Interactions of two endophytic fungi colonizing Atractylodes lancea and effects on the host’s essential oils

Abstract

We investigated interactions between endophytic fungi infecting the same host; Gilmaniella sp. AL12 and Cunninghamella sp. AL4 are endophytes of Atractylodes lancea (Asteraceae). We studied the effect on the host’s essential oils of the single inoculation of one fungus and the mixed inoculation of both. We also observed the effects of these inoculations on endophytic fungal colonization and distribution. Single inoculation included two groups: single inoculation of AL12 (AL12 group), and single inoculation of AL4 (AL4 group). Mixed inoculation included three groups: AL4 inoculation first, followed by AL12 (AL4/AL12), simultaneous inoculation of AL4 and AL12 (AL4_VS_AL12), and AL12 inoculation first, followed by AL4 (AL12/AL4). The control (CK) did not include any fungi. AL4 was observed to prefer to colonize rhizomes, and AL12 to colonize blades of plants. The isolation rate of each fungus in the mixed inoculation group was lower than in the single inoculation group; this may have been due to niche competition. When we inoculated AL12 first and then AL4, the isolation rate of AL4 in stems, leaves, and roots decreased. Similarly, when we inoculated AL4 first and then AL12, the isolation rate of AL12 decreased in the stems and leaves. However, in the roots it appeared that the invasion of AL4 changed the volatile oil levels and that this helped the colonization of AL12; the colonization rate of AL12 following single inoculation was slightly lower than in the AL12/AL4 group and the AL4_VS_AL12 group. This may illustrate that these endophytes have a long history of mutual colonization of roots, and thus have synergies with each other. We used tissue staining to observe inoculated plant tissue. No hyphae were observed intruding into plant cells, and the hyphae of the two fungi were not observed intertwining; meaning that there was no physical interaction between them. However, we found hyphae colonizing the interspaces of leaf tissues and between root cortical cells. These hyphae were single bend fold and unbranched. These mycelia exhibited adaptive morphology compared with those in in vitro pure culture. Transmission electron microscopy (TEM) allowed us to more clearly observe endophytic colonization between root cortical cells. Gas chromatography showed that both single and mixed inoculation significantly increased levels of atractylone and atractylodin in the essential oils of A. lancea. In particular, the relative percentage of atractylodin in groups AL12/AL4 and AL4/AL12 was twice as much as in the control group, and the relative percentage of atractylone in AL4_VS_AL12 was more than three times that of the control. The gas chromatograph of the host’s essential oil was more complex following mixed inoculation than it was in the control.