Demonstrationof Zoospore Activities by Fungi States M. McCarter MANY OF THELOWER FUNGI generally known as Phycomycetes reproduce asexually by producing motile spores called zoospores. Some of these fungi live sapro- Downloaded from http://online.ucpress.edu/abt/article-pdf/40/8/484/36360/4446362.pdf by guest on 01 October 2021 phytically in various habitats; others live parasitically, either as facultative parasites or obligate parasites, on a wide variety of plant species. Plant diseases of importance caused by these organisms include the white rusts, downy mildews, seedling diseases, rots of various plant parts, and blights on the above-ground portions of plants. The ,1< W fungal family Pythiaceae is of great importance because it contains the genera Pythium and Phytophthora, both of which contain species that cause destructive diseases of economically important plants. Pythium spp. are aquatic or terrestrial soil-borne organisms that cause seedling diseases, root rots, and fruitrots on a wide variety of host plants. Phytophthora spp. cause a range of disease types including seedling diseases, root and stem rots, and blights. The most infamous member of the latter genus is P. infestans, the cause of the potato disease known as late blight that was responsible for the potato famine of 1845- 1860 during which 1 million Irish died of starvation and another 11/2 million emigrated to North America (Strobel and Mathre 1970). Both Pythium and Phytophthora spp. produce sporan- gia of various sizes and shapes (fig. 1). Sporangia may germinate directly without the production of zoospores to form a new mycelium or they may produce zoospores. Zoospores may form directlyinside the sporangium or in - side a bubble-likeoutgrowth from the sporangium called FIGURE 1. (A to C). Representative sporangia produced by Phycomy- cetes A) Sporangium (A-1), sporangium with vesicle containing zoo- The author is professor of plant pathology in the spores (A-2), flagellated zoospore (A-3), and a germinating zoospore (A- department of plant pathology and plant genetics, 4) of Phythium aphanidermatum. B) Sporangia types (B-1), sporang- University of Georgia, Athens 30602. McCarter ium with vesicle containing zoospores (B-2), and a zoospore (B-3) of received his Ph.D. degree in plant pathology from W Pythium irregulare. C) Sporangia on branched sporangiophore (C-1) Clemson University (South Carolina) in 1965; his and detached sporangium liberatingzoospores (C-2) of Phytophthora dissertation research was conducted at the Army infestans. Fig. 1 A and 1 B after Middleton (1943) and Fig. 1 C after Biological Laboratories, Ft. Detrick, Maryland, Alexopoulos (1962). where he also served as a research plant pathologist from 1963 to 1965. In 1978, he was the recipient of a vesicle. Pythium spp. normally differentiate their zoo- the Gamma Sigma Delta Distinguishtd Teaching Award, and in 1975, he was named Most Outstanding Teacher, College spores in vesicles. Phytophthora spp. either produce their of Agriculture, University of Georgia. McCarter holds memberships in zoospores directlyin a sporangium without vesicle forma- a number of professional societies, including the American Phy- tion, or in a few cases, pass zoospores into a vesicle after topathological Society and the Georgia Academy of Science; he has published extensively in scientific journals, as well as in popular scienti- their maturation inside the sporangium. Pythium and fic magazines. Phytophthora spp. produce and liberate their zoospores 484 THE AMERICAN BIOLOGYTEACHER, NOVEMBER1978 only in water. Zoospores are fragile, thin-walled structures that are motile by means of two laterallyattached flagella. They swim in a film of water, eventually come to rest, en- cyst, and germinate by means of a germ tube. In case of disease-causing Pythium and Phytophthora spp., zoo- spores are the most important means of spreading from plant to plant during the growing season. Zoospores move at random in the soil or on plant tissue, but they ap- parently may also be attracted to plant parts by chemical secretions (chemotaxis) [Dukes and Apple 1961; Royle and Hickman 1964] or by electrical charges (electrotaxis) VESICLE [Khew and Zentmyer 1974] associated with the host SPORANGIUM plant. Zoospore activities including their production, dis- charge, motility, and attraction to plant parts are highly fascinating to students. The demonstrations and experi- Downloaded from http://online.ucpress.edu/abt/article-pdf/40/8/484/36360/4446362.pdf by guest on 01 October 2021 ments described in this article are appropriate for junior B~~~~~~~~~~~~~~~~~~~~'I high school and higher level students, including beginning students in college biology or botany. Most of the equip- or =_ ment and supplies required should be available in well- ..e. equipped high school or college laboratories.The culture of Pythium aphanidermatum may be provided free by some educational institutions,or it can be purchased from the American Type Culture Collection for approximately $25. Advanced experiments with zoospores can be developed for special research projects and science fairs. ZOOSPORES Procedure and Expected Results FIGURE 2. (A and B). Zoospore production by P. aphanidermatum Several species of Pythium and Phytophthora can be and attractionto tomato roots. (A) An enlarged sporangium and vesicle immediately prior to zoospore liberation. (B) Mass of zoospores at- used to demonstrate zoospore activities. I have found tached to a root of a young tomato plant. Pythium aphanidermatum is the most convenient and predictable organism for classroom use. P. aphanider- be maintained for 3 years or longer at 15-200 C without matum not only readily produces large numbers of zoo- repeated transfer. Cultures can also be maintained by spores but is an important plant pathogen that attacks storing in a refrigerator (5? C) and transferringto fresh more than 80 different plant species (Middleton 1943). media at 4-week intervals. Consequently, the organism can also be used for other Zoospore production. P. aphanidermatum from stock types of demonstrations and projects related to plant cultures is transferred to petri dishes containing freshly diseases. Cultures of P. aphanidermatum can be obtained prepared V-8 A and grown for 48 hours at 30? C. from The American Type Culture Collection, 12301 Mycelium-bearingagar discs (15 mm in diameter) are cut Parklawn Drive, Rockville, Maryland 20852 or from from the periphery of colonies with a sterilized transfer mycologists or plant pathologists at several state educa- needle or cork borer and the discs (four per dish) are tional institutions. placed aseptically in an inverted position in sterile petri P. aphanidermatum is a high temperature (optimun dishes containing 10 ml of sterile distilled water. Prepared 30-35? C) organism that grows rapidly on common dishes are incubated overnight (12-15 hours) at 300 C. laboratory media and survives well in culture (McCarter The water in the dishes is removed the next day and re- and Littrell 1973). Cultures can be maintained between placed with fresh sterile distilled water 3 to 4 hours before experiments in test tubes on slants of V-8 juice agar (V-8 the zoospores are needed for instructional purposes. A, 200 ml of unstrained V-8 juice, 3.0 g CaCo3, 20 g, Zoospore concentrations as high as 2 x 105 zoospores agar, brought to 1 liter with distilled water). Newly trans- per ml can be produced by this procedure. Suspensions ferred cultures are grown on the slants for 48 to 72 hours of zoospores can be observed at the different magnifi- in an incubator at 30-35? C. Culture viabilityand stability cations possible with a stereoscopic microscope. Zoo- can be ensured by completely covering 3-day-old slant spores are observed as small white dots moving in all di- cultures with sterile mineral oil (white mineral oil, rections in the water. Hanging-drop slides (cavity slide sterilized in an autoclave for 30 minutes at 15 p.s.i. and with a cover slip) bearing a drop of the zoospore suspen- heated in an oven at 1 10? C for 1 hour to drive off en - sion can also be observed with low power and high dry trapped bubbles). Cultures prepared in this manner can objectives of a compound microscope for detailed study. ZOOSPORE ACTIVITIES 485 Zoosporesof P. aphanidermatumare about7.5 ,umwide or aggregate(fig. 2B) after 15 to 20 minutes.Zoospores and 12 pm long and reniformin shape withtwo laterally willalso swarmon wounds made by puncturingthe epi- attachedflagella (fig. 1A) [Middleton1943]. The flagella dermis.The root with adheringzoospores may then be are difficultto see when in motionbut can be observedat placed on a glass slide and observedwith the low- and high magnificationwhen the zoospores are moving high-powermagnification of a compound microscope. slowlyor startingto encyst.Zoospores can also be immo- Zoosporescan be observedto encystand germinatewith bilizedwith ultraviolentlight (254 nm) appliedto a drop the germtubes directedtoward the sourceof stimulation. of suspensionfor 4 minutesat 5 cm fromthe lightsource Zoosporescan be observed more clearly after staining or withiodine (1 gramiodine crystals in 100 ml of water, withpositive (cationic) stains such as basicfuchsin, crystal 1 drop per ml of suspension)for countingand detailed violet,fast green, neutral red or safranin0. study. Zoospores are produced in a vesicle.that arises from a filamentous,often-branched, inflated sporangium Possibilitiesfor FurtherExperimentation that may varyin lengthfrom 50 to 1000 pm. Numerous sporangiaand vesicles