Nuclear behavior during basidiospore germination in Cronartium quercuum f. sp. fusiforme P. C. Spaine methods. In this paper, we report on a subsequent 1 ISLIA Forest Service, Southfrn Rrsrarrh Station, 320 mitotic division in C. quercuum f. sp. fusifbrvne basid- (hen Stwet, Athens, Geor@a 30602 iospores that occurred following meiosis. This addi- tional mitotic division was not found to be associated Shigeru Kaneko with different types of basidiospore germination. Laboratory qf Forest Pathology, Forestry and Forest Products Resmnh Institute, Kukizaki, Inashiki-gun, Previous studies of nuclear behavior in basidio- Ibaraki 305, /aFan spores of other rust fungi have reported a binucleate stage occurring after meiosis and before spore rc- lease from sterigmata (Anikster, 1983; Kaneko, 1975). Abstract: Nuclear behavior during basidiospore ger- Kaneko (1975) examined the nuclear behavior of te- mination in Cronartiunz quercuum f. sp. fusiforme was liospores and sporidia in Coleosporium petasitis Cooke examined on glass slides and host seedlings using 4, during indirect and direct germination, and found 6-diamidino-2-phenylindole staining. Mononucleate 97% of basidiospores were binucleate O-2 h after re- basidiospores of Cronartium quercuum f. sp. fusiforme lease from sterigmata. Similar nuclear behavior also normally were produced following meiosis in the te- was observed in C. helianthi and C. vernonia~ by Olive liospore. However, a subsequent mitotic division of- (1942) and in C. sidae by Sanwal (1953). Bauer ten occurred within each basidiospore resulting in a (1986) and Bauer and Oberwinkler (1988) found ba- short-lived binucleate condition. Within 1 h after sidiospores of several rust species, including Cronar- spores were released from the basidium, one of the tiuvn asclepiadeum, (Wilde) Fr., Gymnosporangium cla- two nuclei in most basidiospores began to degener- uarizforme (Pers.) DC., Puccinia maluacearum Bert., ate. Ninety-five percent of basidiospores directly cast Phragmidium uiolaceum (C. F. Shultz) Wint., and (:O- from telia onto seedlings of Pinus taeda germinated leosporium tussilaginis (Pers.) Lev., were binucleate directly forming thin germ tubes. More than 93% of until they formed a hypha, appressorium, or second- germ tubes were mononucleate. On glass slides, how- ary basidiospore, at which time one nucleus degen- ever, 79% of basidiospores germinated indirectly, erated. This condition also was observed in Gymno- forming a secondary basidiospore. During indirect sporangium juni@ri-vircginianae Schwein (Mims and germination, degeneration of the second nucleus oc- Richardson 1989, 1990). curred quickly without exception. No differences in In a previous study of Cronartium quercuum f. sp. nuclear behavior were found between direct and in- fusiforme, Spaine and Kaneko (1993) found that direct germination on host plants or on glass slides. more than 95% of washed basidiospores cast onto However, nuclear movement from basidiospores into water agar germinated directly, forming thin germ germ tubes was faster on the pine seedlings than on tubes. Unwashed basidiospores germinated indirect- glass slides. ly, forming secondary basidiospores. The objective of Key Words: direct germination, indirect germi- this study was to determine whether germination nation, nuclear degeneration, Pinus taeda, Quewus type, direct or indirect, influences or relates to nu- rubra, rust fungus clear behavior. MATERIALS AND METIIODS Fusiform rust disease is a major disease in pine plan- Leaves of Quercus rubra I,. were inoculated with ae- tations of the southeastern United States. Under- ciospores (mixed gall collection, Clarke County, standing the basic biology of the pathogen, Cronar- Georgia) of Cronartium quercuum f. sp. fusiforme. tium quercuum (Berk.) Miyabe ex Shirai f. sp. fusifor- Leaves bearing telia were collected 3 wk after inocu- me, is essential to understanding the disease cycle and lation. To stimulate both germination types, directly the development of successful biological control cast and washed basidio-spores were used (Spaine and Kaneko, 1993). In direct-cast spore treatments, oak leaves with telia 892 SPAINE AND KANEKO: C RONARTZUM N UCLEAR B EHA VIOR 893 were hydrated for 12 h in darkness at 20 C in a petri RESULTS dish containing moist filter paper. This procedure initiated the formation of basidiospores. Leaves were Each cell of a basidium contained one nucleus (FIG. then suspended for 1 h directly over either glass I), but mitosis was observed in immature basidio- slides sprayed with distilled H,O or needles and spores still attached to sterigmata. The binucleate stems of 3-mo-old Pinus taeda seedlings that had condition was short-lived, with most spores becoming been cut off at the stem collar. The P hour incuba- mono-nucleate before germination. Washing spores tion represented spores harvested after a 30-min cast- apparently hastened this process. Nuclear behavior ing period. All other incubation periods followed a was similar during both types of germination, direct, l-h casting period. Slides and seedlings were incu- i.e., formation of a thin germ tube, or indirect, i.e., bated in a petri dish with moistened filter paper for by repeated secondary basidiospore formation. 0, 1, 2, 4, or 6 h. FIG URES 1-8 illustrate the nuclear conditions ob- Washed spores were prepared by a method similar served in the basidium and basidiospores during in- to the one described by Miller (1970). Oak leaves direct and direct germination. Each cell of the basid- were hydrated as described above. Leaves were then ium was mononucleate (FIG. 1). When basidiospores hung for 1 h over a petri dish containing H,O ad- were first cast from the basidium, the majority of justed to pH 2.2 with HCl. Discharged basidiospores spores were binucleate (FIG. 2). However, after a l- were collected on an Advantec’ millipore filter (3.0 2 h period, one of the two nuclei was degenerating km) (Toy0 Roshi, Ltd., Japan), and washed with dis- and fading (FIG. 3). When degeneration of the sec- tilled H,O. Suspensions of collected basidiospores in ond nucleus was complete, basidiospores remained distilled H,O were sprayed onto seedlings of Pinus mononucleate (FIG. 4). In basidiospores that germi- taeda or glass slides in petri dishes containing moist- nated indirectly, the nucleus often was seen migrat- ened filter paper. ing into the sterigma (FIG. 5). The nucleus then mi- All petri dishes of slides and seedlings were kept at grated into the secondary basidiospore where it un- 20 C in darkness for 0, 1, 2, 4, and 6 h. Five slides derwent a second mitotic division (FIG. 6). The bi- and five seedlings were prepared for washed and un- nucleate condition in the secondary basidiospore washed spores for each incubation period. Immedi- also was short-lived, and one nucleus degenerated ately after removal from the petri dishes, slides were quickly leaving a mononucleate spore. During direct air-dried at 50 C. To observe germination of basidi- germination, typically a single nucleus migrated into ospores on seedlings, the needles and stem of a seed- the germ tube (FIG. 7). However, occasionally a bi- ling were placed on a glass slide previously sprayed nucleate condition was observed in the germ tube with distilled H,O, and the basidiospores were (FIG. 8). washed with distilled H,O from the seedlings. These Effects of substrate and of basidiospore washing on slides also were air-dried at 50 C. Five slides were pre- the frequency of nuclear conditions during basidio- pared for unwashed basidiospores, and five for spore germination of C. quercuum f. sp. fusiforme are washed basidiospores. To determine the nuclear be- summarized in FIG. 9. When basidiospores were cast havior within basidiospores, the nuclear conditions in directly onto glass slides, after 30 min in the casting both germination types were examined by 4, 6-diam- chamber and O-h further incubation, 85% of the idino-2-phenylindole (DAPI) staining methods. spores had two nuclei of equal size (FIG. 9, a). How- Before DAPI staining, all dried slides were fixed in ever, after l-h in the casting chamber with O-h further a solution of absolute ethanol and acetic acid (3:l) incubation, only 19% of the spores were binucleate for 10 min, washed in distilled H,O for 10 min five (FIG. 9, a), 76% contained one normal-sized nucleus, times, and air-dried. Slides were stained in DAPI fol- and one smaller nucleus (FIG. 9, b); and 5% were lowing the method described by Kuroiwa and Suzuki mononucleate (FIG. 9, c). Mononucleate basidio- (1980), but changing the DAPI concentration to 0.5 spores increased to 73% after a 1 h incubation period kg/mL. Observations were made with an Olympus (FIG. 9, c). BH-2 epifluorescence microscope. Spores (50 on Basidiospores directly cast onto glass slides began each of five slides, 250 total) were examined for each to develop germlings after a l-h incubation period. of the 25 treatments. Nuclear conditions in basidia However, 65% of the spores germinated indirectly, developed from teliospores were examined by stain- producing secondary basidiospores within a 6h in- ing germinating telia with DAPI. cubation (FIG. 9, i-q) period. Only one nucleus was observed (FIG. 5) in 98% of primary spores or sec- ondary sterigmata from the I- to 6-h incubation pe- 1 The use of trade or firm names in this publication is for reader information and does not imply endorsement by the U.S. Depart- riod (FIG. 9, l-o). This suggests nuclear degeneration ment of Agriculture of any product or service. occurred rapidly during indirect germination. After 894 FIGS. 1-8. Fluorescence micrographs of Cronartium quewuum f. sp. f usif orme illustrating different nuclear conditions observed in the basidium and basidiospores during indirect and direct germination. P = primary basidiospore; S = secondary basidiospore. Bar = 20 pm for all figures. 1. Basidium with one nucleus in each cell. 2. Binucleate basidiospores. 3. Binucleate basidiospores, with one nucleus degenerating. 4. Degeneration of second nucleus nearly complete.