Hello,

I am an entomology graduate research assistant from the University of Arkansas currently working on the biological control of Amblyomma americanum ticks with cultures of Metarhizium anisopliae isolated from soil in Arkansas. I have been maintaining these cultures for about a year now and I have subcultured the fungal isolates about 4-5 times. Presently I have observed that cultures of one isolate have taken an orange, wrinkly, appearance and are not sporulating. Cultures of another M. anisopliae isolate in my possession have taken a bright yellowish color and have shown a reduction in sporulation from previous cultures. Previously these cultures had white mycelia with profuse amounts of sporulation. Both of these isolates were inoculated onto plates of Sabouraud Dextrose agar (SDA) supplemented with 1% yeast extract and 0.5 ml of gentamicin sulfate(10g/ml ). Fungal isolates were inoculated onto the plates with a solution of sterile deionized water, 0.05% Tween 80 and streptomycin (5mg/ml). The cultures are being held at a temperatures 24-25 degrees Celsius and relative humidities of 92-93%. Both of these cultures were used in a bioassay against A. americanum ticks and hardly any mortality of the ticks was seen until 5 weeks post-inoculation. With all this being said, is it possible that the multiple serial transfers of the fungi to SDA are causing them to lose their virulence and ability to sporulate? Are the holding conditions of the fungi that I mentioned above have a role to play in this? Also, what are some suggestions on how I can properly maintain the cultures to where they are able to properly maintain their sporulation and virulence properties?