An artificial wax substrate for the in-vitro study of phylloplane microorganisms

An artificial substrate consisting of a wax-based medium amended with nutrients has been developed for the in vitro study of microbial interactions on the phylloplane. Inoculation of bacteria and yeasts onto the surface demonstrated that these micro-organisms remain viable for up to 13 days (e.g. Aureobasidium pullulans) and proliferate on the surface under conditions of low humidity. The growth rate was slower (doubling time 1.2 days) than on nutrient agar or in nutrient broth. The artificial leaf wax substrate supported a lower bacterial population (10(6)-10(7) c.f.u. plate(-1)) than conventional nutrient substrates. Inoculation with Aureobasidium pullulans in high (10(9) c.f.u. plate(-1)) or low (10(4) c.f.u. plate(-1)) numbers indicated that the surface could support between 16 x 10(4)-16 x 10(5) c.f.u. per cm(2). In the absence of incorporated nutrients, yeast numbers were found not to increase. Spore germination and mycelial elongation of filamentous fungi also occurred on the artificial leaf wax substrate but rates were reduced in comparison with those on nutrient agar. Inoculation of the artificial leaf substrate with a mixed population of a soil yeast and a phyllosphere-specific yeast resulted in competition between species such that the phyllosphere yeast became dominant.