Bot. Bull. Acad. Sin. (1997) 38: 41_44

Ho and Ko Single-spore isolation of fungi

A simple method for obtaining single-spore isolates of fungi

Wang-Ching Ho1,2 and Wen-Hsiung Ko1,3

1Department of Plant Pathology, Beaumont Agricultural Research Center, University of Hawaii at Manoa, Hilo, Hawaii 96720, USA

(Received June 17, 1996; Accepted October 18, 1996)

Abstract. A new method of single spore isolation was developed. A 0.1 l spore suspension was placed on water agar above one of 50_100 circles marked on the bottom of a plate. After 12_24 h, the number of spores in each circle was counted. Single germinating spores in each circle were transferred separately. For all six species of fungi tested, the number of spores in each micro-drop ranged from 0 to 4. More than 50% of the micro-drops contained a single spore with an unbranched germ tube. This method made it easy to locate well separated spores for single-spore isolation, shortened the isolation time by half, and reduced the incubation period from two days to one.

Keywords: Single-spore isolation; Zoospores; Conidia.

Introduction

Establishing a large number of single-spore isolates is essential for studies of variation, mutation, and segregation in fungi. The conventional method of isolating well separated spores streaked on the agar medium under a stereoscopic microscope (Tuite, 1969) is tedious and time consuming. While studying the mating type change in Phytophthora parasitica Dastur, Ko (1981) developed a relatively easy method for mass isolation of single-zoospore isolates. The method consisted of streaking encysted zoospores on water agar. After incubation at 24C for 48 h, separated visible colonies were marked and then observed under a light microscope at 100 magnification with 10 objective. Those colonies originating from single zoospores were transferred to V-8 agar plates. This method also has been used to study chemical regulation of mating type in various species of Phytophthora (Ko et al., 1986; Ann and Ko, 1989; Chang and Ko, 1990), to compare the enzyme activity between asexual and sexual progenies of P. parasitica (Ann and Ko, 1990a), to obtain genetic evidence of diploidy for this fungus (Ann and Ko, 1990b), and to document variation in growth rate and colony morphology in P. parasitica induced by exposure to metalaxyl (Chang and Ko, 1992). The method was also used to detect mating type changes during long-term storage of Pythium splendens Braun (Guo and Ko, 1991), to

determine whether this fungus was diploid (Guo and Ko, 1994), and to document continuing variation in successive asexual generations of Py. splendens following sexual reproduction (Guo and Ko, 1995). The method developed by Ko (1981) requires time to locate the origin of each colony under the microscope to confirm that the colony originated from a single spore (Figure 1). In this report we describe a simple procedure that eliminates the need to locate the colony origins and, thereby, shortens the time needed to identity single-spore isolates.

2Present address: Department of Plant Protection, National Pingtung Polytechnic Institute, Pingtung, Taiwan, Republic of China.

3Corresponding author. Fax: (808) 969-7923.

Figure 1. A small colony developed from a single zoospore (arrow) of Phytophthora parasitica on water agar 48 h at 24C (320).


Botanical Bulletin of Academia Sinica, Vol. 38, 1997

Materials and Methods

Microorganisms

Cultures of Phytophthora megasperma Drechsler, P. parasitica, Pythium carolinianum Matthews and Py. myriotylum Drechsler were each originated from a single zoospore. Botryodiplodia theobromae Pat., and Colletotrichum gloeosporioides (Penzig) Sacc. originated from single conidia.

Production of Spores

Phytophthora parasitica, P. megasperma, Py. carolinianum, and Py. myriotylum were individually grown on 5% V-8 agar (5% V-8 juice, 0.02% CaCO3 and 2% Bacto agar) at 24C for 3 days. Sporangia of each fungus were induced by incubating three pieces (ca. 5 5 5 mm) of the agar culture in 10 ml sterile distilled water in a plastic Petri plate (60 mm diam.) for 2 days at 24C under cool white fluorescent light (1,000 lux). Zoospores were released from sporangia by chilling at 5C for 15 min and were induced to encyst by agitating the zoospore suspension in a test tube for 1 min with a Vortex mixer. For production of conidia, B. theobromae and C. gloeosporioides were grown on 10% V-8 agar (10% V-8 juice, 0.02% CaCO3 and 2% Bacto agar) at 24C for 2 weeks under light. The conidial suspension was prepared by transfering a loopful of conidial mass into sterile distilled water in a test tube.

Isolation of Single-Spore Isolates

The concentrations of the spore suspensions were adjusted to approximately 10 spores/l with a Pipetman

microliter pipet (P-20D, West Coast Scientific Inc., Oakland, California, U.S.A) (Ko et al., 1973). A marking pen was used to draw 50_100 circles (ca. 3 mm diam.) on the bottom of each water agar (2% Bacto agar) plate (9 cm diam.). A 0.1 l drop of the spore suspension was placed on the surface of the water agar above each circle. After incubation at 24C for 12_24 h, each circle was inspected under a microscope at 100 magnification from the bottom of the plate. Those circles containing a single germinating spore were marked and spores in those circles were individually transferred to 10% V-8 agar plate.

Figure 2. A zoospore of Phytophthora parasitica with an unbranched germ tube on water agar after 12 h at 24C (320).

Table 1. Ratios of spore number in mico-drops of spore suspensions adjusted to about 10 spores/l.

Fungus Spore type No. of micro-dropsa containing

0 1 2 3 4 (spores)

Botryodiplodia theobromae Conidia 25 57 14 4 0

Colletotrichum gloeosporioides Conidia 25 55 14 4 2

Phytophthora megasperma Zoospores 31 51 12 4 2

P. parasitica Zoospores 28 55 13 3 1

Pythium carolinianum Zoospores 32 51 15 2 0

Py. myriotylum Zoospores 27 52 17 3 1

aEach micro-drop contained 0.1 l of spore suspension.

Table 2. Time required for isolation of 50 single-spore isolates from different fungi using the new method and the previous methoda.

Time required (min)

Fungus Present method Previous methoda

Botryodiplodia theobromae 51 137

Colletotrichum gloeosporioides 52 130

Phytophthora megasperma 62 133

P. parasitica 72 165

Pythium carolinianum 65 131

Py. myriotylum 64 127

aKo (1981).


Ho and Ko Single-spore isolation of fungi

Results and Discussion

When the spore concentration was adjusted to about 10 spores/l, the number of spores in each micro-drop ranged from 0 to 4 (Table 1). More than 50% of the micro-drops contained a single spore, and 35% of the drops contained no spores. Approximately 14, 3, and 1% of the micro-drops contained 2, 3, and 4 spores, respectively. For example, when conidia of B. theobromae were tested, 57% of the drops contained a single spore, while 25, 14, and 4% of the drops contained 0, 2, and 3 spores, respectively. Within 12 to 24 h of incubation, most spores produced an unbranched germ tube making them easy to locate (Figure 2). Therefore, the time needed to identify a single-spore isolate was greatly shortened. For all six fungal species tested, the time required to obtain 50 single-spore isolates was about 60 min with the new method versus over twice that long with the old method (Table 2).

The method described in this study has the advantage of being easy to locate well separated spores for single-spore isolation. It not only shortened the isolation time by half, but also reduced the incubation time from two days to one.

Acknowledgments. We thank Drs. M. Aragaki and W. Nishijima of the University of Hawaii, and Dr. J. L. Lockwood of the Michigan State University for cultures of Py. carolinianum, C. gloeosporioides, and P. megasperma, respectively. Dr. W. C. Ho was supported by a postdoctoral fellowship from the National Science Council, the Republic of China. Journal Series Paper No. 7221 of the Hawaii Institute of Tropical Agriculture and Human Resources.

Literature Cited

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Ann, P. J. and W. H. Ko. 1990a. Survey of enzyme activity on solid media in Phytophthora. Can. J. Bot. 68: 139_143.

Ann, P. J. and W. H. Ko. 1990b. Growth rate and colony morphology of progenies of zoospoes and selfed oospoes of Phytophthora parasitica. Mycologia 82: 693_697.

Chang, T. T. and W. H. Ko. 1990. Effect of metalaxyl on mating type of Phytophthora infestans and P. parasitica. Ann. Phytopathol. Soc. Japan 56: 194_198.

Chang, T. T. and W. H. Ko. 1992. Variation in growth rate and colony morphology of Phytophthora parasitica induced by metalaxyl. Ann. Phytopathol. Soc. Japan 58: 72_77.

Guo, L. Y. and W. H. Ko. 1991. Hormonal regulation of sexual reproduction and mating type change in heterothallic Pythium splendens. Mycol. Res. 95: 452_456.

Guo, L. Y. and W. H. Ko. 1994. Growth rate and antibiotic sensitivities of conidium and selfed-oospore progenies of heterothallic Pythium splendens. Can. J. Bot. 72: 1709_1712.

Guo, L. Y. and W. H. Ko. 1995. Continuing variation in successive asexual generation of Pythium splendens following sexual reproduction. Mycol. Res. 99: 1339_1344.

Ko, W. H. 1981. Reversible change of mating type of Phytophthora parasitica. J. Gen. Microbiol. 125: 451_454.

Ko, W. H., L. L. Chase, and R. K. Kunimoto. 1973. A microsyringe method for determining concentration of fungal propagules. Phytopathology 63: 1206_1207.

Ko, W. H., C. J. Lee, and H. J. Su. 1986. Chemical regulation of mating type in Phytophthora parasitca. Mycologia 78: 134_136.

Tuite, J. 1969. Plant Pathological Methods - Fungi and Bacteria. Burgess Publ. Co., Minneapolis, Minnesota, 239 pp.