these isolates produced oogonia with very few protrusions
(Table 3, Figure 2). Moreover, 75 to 85% of the oogo-
nia produced by isolates H1028 and H1026 did not have
any protrusions and were indistinguishable from those
produced by P. heveae (Figure 2D). The ITS sequence
similarity between P. heveae and P. katsurae (98.8%) was
high in comparison with their relationships to other spe-
cies tested (79.2 to 89.8%) (Table 4). These results clearly
indicate that the two taxa share a recent common ancestor
and that the presence of oogonial protuberances is a trait
that distinguishes them.
Acknowledgements. We thank Drs. T. H. Chen and T. T.
Chang of Taiwan Forestry Research Institute for assistance
in collection of soil samples, Dr. Janice Uchida of the
University of Hawaii for supplying the coconut isolates of
P. katsurae, and Dr. Z. G. Zhang of Nanjing Agricultural
University for ITS sequence similarity analysis.
LITERATURE CITED
Chee, K.H. and F.J. Newhook. 1965. Improved methods for use
in studies on Phytophthora cinnamomi Rands and other
Phytophthora species. New Zealand J. Agric. Res. 8: 88-95.
C he rn, L .L ., C.S . Ta ng, an d W.H. Ko . 19 99. Che m ic al
characterization of
a
hormones of Phytophthora parasitica.
Bot. Bull. Acad. Sin. 40: 79-85.
Cooke, D.E.L., A. Drenth, J.M. Duncan, G. Wagels, and C.M.
Brasier. 2000. A molecular phylogeny of Phytophthora and
related oomycetes. Fung. Genet. Biol. 30: 17-32.
Erwin, D.C. and O.K. Ribeiro. 1996. Phytophthora Diseases
Worldwide. APS Press, St. Paul, Minnesota.
Grim en, G.R. and A.F. Alexander. 1973. Citrus leaf pieces
as traps for Phytophthor a parasitica from soil slurries .
Phytopathology 63: 540-541.
Ho , H. H., P. J . An n, an d H .S . C ha ng . 1 995 . T he ge nu s
Phytophthora in Taiwan. Institute of Botany, Academia Si-
nica Monograph Series 15: 1-81.
Ho, H., H. Zeng, and F. Zheng. 2005. A survey of Pythiaceous
fungi on Hainan Island of South China. Phytopathology 95:
S43.
Jee, H.J., C.S. Tang, and W.H. Ko. 2002. Characterization of
phytochemica ls s timulat ory to s exual reproducti on in
Phytophthora cactorum and P. parasitica. Bot. Bull. Acad.
Sin. 43: 203-210.
Katsura, K. 1976. Two new species of Phytophthora caus ing
damping-off of cucumber and trunk rot of chestnut. Trans.
Mycol. Soc. Japan 17: 238-242.
Kliejunas, J.T. and W.H. Ko. 1976. Dispersal of Phytophthora
cinnamomi on the island of Hawaii. Phytopathology 66:
453-470.
Table 4. Comparison of similarity levels of ITS sequences
among Phytophthora heveae, P. katsurae, P. humicola, and P.
palmivora
a
.
Species paired
Similarity (%)
P. heveae
P. katsurae
98.8
P. heveae
P. palmivora
89.5
P. heveae
P. humicola
80.4
P. katsurae
P. palmivora
89.8
P. katsurae
P. humicola
83.1
P. humicola
P. palmivora
79.2
a
ITS sequences were retrieved from GenBank.
Figure 2. Comparison of oogonial morphology between Phy-
tophthora katsurae (A) and P. heveae (B) from Taiwan, and P.
katsurae from Hawaii (C, D). X 1000.
pg_0005
KO et al. X
Phytophthora
in Taiwan
277
Ko, W.H. and H.S. Chang. 1979. Phytophthora katsurae, a new
name for P. castaneae. Mycologia 71: 840-844.
Ko, W.H., H.S. Chang, and H.J. Su. 1978. Isolates of Phytoph-
thora cinnamomi from Taiwan as evidence for an Asian
origin of the species. Trans. Br. Mycol. Soc. 71: 496-499.
Ko, W.H. 1998. Chemical stimulation of sexual reproduction in
Phytophthora and Pythium. Bot. Bull. Acad. Sin. 39: 81-86.
Kroon, L.P.N.M., F.T. Bakker, G.B.M. van der Bosch, P.J.M.
Bonants, and W.G. F lier. 2004. Phylogenetic analysis of
Phytophthora species based on mitochondrial and nuclear
DNA sequences. Fung. Genet Biol. 41: 766-782.
Liya na ge, N.I.S. and B.E.J. Wheel er. 1989. Phytophthora
katsurae from cocoa. Plant Pathol. 38: 627-629.
Oudemans, P. and M.D. Coffey. 1991. A revised systematics of
twelve papillate Phytophthora species based on isozyme
analysis. Mycol. Res. 95: 1025-1046.
Stamps, D.J . 1985. Phytophthora katsurae. Descriptions of
Pathogenic Fungi and Bacteria No. 837. Commonw. Mycol.
Inst., UK.
Stamps, D.J., G.M. Waterhouse, F.J. Newhook, and G.S. Hall.
1990. Revised tabular key to the species of Phytophthora.
Mycol. Pap. 162: 1-28.
Uchi da, J .Y., M. Ara gaki, J . Ooka , and N. Na gata . 1992.
Phytophthora fruit and heart rots of coconut in Hawaii.
Plant Dis. 76: 925-927.
Zentmyer, G.A. 1979. Origin of Phytophthora cinnamomi:
evidence that it is not an indigenous fungus in the Americas.
Phytopathology 67: 1373-1377.
Zhang, K.M., H.H. Ho, and J.V. Lu. 1995. The occurrence of
Phytophthora heveae in mainland China. Mycopathologia
129: 87-90.
Zhou, X.G., Z.Y. Zhou, C.P. Lu, S.J. Wang, and W.H. Ko. 1992.
Phytophthora cinnamomi in Shangha i and its possible
origin. Mycopathologia 120: 29-32.
Wu, H., X.-B. Zheng, and W.H. Ko. 2003. Effect of culture
origin on chemical stimulation of sexual reproduction
in Phytophthora and Pythium. Bot. Bull. Acad. S in. 44:
323-328.
pg_0006