Mycologia, 96(3), 2004, pp. 647–649. q 2004 by The Mycological Society of America, Lawrence, KS 66044-8897
Pythium sukuiense, a new species from undisturbed natural forest in Taiwan
7 mm), unis in quoque oogonio, paragynis, monoclinis, raro diclinis.
Wen-Hsiung Ko1 Department of Plant and Environmental Protection Sciences, Beaumont Agricultural Research Center, University of Hawaii at Manoa, Hilo, Hawaii 96720
ETYMOLOGY: sukuiense (four seasons), referring to the location name in Taiwanese where the pholotype was found. HOLOTY PE: TAI 245297 (dried culture), Herbarium of Department of Botany, National Taiwan University, Taipei, Taiwan. A living culture from the holotype has been deposited at the Centraalbureau voor Schimmelcultures, Utrecht, Netherlands (CBS 110030). Pythium sukuiense was isolated from a soil suspension by baiting with azalea (Rhododendro simsii) leaf disks (Zhou et al 1992). A 50 g soil sample was mixed with 150 mL distilled water in a 400 mL beaker, and 10 azalea leaf disks (9 mm diam) were floated in each beaker. After 3 d at 24 C, baits were removed from beakers, blotted dry and placed on a selective medium consisting of 5% V-8 juice, 0.02% CaCO3 and 2% agar. After autoclaving, 100 mg of ampicillin, 50 mg of nystatin and 10 mg of pentachloronitrobenzene per liter were added (Ko et al 1978). Plates were incubated on the laboratory bench at 24 C. Two singlehyphal tip isolates with identical morphological characteristics were obtained from separate soil samples collected from an undisturbed forest reserve at Sujih of Ilan County in northern Taiwan in Dec 2001. Isolate Py-sk-1 used in this study was a single-zoospore isolate derived from one of these two isolates. Pythium sukuiense is a fast growing organism compared to other fungi. It grew 18 mm/d at 24 C in the dark on 10% V-8 agar consisting of 10% V-8 juice, 0.02% CaCO3 and 1.5% agar. After incubation for 12 d, the isolate produced sexual structures in V-8 agar. Oogonia were spherical and smooth. About 60% of them were terminal (FIG. 1A), while the other 40% were intercalary (FIG. 1B). The diameter of oogonia was 10–14 mm, with an average of 13 mm. Attached to each oogonium was a club-shaped monoclinous antheridium (8–10 3 6–8 mm, average 9 3 7 mm) originating from the oogonial stock below the oogonium (FIG. 1) or, less frequently, from the parent hyphae. On rare occasions, the fungus also produced diclinous antheridia. Oospores, which nearly filled oogonia in most cases, were spherical and smooth with a wall thickness of 1–1.6 mm, with an average of 1.2 mm. The size of oospores were 9–13 mm, with an average of 11 mm.
Shin-Yuan Wang Pao-Jen Ann Department of Plant Pathology, Taiwan Agricultural Research Institute, Wufeng, Taichung, Taiwan
Abstract: A new species, Pythium sukuiense, was isolated from an undisturbed natural forest in northern Taiwan. The fungus produces sporangia indistinguishable from hyphae and very small oogonia and oospores. Oogonia were smooth and terminal or intercalary and attached with a single antheridium. Oospores were aplerotic, with an average size of only 11 mm. Key words: forest soil, indigenous fungus, Oomycota, taxonomy
During a recent survey of the distribution of pythiaceous species in undisturbed natural forests in Taiwan, two isolates of a previously undescribed Pythium species were obtained from soil samples. The fungus produced small oogonia and oospores, and there were no discernable sporangia. Sometimes, a few strange, spherical- to oval-shaped, structures without walls were produced in agar medium. These structures were found to be vesicles produced on tips of hyphae functioning as sporangia. The characteristics of this Taiwainese isolates are different from recognized species of Pythium (Waterhouse 1967, 1968, Plaats-Niterink 1981, Dick 1990). The organism, therefore, is described as a new species. Pythium sukuiense Ko, Wang et Ann, sp. nov.
Myceliis hyalinis, usque ad 6.7 mm crasis. Sporangiis filamentosis. Vesiculis zoosporis 12–30 continentibus. Zoosporiis reniformibus, biciliatis, circiter 10 3 7 mm. Oogoniis terminalibus, vel intercalaribu, globosis, 10–14 mm diam (med. 13 mm). Oosporis sphaericis, apleroticis, 9–13 mm diam (med. 11 mm), membrana 1–1.6 mm diam (med.1.2 mm) crassa. Antheridiis clavatis, 8–10 3 6–8 mm(med. 9 3
Accepted for publication February 4, 2004. 1 Corresponding author. E-mail:
[email protected]
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MYCOLOGIA
FIG. 1.
Sexual structures produced by Pythium sukuiense, 7503. A, terminal oogonium. B, intercalary oogonium.
FIG. 2. The process of zoospore release from a hyphal tip of Pythium sukuiense, 2503. A, a small swelling on the tip of a hypha. B, a vesicle resulting from the enlargement of the swelling. C, a vesicle before zoospore differentiation. D, formation of spherical aggregates during the later stage of zoospore differentiation. E, dispersal of zoospores after disintegration of vesicle wall. F, an empty opening (arrow) left on the hyphal tip after zoospore dispersal.
KO
ET AL:
NEW PYTHIUM
To induce formation of zoospores, an agar block (ca. 8 3 8 3 2 mm) half-colonized with hyphae was obtained from the advancing margin of a young colony growing on 10% V-8 agar for 24 h at 24 C in the dark and transferred to 3 mL of sterile distilled water in a small Petri plate (60 mm diam). After incubation at 24 C under light for 24 h, the 24 C water was replaced with 3 mL of sterile cold (8 C) distilled water and incubated at 8 C for 30 min. The culture then was returned to 24 C. Within 70 min, small vesicles appeared at the tips of some hyphae (FIG. 2A). When observed under a microscope, the cytoplasm in the hyphae could be seen moving rapidly into the vesicles and causing their enlargement (FIG. 2B). When the vesicle stopped enlarging, its contents began to show a slight irregular movement (FIG. 2C). Spherical aggregates formed slowly (FIG. 2D), and the cytoplasmic movement became faster and faster, resulting in delimitation of zoospores. Each vesicle contained 12– 30 and sometimes even more zoospores, which measured approximately 10 3 7 mm. Finally, the wall of the vesicle disappeared and zoospores swam away, leaving an empty opening on each hyphal tip (FIG. 2F). The whole process took about 17 min. Pythium sukuiense bears some resemblance to Pythium aquatile Ho¨huk in the production of filamentous sporangia indistinguishable from hyphae (Waterhouse 1968, Plaats-Niterink 1981). However, the average oogonium size of P. aquatile (346 mm3) (PlaatsNiterink 1981) was about 2.6 times larger than that of P. sukuiense (133 mm3). The average oospore size of the former (252 mm3) (Plaats-Niterink 1981) also was about 2.6 times larger than that of the latter (95 mm3). Four other described Pythium species have an av-
SPECIES FROM
TAIWAN
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erage oospore size smaller than 15 mm (Waterhouse 1968, Plaats-Niterink 1981). Pythium adhaerens Sparrow with 14.5 mm oospores is different from P. sukuiense in the formation of 1–4 antheridia encircling an oogonium. Pythium dissimile Vaartaja with 13.8 mm oospores produces sporangia consisting of irregular hyphal swellings and oogonia lacking antheridia. Pythium sulcatum Pratt & Mitchell, with 14.5 mm oospores, forms 1–3 large antheridia encircling an oogonium. Pythium tenues Gobi with 8–9 mm oospores produces sporangia consisting of more or less swollen hyphae and oogonia each containing 1–2 antheridia. ACKNOWLEDGMENTS
This research was supported in part by a grant from the National Science Council of Taiwan. We thank Dr. Arthur de Cock of CBS for the confirmation of the new species and Dr. Yu-Ming Ju for assistance in Latin diagnosis. LITERATURE CITED
Dick MW. 1990. Keys to Pythium. Reading, UK: Published by the author. 64 p. Ko WH, Chang HS, Su HJ. 1978. Isolates of Phytophthora cinnamomi from Taiwan as evidence for an Asian origin of the species. Trans Br Mycol Soc 71:496–499. Plaats-Niterink AJ van der. 1981. Monograph of the genus Pythium. Stud Mycol 21:1–242. Waterhouse GM. 1967. Key to Pythium Pringsheim. Mycol. Pap. 109:1–15. . 1968. The genus Pythium Pringsheim. Diagnoses (or descriptions) and figures from the original papers. Mycol Pap 110:1–50. Zhou XG, Zhu ZY, Lu CP, Wang SJ, Ko WH. 1992. Phytophthora cinnamomi in Shanghai and its possible origin. Mycopathologia 120:29–32.
