Phytopath. Z., 108,267-280 (1983) © 1983 Verlag Paul Parey, Berlin und Hamburg ISSN 0031-9481 / InterCode: PHYZA3 Lehrstuhl fur Phytopathologie der Universitdt Konstanz, Fakultdt fur Biologie Activation of Teliospore Germination 1 In Uromyces appendiculatus var. appendiculatus ) I. Aging and Temperature By 2 R. E. GOLD and K. MENDGEN ) With 8 figures Received April 29, 1983 Abstract The effects of aging and temperature on teliospore germination in Uromyces appendi­ culatus var. appendiculatus were studied. 1. Indoor storage was good at ~ 4 QC in the dirk. Under these conditions spores remained dormant for ~ 9 mo, but thereafter germination gradually increased to a maximum (~ 63 %) after 36 moo Spores .9tored outdoors showed a marked increase in germination after ~ 4 mo and reached maximum (~ 73 %) at 7-8 moo 2. The optimum temperature for germination was at 18 QC. While freezing (-18 QC) and thawing (20 QC) had no beneficial effect on .pore germination, heat treatments from 30-32 QC for 3-4 d proved effective in activating teliospore germinaJtion. Heat treatments caused an increase in spore mortality. Ba,sidiospore release from heM-treated teliospores was markedly reduced compared to control spores. 3. In all experiments the emergence of metabasidia was preceded by an obligatory 3-5 d lag period regardless of the treaJtment given, the age or level of germinability of treated and control spores. 1) This study represents a portion of a dissertation submitted by the senior author to the Faculty of Biology of ,the University of Kon~tanz in March, 1983, in pantial fulfillment of the requirements for the degree of Doctor of Natur.al Sciences (Dr. rer. nat.). 2) Author to whom reprint request> should be addressed. U.S. Copyright Clearance Cemer Code Statement: 0031-9481/83/0804-0267$02.50/0 268 GOLD and MENDGEN Zusammenfassung Die Aktivierung der Teleutosporenkeimung bei Uromyces appendicalatas var. appendicalatas durch Lagerung und Warmel:rehandlung Es wurde der EinfluB von Sporenlagerung und Temperarur auf die Teleuwsporen­ keimung bei Uromyces appendiculatus var. appendiculatus untersucht. 1. Gute Lagerungsbedingungen fur die Sporen waren bei 4°C im Dunkeln gegeben (Beobachtungszeitraum 4 Jahre). Hierbei blieben die Sporen ~ 9 Monate im Ruhestadium, danach stieg ihre Keimfahigkeit allmahlich auf ein Maximum (~ 63 %) nach 36 Monaten an. Im Gegensatz dazu waren Sporen, die auf dem Feld uberwin.terten, schon nach 4 Monaten keimfahig und erreichten ihr Maximum (~ 73 %) nach 7-8 Monaten. 2. Das Temperaturoptimum der Teleutosporenkeimung Jag bei 18°C. Der EinfluB von Kiilte- und Warmebehandlungen auf die Sporenkeimung wurde eingehend untersucht. Obwohl mehrmaliges Einfrieren und Auf~auen keine wesentliche Ak.tivierung der Keimung ergab, waren Warmebehandlungen bei 30-32°C fUr 3-4 d sehr wirksam. Eine geringe Erhohung der Mortalitatsrate und eine starke Beeintrachtigung der Abschleuderung von Basidiosporen waren weitere Folgen der Warmebehandlung. 3. In alien Versuchen, unabhangig von der gegebenen Behandlung, dem Alter oder der Keimfahigkeit der Teleutosporen, wurde beobachtet, daB nach einer obligatorischen Lagphase von 3-5 d das Metabasidium die Keimpore durchbrach und sich dann voll ausbildete. The teliospores of many agriculturally important rusts (e. g. Melampsora lini, Puccinia graminis, Uromyces appendiculatus var. appendiculatus) remain dormant for several months after formation. Problems associated with acti­ vating such spores to germinate readily have hindered studies of taxonomy, pathogenicity, and microscopy of the initial stages of basidiospore penetration and infection (see MENDGEN 1983). Many studies on teliospore germination have been conducted, but very few provide quantitative and/or qualitative information for species possessing dormant teliospores (BINDER et al. 1977, BLANK and LEATHERS 1963, HORNER 1963, KUSIEWICZ 1977, NWHAUS 1969). Various methods of stimulating germination of dormant tdiospores have been reported, including: cool laboratory storage, natural weathering, wetting and drying and/or freezing and thawing (see G0l-:D and STATLER 1983, MENDGEN 1983), chemical treatment (BINDER et al. 1977), application of host substances (KLISIEWICZ 1972, 1973), prolonged incubation on agar or water (GROTH and MOGEN 1978), and control of light (NEUHAUS 1969). Unfortunately, the reproducibility of these methods with similar isolates and their applicability to other rust species is unsatisfactory or unknown. Teliospores of Uromyces appendiculatus var. appendiculatus'l) (bean rust) were chosen for the present study. In the first part of this study, the effects of aging and temperature on the activation and pattern of teliospore ger­ mination were studied quantitatively and qualitatively. Preliminary reports of this research have been published earlier (GOLD and MENDGEN 1981 a, b). 3) Synonyms: Uromyces phaseoli (Pers.) Wine. and Uromyces phaseoli (Pers.) Wint. var. typica Anh. See BOEREMA and VERHOEVEN (1979) and CUMMINS (1978) for an accurate nomenclatural account of these and related species. Activation of Teliospore Germination. 1. 269 Material and Methods 1. Fungal isolates The bean rust isolates are listed in direct relation to the extent in which ~hey were experimentally ~udied: Fungus Origin Symbol U. appendiculatus var. Lahr Valley, Black Forest, SWBR appendiculatus W. Germany U. appendiculatus var. Gottingen, W. Germany GBR appendiculatus U. appendiculatus var. Reichenau Island, RBR 1 appendiculatus Lake Constance U. appendiculatus var. Con~ance, W. Germany KBR 1 appendiculatus The following isolates were only briefly studied for comparison: Phragmidium mucronatum Kerry County, Ireland Phragmidium violaceum Mainau Island, Lake Constance, W. Germany Puccinia carthami (1) Davis, California, USA Puccinia sorghi (2) Ziirich, Switzerland Uromyces dianthi (3) Liutlehampwn, England Uromyces scuttelatus Constance, W. Germany The original uredinial or telial culture was kindly provided by: 1. Dr.]. M. KLISIEWICZ, University of California-Davis. 2. Mr. T. ULLMAN, Eidgenossische Technische Hochschule, Ziirich. 3. Dr. D. M. SPENCER, Glasshouse Crops Research Institute, Littlehampton. 2. General The fungus, Uromyces appendiculatus var. appendiculatus, was increased for all exper­ iments on susceptible garden bean (Phaseolus vulgaris L., cv. Favor't). The plants were grown in a growth chamber held:Lt 18±0.50C, ~70%RH under 10000lx (16h photoperiod). Fourteen day old primary leaves were inoculated with an urediniospore suspension (~ 30 mg/ 100 ml distilled water with 0.05 % Tween 20) and incubated under dim light or in the dark a~ 18-20 °C and 100 % RH for 20-24 h. Secondary growth of the bean plants was removed biweekly. A.t 7-10 d after inoculation, uredinia were present on both ad- and abaxial leaf surfaces and heavy sporulation began. From 2-3 wk after initial sporulation, teliospore pro­ duction gradually replaced urediniospore formation in uredinia accompanied by teliospore production in separately developing telial sori. Mature teliospores were colle~ed 2-3 ,times weekly by gently brushing the infected leaves over aluminium foil. Teliospores were swred in closed glass vials in a refrigerator held at ~ 4°C and ~ 70 % RH in the dark. For the germin;l!tioll experiments teliospore-s were evenly brushed ontO a hydrophilic cellulose nitrate filter (Sartorius, 8,um pores) on purified 2 % glass-distilled water agar (Merck) in 6 or 9 cm plastic Petri dishes (Greiner). The rapidly evaporating fluorochemical liquid FC-75 (3M Brand Inert Fluorchernical Liquid) was used as carrier for the spores. The evenly spread spore ma-ss (~ 200 teliospores/mm2) appeared as a light brown monolayer. The plates were kept at 18 ± 0.5 °C under 1000 Ix (16 h photoperiod = 06.00-22.00 h, light; 22.00-06.00 h, dark). 270 GOLD and MENDGEN 3. Sampling and statistical analysis In all experiments, 200-300 spores were tallied in each of 3-5 replicates for each treatment per examina.tion date. Each experimel1Jt was repeated a minimum of 2-3 times. Observations were made at X 500 or X 640 using a Zeiss Universal microscope equipped with illlterference-contrast optics. Spore 5amples were scored for germination, mortali·ty, and in some cases for vacuolation. Spores were considered to be germinated when the metabasidium or por,tions ,therefrom were distinguishable beyond the limits of the apical germination pore and dead when the cytoplasm appeared anucleate and disorganized. All values represent total percenuge germination, mortality, or vacuolation, respectively. The results were analysed using Wilcoxon's rank test (Figs. 2, 4, 7, 8b) or Chi-square (X 2) from fourfold tables (Figs. 5, 8). Statistical differences with a significance level of P < 0.05 are indicated with an star (*). 4. Influence of aging on te1iospore germination. Teliospores of SWBR were produced in the fall and wimer of 1978 in growth chambers and stored a.t 4 DC as described above. As comparison, naturally overwintered spores of RBR 1 from field grown pole beans (1981) were tested. In this experiment, 2 alterna.te methods of overwintering were employed. In the first case telia-laden trifoliate leaves were collected randomly from the plants and air dried for 7 d in a cool greenhouse. The dried leaves were placed into a jute sack that was hung on ,the out5ide of a shed. The sack was not in direct sunlight and was protected from rain- and snowfall. The second alternative was simply to leave the infected pole beans standing with no protection or special precautions to minimize na.tural weathering.