Evaluation of Tetrazolium Bromide as a Vital Stain for Fungal Oospores

Sutherland, E. D., and Cohen, S. D. 1983. Evaluation of tetrazolium bromide as a vital stain for fungal oospores. Phytopathology 73:1532-1535. Staining of fungal oospores with tetrazolium bromide (MTT) is proposed The number of oospores of P. megasperina f. sp. glycinea race I stained with as a test for estimating oospore viability and as a technique for enhancing MTT in phosphate buffer did not differ significantly from the number detection of oospores in root tissue. More than 80% of the oospores of stained with MTT in water. Viability results as indicated by staining and Phvtophthora cactorum, P. niegasperma f. sp. glycinea races I and 3, and germination were comparable when oospores were killed by autoclaving. Pithium aphanidermatum stained a rose color with 0. 1% MTT after 24 hr When oospores of P. megasperma f. sp. glycinea race I were stored in sterile of incubation at 35 ± 2 C. Very few oospores of Aphanomrnces cochlioides, water for I, 2, and 3 wk at 23 ± 2 C, oospore germination decreased, and the A. euteiches, or Ptrhiumn ultimum stained under these same conditions. percentage of oospores which stained rose showed a corresponding Staining of most fungal species was better at 35 ± I C than 23 + 2 C. decrease. Dormant spores stained rose and activated and germinated spores Oospore staining of P. megasperma f. sp. gltcinea race I by MTT did not stained blue when MTT was added to germination plates. Oospores were change sigriificantly when the oospore concentration was 103, 10 4 , or 10 detected easily in infected soybean root tissue stained with MTT. oospores per milliliter, or when MTT concentration was 0.05, 0.5, or 5%. Additional key words: oospore germination. To study the survival of oospores of Phyitophthora megasperma Michigan State University, East Lansing; A. euteiches Drechs. Drechs. f. sp. g/ycinea Kuan and Erwin [= P. megasperma Drechs. isolate L30 from W. F. Pfender, University of Wisconsin, Madison; var. sojae Hildb.] that have been exposed to various environmental Phy'tophthora cactorum (Leb. and Cohn) Schroet. from J. E. conditions, germination has been used as a measure of spore Mitchell, University of Wisconsin, Madison; P. megasperma f. sp. viability, but results from this method are complicated by the glycinea races I (ATCC 44032) and 3 from A. F. Schmitthenner, dormancy of the oospores (19). The use of vital stains, such as Ohio Agricultural Research and Development Center, Wooster; tetrazolium salts, offers an alternative to oospore germination for and Py'thium aphanidermatum (Edson) Fitzpatrick from R. D. assessing viability. Nelson and Olsen ( 1i) used tetrazolium chloride Lumsden, USDA, Soilborne Diseases Laboratory, Beltsville, M D. (TTC) to locate sporangia of Sy'nchy'trium endobioticum in potato Pithiurn monospermum Pringsheim, a hyperparasite of P. tissue and verified the vital staining property of TTC by reducing megasperma f. sp. glycinea (5), and P. ultimum Trow were isolated both viability and staining of sporangia with heat and chemical previously in our laboratory. treatments. Pathak et al (12) detected oospores of Peronospora To produce oospores, A. cochlioides and A. euteiches were manshurica in soybean seeds with TTC, and correlated the grown for 40 days in oatmeal broth (15), and P. cactorum and P. proportion of stained oospores with germination. Shetty et al (16) megasperma f. sp. gly'cinea were grown for 30 days in modified V-8 utilized TTC to locate oospores of Sclerospora graminicola on juice broth supplemented with cholesterol (2). P. aphanidermatum, pearl millet seed. Workers (18) have questioned TTC as a vital P. monospermum, and P. ulhimum were grown in clarified V-8 oospore stain for Sclerospora graminicola, because oospore juice broth for 3 wk. Clarified V-8juice was prepared by adding l0 g staining varied greatly with presoaking time, incubation time and calcium carbonate to I L of V-8 juice (Campbell Soup Co., temperature, and stain and oospore concentrations. Another Camden, NJ 08101), heating the mixture to 80 C, and then tetrazolium compound, tetrazolium bromide, was used to test for centrifuging at 13,200 g for 10 min. One hundred milliliters of the viability of oospores of Phitophthora infestans in vitro (14), and supernatant plus 30 mg of cholesterol dissolved in 2 ml of 95% was also recommended for other Phytiophthora spp. (13). The ethanol were added to 900 ml of distilled water. chemistry of tetrazolium salts was reviewed by Altman (1). Briefly, Cultures of A. cochlioides, A. euteiches, P. cactorum, and P. the tetrazolium staining reaction occurs when the salt is reduced by megasperma f. sp. g/iycinea were frozen at 12 C for I hr to aid in a cellular dehydrogenase to form its colored formazan product separating the oospores from the mycelium. Mycelial mats of P. (4,8). aphanidermatum were transferred to sterile petri dishes containing The purpose of this study was to further investigate the use of filter paper to dry and store the mats prior to oospore extraction. tetrazolium bromide as a vital stain for oospores of several fungal Oospores were dislodged from mycelia by comminution of the species, to determine optimal staining conditions for oospores, and cultures suspended in water in a Servall Omni-mixer (Ivan Sorvall, to describe the relationship of oospore staining to germination. N orwalk, CT 06856) at a rheostat setting at 70% of line voltage ( 117 v) with ten 5-sec pulses for Aphanomyces and Pythium spp., and at MATERIALS AND METHODS a rheostat setting of 40% of line voltage (117 v) for 10 min for Production and isolation of fungi. The fungal species used were Phyjtophthora spp. The fragments were reground in the OmniAphanomyices cochlioides Drechs. obtained from C. L. Schneider, mixer to release more oospores. The liquid containing the oospores was centrifuged for 15 sec at 3,100 g. The supernatant liquid was The publication costs of this article were defrayed in part by page charge payment. This removed by suction, leaving the oospores concentrated in the article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. § pellet. Centrifugation was repeated until the oospore suspension 1734 solely to indicate this tact. 1734 __solelytoindicatethisfact._ was essentially free from hyphal fragments. Oospores were stored ©1983 The American Phytopathological Society in sterile distilled water at 4 ± 2 C for up to I wk prior to use.