The pathogenic fungus Neozygites tanajoae, one of the most efficient natural enemies of the cassava green mite (CGM) Mononychellus tanajoa in Brazil, was introduced experimentally in Benin in 1998/1999 for the control of CGM. Isolation methods and culture media for in vitro production of N. tanajoae are reported for the first time in this study. Continuous growth of N. tanajoae was achieved using medium NT-1 (IPL-41+5-10% fetal bovine serum+0.3% lactalbumin hydrolysate+0.3% yeastolate). This medium supported production of N. tanajoae up to 1.53 (+ or - 0.08) x 10(7) hyphal bodies/mL after 8 days. The growth of N. tanajoae from Cruz das Almas, Brazil, was compared to the growth of two Neozygites floridana isolates with wider host ranges from North Carolina, US, and Palmira, Colombia, in 11 cell culture media. We demonstrated that differences in nutritional requirements exist between N. tanajoae and the similar species, N. floridana. N. tanajoae is a particularly fastidious species highly specific to CGM and grows well in few media while N. floridana which is less host specific, grows in a broader range of media, including serum free media. N. floridana isolates produced more than 2 x 10(6) hyphal bodies/mL in > or =7 of the 11 media tested. However, the N. tanajoae isolate reached the same final concentration in only 3 media. Cell densities of N. tanajoae also increased slower than in N. floridana isolates in most media. N. tanajoae differed morphologically from the two N. floridana isolates in vitro. Hyphal bodies of eight N. tanajoae isolates are shorter than hyphal bodies of the two N. floridana isolates. The distinction of these two species was initially proposed based on host specificity, genetic and physiological patterns and is supported by the results presented in this study.