Content determination and microscopic observation proved that dendrobine accumulation in the stem of Dendrobium nobile Lindl. increased after infection with mycorrhizal fungus MF23 (Mycena sp.). Large-scale transcriptome sequencing of symbiotic and asymbiotic D. nobile revealed that 30 unigenes encoding proteins were possibly related to the biosynthesis of dendrobine sesquiterpene backbone. A qRT-PCR experiment of 16 unigenes, selected randomly, proved that there were significant changes in the expression levels of AACT, MVD, PMK and TPS21 at 9 weeks after inoculation. These results implied that MF23 might stimulate dendrobine biosynthesis by regulating the expressions of genes involved in the mevalonate (MVA) pathway. The biogenetic pathway of dendrobine was suggested systematically according to the structural features of dendrobine alkaloids and their sesquiterpene precursors, which implied that post-modification enzymes might play a major role in dendrobine biosynthesis. Thus, genes encoding post-modification enzymes, including cytochrome P450, aminotransferase and methyltransferase, were screened for their possible involvement in dendrobine biosynthesis. This study provides a good example of endophytes promoting the formation of bioactive compounds in their host and paves the way for further investigation of the dendrobine biosynthetic pathway.

Plant growth-promoting fungi (PGPF) have attracted considerable interest as bio-fertilisers due to their multiple beneficial effects on plant quantity and quality and their positive relationship with the ecological environment. Advancements in the development of PGPF for crops and economic plant cultivation applications have been achieved, but such improvements for the use of PGPF with popular medicinal herbs, such as Salvia miltiorrhiza, are rare. In this study, we collected S. miltiorrhiza specimens inhabiting wild, semi-wild, farmland and pot-cultured areas in the Henan province of China and isolated endophytes from the roots, shoots and leaves of these samples. Twenty-eight strains of the dominant genus Alternaria were identified and selected as candidate PGPF. Under greenhouse conditions, Alternaria sp. A13 simultaneously enhanced the dry root biomass and secondary metabolite accumulation of S. miltiorrhiza as the optimal PGPF of the 28 candidate isolates. To further assess the interaction between S. miltiorrhiza and Alternaria sp. A13, the effects on seedlings growth, active ingredient accumulation, and the activity of key enzymes for effective biosynthetic pathways were investigated over a period of six months under field conditions. Compared to uninoculated seedlings, S. miltiorrhiza seedlings colonised by Alternaria sp. A13 showed significant increment of 140% in fresh weight, 138% in dry weight, and enhancement in the contents of total phenolic acid, lithospermic acids A and B (LAA and LAB, respectively) of 210%, 128% and 213%, respectively. Examination of the related enzyme activities showed that the elicitation effect of A13 on LAB accumulation correlated with cinnamic acid 4-hydroxylase (C4H) activity in the phenylpropanoid pathway under field conditions. Our results confirmed that Alternaria sp. A13 not only contributes to the stimulation of S. miltiorrhiza root growth, but also boosts the secondary metabolism, thus demonstrating its application potential as a bio-fertiliser for S. miltiorrhiza cultivation, especially in areas outside of its native growth regions.

Anoectochilus formosanus, commonly known as “Jewel Orchid”, is a Chinese folk medicine used to treat hypertension, diabetes, and heart disease. The existence of A. formosanus is currently threatened by habitat loss, human and animal consumption, etc. The highly potent medicinal activity of A. formosanus is due to its secondary metabolites, especially kinsenosides and flavonoids. This orchid also has a unique mycorrhizal relationship. Most adult orchids rely on endophytes for mineral nutrition and have complex interactions with them, which are related to plant growth, yield and changes in secondary metabolites. This study investigated the promoting role of F-23 fungus (genus Mycena) on the biomass and contents of kinsenosides and flavonoids of A. formosanus in pot culture. The following were observed after 10 weeks of symbiotic cultivation: increased shoot height, shoot dry weight, and leaf numbers by 16.6%, 31.3%, and 22.5%, respectively; increased contents of kinsenosides, isorhamnetin-3-O-β-d-rutinoside, and isorhamnetin-3-O-β-d-glucopyranoside by 85.5%, 226.1%, and 196.0%, respectively; some hyphae in epidermal cells dyed red and/or reddish brown by safranine; and, significantly reduced number of starch grains in cortical cells. Moreover, F-23 fungus significantly improved the kinsenoside and flavonoid contents of A. formosanus. These findings supported the reports that endophytes can alter the production of secondary metabolites in their plant hosts, although further physiological, genetic and ecological analyses are warranted.

Anoectochilus roxburghii (Wall.) Lindl. (Orchidaceae) is an endangered medicinal plant in China, also called “King Medicine”. Due to lacking of sufficient nutrients in dust-like seeds, orchid species depend on mycorrhizal fungi for seed germination in the wild. As part of a conservation plan for the species, research on seed germination is necessary. However, the molecular mechanism of seed germination and underlying orchid-fungus interactions during symbiotic germination are poorly understood. In this study, Illumina HiSeq 4000 transcriptome sequencing was performed to generate a substantial sequence dataset of germinating A. roxburghii seed. A mean of 44,214,845 clean reads were obtained from each sample. 173,781 unigenes with a mean length of 653 nt were obtained. A total of 51,514 (29.64%) sequences were annotated, among these, 49 unigenes encoding proteins involved in GA-GID1-DELLA regulatory module, including 31 unigenes involved in GA metabolism pathway, 5 unigenes encoding GID1, 11 unigenes for DELLA and 2 unigenes for GID2. A total of 11,881 genes showed significant differential expression in the symbiotic germinating seed sample compared with the asymbiotic germinating seed sample, of which six were involved in the GA-GID1-DELLA regulatory module, and suggested that they might be induced or suppressed by fungi. These results will help us understand better the molecular mechanism of orchid seed germination and orchid-fungus symbiosis.

Background: Orchids maintain a symbiotic relationship with mycorrhizal fungi in the lifecycle. Previous reports indicated that diverse mycobionts may have different roles during orchid growth and development. Although various mycorrhizal fungi have been isolated fromDendrobiumroots and protocorms, little is known about their specific effects on seed germination and seedling growth. To understand the specific role of isolated fungal strains (i.e.,TulasnellaandSebacina), we used symbiotic culture to compare the effect of 6 fungal strains on seed germination and seedling growth ofDendrobium officinale, an important Chinese medicinal orchid.Results: In symbiotic germination tests, 6 fungal strains (4Tulasnellastrains and 2Sebacinastrains) promoted seed germination with different efficiencies. Seeds inoculated withTulasnellastrains S6 and S7 conferred higher germination percentage and faster protocorm development than other fungal strains. In symbiotic cultures, seedlings inoculated withSebacinastrain S3 had optimal fresh and dry matter yield. Also,Tulasnellastrains S6 and S7 promoted seedling growth with good fresh and dry matter yield.Sebacinastrain S2 inoculation greatly enhanced root and tiller production and the content of total crude polysaccharides, although seedlings were smaller with less fresh and dry matter yield than other seedlings.Conclusions: TulasnellaandSebacinastrains could promote seed germination and seedling growth ofD. officinalewith different efficiencies. Our results suggest a non-specific mycorrhizal association and development-dependent preference. Our data provide the basic knowledge for use of different fungal strains in conservation and/or production practices ofD. officinale.