Table 1.
Metarhizium spp. isolates used in this study, including their hosts and origins (state and country).
Figure 1.
Destruxin (DTX) production by 12 Metarhizium spp. isolates in vitro.
DTXs production is represented by mean values ± standard error after 5 days in submerged shaken cultures. Production of DTXs in supernatant of cultures was determined by quantitative HPLC analysis of the major components, viz., DTXs A, B and E. Cultures and assays were repeated 3 times.
Figure 2.
Time course of in vitro production of DTXs A, B, and E by Metarhizium anisopliae s.l. ARSEF 759.
Destruxin concentrations in supernatants of submerged liquid cultures were determined by quantitative HPLC-UV analysis of the major components, viz., DTXs A, B and E. Values are expressed in mg DTXs per g dry weight mycelium.
Figure 3.
Time course of in vitro production of DTXs A, B, and E by Metarhizium robertsii ARSEF 2575.
Destruxin concentrations in supernatant of submerged liquid cultures were determined by quantitative HPLC analysis of the major components, viz., DTXs A, B and E. Values are expressed in mg DTXs per g dry weight mycelium.
Figure 4.
Re-isolation of Metarhizium robertsii or M. acridum after their endophytic colonization of cowpeas (Vigna unguiculata) and cucumber (Cucumis sativus).
Control plants with no fungus inoculation (A, D, G, and J); M. robertsii growing from surface sterilized roots (B) and leaves (E) of cowpeas; M. robertsii growing from surface sterilized roots (H) and leaves (K) of cucumber. M. acridum growing from surface sterilized roots (C) and leaves (F) of cowpeas; and M. acridum growing from surface sterilized roots (I) and leaves (L) of cucumber. Note that the characteristic brownish-green conidia of M. robertsii were obscured by a layer of white mycelium, whereas the dark green conidia of M. acridum were more visible due to very little mycelial overlay.
Figure 5.
HPLC-MS analysis of cowpea extracts for destruxin (DTX) production.
(A) Analysis of not colonized (free of fungus) plants (negative control); (B) plants endophytically colonized by Metarhizium robertsii ARSEF 2575; and (C) not-colonized plants spiked with DTX standards (positive control). The cowpea seeds, both fungus-inoculated and control (not colonized) were incubated on moist filter paper under optimal light (16L∶8D) and temperature (25°C) conditions for 12 days at which time the germlings had developed roots, stems, cotyledons and two true leaves. DTXs were extracted from entire plants using methanol 100% and SPE-C18 cartridges.
Table 2.
Mean mortality (%) ± standard error of Tenebrio molitor larvae 5 days after treatment, and Galleria mellonella 3 days after treatment.