Standard deviation bars denote averages from three independent experiments. *: significant difference, p <0.05; **: significant difference, p <0.01. Figure 5 Exogenous addition of 8-Br-cAMP to the AC-RNAi mutant results in increased growth rates. The morphology of the wild type, knockdown control and AC-RNAi mutant colonies grown in the presence of 8-Br-cAMP (5 mM) were inoculated on PDA medium. These cultures were grown for 5 d prior to documentation. Scale bar: 0.5 cm. MaAC is required for in vivo virulence and growth Differences in virulence and invasive
growth inside insects were also compared between the wild type and RNAi mutant. Figure 6A shows selleck chemical that, 5 days post-inoculation on the pronotum, locusts infected selleck inhibitor by the wild type fungus began to die, while those infected by the RNAi mutant died 1 day later. Figure 6B shows that when the insects were inoculated by the injection of conidia into abdominal segments, the locusts began to die 4 days after injection of the wild type, and again the insects treated with the conidia of RNAi mutant died 1 day later. Accordingly, the lethal time value for 50% mortality (LT50) by topical inoculation and
injection of the RNAi mutant was significantly higher than that of the wild type (p <0.05) (Figure 6C), which indicated that MaAC is required for M. acridum virulence. Figure 6 The virulence and fungal growth in the haemolymph of locust in vivo and in vitro . A. Topical application with 5 μL suspensions of 1 × 107 conidia/mL of wild type and RNAi mutant (control insects were inoculated with 5 μL cottonseed oil). B. Survival of the locusts by injection with 5 μL suspensions of 2 × 106 conidia/mL (control insects were injected with 5 μL sterile water). C. Lethal time for 50% mortality (LT50) values of Locusta migratoria treated with the wild type or AC-RNAi
mutant. Error bars denote standard deviations obtained from five Epacadostat nmr trials. D. DNA concentration of AC-RNAi and wild type in the hemolymph of locusts 48 h after injection. E. Photomicroscopy of the development of conidiation patterns of M. acridum in the hemolymph of locusts. After 4 d of infection on the pronotum, the conidiation of the RNAi mutant strain grew slower than the wild type strain. The conidiation of the RNAi mutant strain grew also slower than the wild type Chloroambucil strain 3 d after injection into abdominal segments. F. Photomicroscopy of the development of conidiation patterns of M. acridum in the hemolymph of locusts in vitro. After they were cultured for 24 h, the conidiation of the RNAi mutant strain grew slower than the wild type strain. Scale bar: 20 μm. Error bars are standard deviations of five trials. *: significant difference, p <0.05, **: significant difference, p <0.01. To confirm the effect of MaAC on virulence, fungal growth in vivo was observed by photomicroscopy and quantified by real-time PCR. The M.