title: Determination of viability of Phytophthora capsici oospores with the tetrazolium bromide staining test versus a plasmolysis method.
abstract: BACKGROUND
  Oospores are the most persistent propagules of Phytophthora capsici. The viability of oospores is determined by staining methods such as the tetrazolium bromide (MTT) test.
  
  
  OBJECTIVE
  The aim was to assess the MTT test and a plasmolysis method for their utility in determining viability of oospores of P. capsici.
  
  
  METHODS
  For either procedure the reactions of non-treated (viable) and lethally heat-treated (non-viable) oospores were assessed and compared with different matings of isolates.
  
  
  RESULTS
  The plasmolysis method provided lower estimates of oospore viability relative to MTT in almost all cases. Viability of autoclaved oospores determined by MTT ranged from 26 to 35%, while in the plasmolysis method, the false positive rate was null. Data of non-treated (viable) oospores determined by both techniques showed a significant positive correlation (r=0.998, P=0.0001). Viability assessments in the plasmolysis method of heated oospores in sterile water at 52°C (0-2-4-20 hours) showed viability values of 81-4-0.7-0% while MTT values were 78-15.3-9-5.7% respectively. They both exhibited different values for the other tested mating (57.7-1-1-0% and 50-7.3-7.7-9% respectively). Data of autoclaved, heat-treated (52°C), and non-treated oospores determined by both techniques showed a significant positive correlation (r=0.946, P<0.0001). However, MTT always overestimated viability rates when values were near zero.
  
  
  CONCLUSIONS
  MTT staining was non-objective, unstable and with a high rate of false positives. In contrast, reliable results were obtained using the plasmolysis method. MTT should be used in combination with other techniques such as the plasmolysis method for determining viability of P. capsici oospores.
authors.affiliation: NEIKER-Tecnalia, Basque Institute of Agricultural Research and Development, Derio, Spain.
authors.forename: Aitzol
authors.initials: A
authors.lastname: Etxeberria
authors.forename: Sorkunde
authors.initials: S
authors.lastname: Mendarte
authors.forename: Santiago
authors.initials: S
authors.lastname: Larregla
mesh_terms: D003116:Color; D004396:Coloring Agents; D005189:False Positive Reactions; D006358:Hot Temperature; D006982:Hypertonic Solutions / Q000494:pharmacology; D050296:Microbial Viability; D009172:Mycology / Q000379:methods*; D009997:Osmotic Pressure; D010838:Phytophthora / Q000166:cytology*; D012988:Soil Microbiology; D013172:Spores, Fungal / Q000187:drug effects; D013194:Staining and Labeling; D013778:Tetrazolium Salts; D013844:Thiazoles
chemical_list: D004396:Coloring Agents; D006982:Hypertonic Solutions; D013778:Tetrazolium Salts; D013844:Thiazoles; C022616:thiazolyl blue