Shun-Xing Guo

Isolation and identification of endophytic and mycorrhizal fungi from seeds and roots of Dendrobium (Orchidaceae)

The seed germination of orchids under natural conditions requires association with mycorrhizal fungi. Dendrobium nobile and Dendrobium chrysanthum are threatened orchid species in China where they are considered medicinal plants. For conservation and application of Dendrobium using symbiosis technology, we isolated culturable endophytic and mycorrhizal fungi colonized in the protocorms and adult roots of two species plants and identified them by morphological and molecular analyses (5.8S and nrLSU). Of the 127 endophytic fungi isolated, 11 Rhizoctonia-like strains were identified as Tulasnellales (three strains from protocorms of D. nobile), Sebacinales (three strains from roots of D. nobile and two strains from protocorms of D. chrysanthum) and Cantharellales (three strains from roots of D. nobile), respectively. In addition, species of Xylaria, Fusarium, Trichoderma, Colletotrichum, Pestalotiopsis, and Phomopsis were the predominant non-mycorrhizal fungi isolated, and their probable ecological roles in the Dendrobium plants are discussed. These fungal resources will be of great importance for the large-scale cultivation of Dendrobium plants using symbiotic germination technology and for the screening of bioactive metabolites from them in the future.

Determination of optimal carbon source and pH value for sclerotial formation of Polyporus umbellatus under artificial conditions

Polyporus umbellatus is one of the precious medicinal fungi, with sclerotia used as a diuretic agent and antidote in China for many years. This has led to the present interest in producing sclerotia of P. umbellatus in the laboratory due to a decreased abundance in natural sources. Here, we investigated the determining factors for sclerotial formation in P. umbellatus. Five carbon sources, namely, maltose, fructose, glucose, sucrose and soluble starch with different initial pH values were evaluated for their effects on mycelial growth and sclerotial development of P. umbellatus. Maltose, fructose and glucose could induce sclerotial formation of P. umbellatus. Sucrose and soluble starch could stimulate growth of the fungus but had no effect on sclerotial formation. The most efficient sclerotial production occurred with maltose followed by fructose and a pH of 5. In addition, different macroscopically evident characteristics of sclerotial development of P. umbellatus induced by different carbon sources were also observed. Our findings could provide new insights into further research on sclerotial production in P. umbellatus under artificial cultivation.

Ultrastructure of symbiotic germination of the orchid Dendrobium officinale with its mycobiont, Sebacina sp.

Dendrobium officinale is an endangered epiphytic orchidaceous medicinal plant. Similar to other orchid plants, the seed germination of D. officinale under natural conditions depends nutritionally upon mycorrhizal fungi. The compatible fungi have been isolated from D. officinale protocorms using in situ seed baiting technique in our previous studies. However, the interaction between seed germination of D. officinale and its mycobiont is still unclear. In the present study, we investigated the morphological changes of seed and fungus during the symbiotic germination using a light microscope and transmission electron microscope. Seeds of D. officinale have no conspicuous suspensor cells. The fungus enters into the embryo cell through the posterior end of the embryo and colonises the cortical cell in the first stage of germination (Stage 1). Then, the hyphae form pelotons with the protocorm development (Stages 1–3). After protocorm formation, the reinvaded fungal hyphae conspicuously decrease. Invaded hyphae lose bioactivity, form clumps and start degeneration at Stage 4 or 5 (seedling development). When penetrating the neighbouring cortical cell, the fungal hyphae constrict to collar shape at the contacted site and follow by swelling in the apex. Our study suggested that fungi trigger protocorm development and concomitant reserve utilisation during the symbiotic germination.

Identification of Hortaea werneckii Isolated from Mangrove Plant Aegicerascomiculatum Based on Morphology and rDNA Sequences

Hortaea werneckii is a black yeast-like ascomycetous fungi associated with the human superficial infection tinea nigra, which commonly occurs in tropical and subtropical countries. Now, this fungus has been found in the halophilic environment all over the world and recognized as a new model organism in exploring the mechanisms of salt tolerance in eukaryotes. During a survey of endophytic fungi of mangrove forest at South China Sea, two isolates of H. werneckii were recovered from medicinal plant of Aegicerascomiculatum. The isolates were identified by morphological characters and phylogenetic analyses (e.g., ITS rDNA, LSU rDNA and translation elongation factor EF1α). Some physiological tests such as thermotolerance, acid tolerance (pH) and NaCl tolerance as well as pathogenicity test in vitro for the strains of Hortaea were performed. It is the first report that H. werneckii was isolated from medicinal plant of A.comiculatum in south sea of China as the endophytic fungi.

A new method to induce sclerotial differentiation in Polyporus umbellatus by split-plate culture

Polyporus umbellatus is one of the most valuable medicinal fungi, and its sclerotium has been used as a diuretic agent and an antidote in traditional Chinese medicine. In nature, Polyporus umbellatus has almost been depleted because of over-exploitation and lack of natural habitats. Thus, artificial sclerotia production has increased. This study aimed at finding an effective method to induce sclerotia, and selected the split-plate culture method. One side contained fructose agar medium (FAM), while the other side contained nutrient-limited medium. It was observed that sclerotia were only formed on the nutrient-limited medium side but scarcely emerged on the FAM side, even when the fructose concentration on both sides were the same. The sclerotial differentiation rate was 100% and the sclerotial yield was 106% higher than in the conventional way. In conclusion, the split-plate culture method is an effective way to induce P. umbellatus sclerotia in the laboratory.