Evaluation of Tetrazolium Bromide as a Vital Stain for Fungal Oospores

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Abstract
Staining of fungal oospores with tetrazolium bromide (MTT) is proposed as a test for estimating oospore viability and as a technique for enhancing detection of oospores in root tissue. More than 80% of the oospores of Phytophthora cactorum, P. megasperma f. sp. glycinea races 1 and 3, and Pythium aphanidermatum stained a rose color with 0.1% MTT after 24 h of incubation at 35.degree. .+-. 2.degree. C. Very few oospores of Aphanomyces cochlioides, A. euteiches or Puthium ultimum stained under these same conditions. Staining of most fungal species was better at 35.degree. .+-. 1.degree. C than 23.degree. .+-. 2.degree. C. Oospore staining of P. megasperma f. sp. glycinea race 1 by MTT did not change significantly when the oospore concentration was 103, 104 or 105 oospores per milliliter or when MTT concentration was 0.05. 0.5 or 5%. The number of oospores of P. megasperma f. sp. glycinea race 1 stained with MTT in phosphate buffer did not differ significantly from the number stained with MTT in water. Viability results as indicated by staining and germination were comparable when oospores were killed by autoclaving. When oospores of P. megasperma f. sp. glycinea race 1 were stored in sterile water for 1.2 and 3 wk at 23.degree. .+-. 2.degree. C, oospore germination decreased, and the percentage of oospores which stained rose showed a corresponding decrease. Dormant spores stained rose and activated and germinated spores stained blue when MTT was added to germination plates. Oospores were detected easily in infected soybean root tissue stained with MTT.

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