Metagenomics and In Vitro Growth-Promoting Experiments Revealed the Potential Roles of Mycorrhizal Fungus Humicolopsis cephalosporioides and Helper Bacteria in Cheilotheca humilis Growth

In mycorrhizal symbiotic relationships, non-photosynthetic myco-heterotrophic plants are unable to supply photosynthates to their associated fungi. On the contrary, they rely on fungal carbon to sustain their own growth. Mycorrhizal fungi can mediate plant interactions with the rhizosphere microbiome, which contributes to the promotion of plant growth and nutrient uptake. However, the microbial community and key microbial species that function during the growth of the myco-heterotrophic plant <i>Cheilotheca humilis</i> remain unclear. In this study, we evaluated the microbial community associated with <i>Cheilotheca humilis</i>, which was confirmed via morphological characteristics typical of this plant species. Metagenomic analysis showed that the <i>Afipia carboxidovorans</i> was dominant at species level. Based on the LDA score, <i>Bradyrhizobium ottawaense</i> exhibited the higher abundance in the CH-B group (related to bud) while <i>Afipia carboxidovorans</i> was identified from the CH-F group (related to flower). Microbial co-occurrence networks showed that the <i>Rhizobium</i> genus, <i>Herbaspirillum</i> genus, and Cyanobacteriota were defined as core functional microbial species. To explore the potential microorganisms, metagenome-assembled genomes (MAGs) of the rhizosphere microbiome identified 14 medium- and high-quality MAGs, mainly involved in carbon fixation, nitrogen transformation, and phosphorus metabolism, possibly providing nutrients for the plant. Furthermore, a total of 67 rhizospheric and 66 endophytic microorganisms were isolated and obtained. In vitro experiments showed that the mycorrhizal helper bacteria (MHBs) <i>Rhizobium</i> genus and <i>Pseudomonas</i> genus possessed the ability of nitrogen fixation, phosphate solubilization, and siderophores production. Most importantly, the mycorrhizal fungus <i>Humicolopsis cephalosporioides</i> was obtained, which could potentially produce cellulase to supply carbohydrates for host. The findings suggest the mycorrhizal fungus <i>Humicolopsis cephalosporioides</i> and helper bacteria have great potential in the growth of the myco-heterotrophic plant <i>Cheilotheca humilis</i>.