Isolation of endophytic Frankia from root nodules of Casuarina equisetifolia and infectivity of the isolate to host plants

Abstract

Attempts were made to isolate endophytic Frankia from the root nodules formed on Casuarina equisetifolia grown in a mixture of vermiculite and field soil obtained from the Okayama University Campus. The isolation of Frankia was performed by macerating a nodule lobe directly in a test tube containing modified Qmod medium with casamino acids and sodium pyruvate as nitrogen and carbon sources. One colony, which showed hyphal and sporangial characteristics similar to those of Frankia, appeared in a test tube after 1-month incubation. The root nodules were successfully formed on the roots of the host plants by inoculation of the isolate which was multiplied using the original Qmod medium containing yeast extract, peptone and propionate. The acetylene reduction activity (ARA) obtained was 6.29 ± 1.57 µmol C2H4 h−1 (g F.W. nod.)−1. Accordingly, we identified the isolate as an effective Frankia strain for C. equisetifolia and designated it as Ceq1. Since this strain also formed root nodules on Alnus sieboldiana and Myrica rubra, it was considered to have a wider host range than that of Frankia strains which had been previously isolated from Casuarina nodules. However, the values of the nodule fresh weight and ARA in A. sieboldiana and M. rubra were much lower than those in C. equisetifolia and only half of the plants tested were nodulated in M. rubra. This strain did not form root nodules on Elaeagnus macrophylla. Frankia vesicles, which are the specialized structures where nitrogenase is located, were not observed in the free-living culture grown in Qmod medium containing abundant nitrogen and in the nodules newly formed by inoculation of the Ceq1 strain.