Biocontrol fungi induced stem-base rot disease resistance of Morinda officinalis How revealed by transcriptome analysis.

Introduction: Morinda officinalis How (MO) is a Rubiaceae plant, and its medicinal part is dried root, which is one of the "Four Southern Medicines" in China. At present, the plant MO breed seedlings mainly by cutting methods. Long-term asexual propagation makes pathogenic fungi accumulate in MO, leading to stem-base rot, which is caused by Fusarium oxysporum (Fon). Methods: In this study, we used Trichoderma harzianum and Pestalotiopsis sp. as biocontrol fungi to investigate their antagonistic ability to Fon through in vitro antagonism and pot experiments, and combined with transcriptome sequencing to explore the mechanism of biocontrol. Results: The results showed that both Trichoderma harzianum and Pestalotiopsis sp. could inhibit the growth of Fon. In addition, Trichoderma harzianum and Pestalotiopsis sp. could also enhance the basic immunity to Fon by increasing the activities of defensive enzymes such as POD and SOD, chlorophyll content, soluble sugar content, and oligosaccharide content of MO. The mechanism of biological control of stem-base rot of MO was discussed by transcriptome technology. MO was treated with two treatments, root irrigation with biocontrol fungi or inoculation with Fon after root irrigation with biocontrol fungi. Transcriptome sequencing revealed that nearly 11,188 differentially expressed genes (DEGs) were involved in the process of inducing MO systemic resistance to Fon by biocontrol fungi. Meanwhile, Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, as well as transcription factor (TFs) prediction showed that there were significant differences in the expression levels of MO roots under different treatments. Also, the genes of the "MAPK signaling pathway" and "plant hormone signaling pathway" were analyzed, in which the ERFs gene of the ethylene signal transduction pathway participated in the metabolism of glycosyl compounds. It is speculated that the ethylene signal may participate in the immune response of the sugar signal to the infection of Fon. After qRT-PCR verification of 10 DEGs related to the ethylene signal transduction pathway, the expression trend is consistent with the results of transcriptome sequencing, which proves the reliability of transcriptome sequencing. Discussion: In conclusion, this study preliminarily identified the molecular mechanism of the biological control of MO stem-base rot and provided a scientific basis for further research on the prevention and control mechanism of MO stem-base rot.

Nutritional Value, Volatile Components, Functional Metabolites, and Antibacterial and Cytotoxic Activities of Different Parts of Millettia speciosa Champ., a Medicinal and Edible Plant with Potential for Development.

Highly nutritious traditional plants which are rich in bioactive substances are attracting increasing attention. In this study, the nutritional value, chemical composition, biological activities, and feed indices of different parts of Millettia speciosa were comprehensively evaluated. In terms of its nutritional value, this study demonstrated that the leaves, flowers and seeds of M. speciosa were rich in elements and amino acids; the biological values (BVs) of these ingredients ranged from 85% to 100%, showing the extremely high nutritional value of this plant. GC-MS analysis suggested that the main chemical components of the flower volatile oil were n-hexadecanoic acid (21.73%), tetracosane (19.96%), and pentacosane (5.86%). The antibacterial activities of the flower and seed extracts were significantly stronger than those of the leaves and branches. The leaf extract displayed the strongest antifungal activities (EC50 values: 18.28 ± 0.54 µg/mL for Pseudocryphonectria elaeocarpicola and 568.21 ± 33.60 µg/mL for Colletotrichum gloeosporioides) and were the least toxic to mouse fibroblasts (L929) (IC50 value: 0.71 ± 0.04 mg/mL), while flowers were the most toxic (IC50 value: 0.27 ± 0.03 mg/mL). In addition, the abundance of fiber, protein, mineral elements, and functional metabolite contents indicated the potential applicability of M. speciosa as an animal feed. In conclusion, as a traditional herbal plant used for medicinal and food purposes, M. speciosa shows potential for safe and multifunctional development.

Diversity of Fungal Communities on Diseased and Healthy Cinnamomum burmannii Fruits and Antibacterial Activity of Secondary Metabolites.

The composition and structure of fungal communities on healthy and diseased fruits of Cinnamomum burmannii (Nees and Nees) Blume were characterized, with evaluation of the antibacterial activity of secondary metabolites from culturable fungi following the first identification of secondary metabolites in the fungus Medicopsis romeroi (Esf-14; GenBank accession number OK242756). These results are significant for understanding the functional variation in bioactivity in fungal communities and developing a broader range of bioactive resources. High-throughput sequencing results indicated that the fungal community in diseased fruit differed from that in healthy fruit at the phylum, class, order, or genus level, with significant differences in the species and relative abundance of the dominant flora. A total of 49 (healthy fruit) and 122 (diseased fruit) artificially cultivable endophytic fungi were isolated, and 41 different strains (11 from healthy fruit and 30 from diseased fruit) were successfully identified by morphological and molecular biological analyses, which were classified into 8 groups and 23 genera by phylogenetic tree analysis, with Pleosporales, Glomerellales, and Hypocreales being the dominant groups at the order level and Colletotrichum being the dominant group at the genus level. The results of the antibacterial assay demonstrated that the secondary metabolites of all strains had different degrees of antibacterial activity, while the secondary metabolites of endophytic fungi from diseased fruit were generally stronger than those of fungi from healthy fruit, with the active secondary metabolites dominated by small and moderately polar compounds. Combined analysis of fungal communities, phylogenetic tree analysis, and bioactivity analysis of culturable strains revealed strong antibacterial activity of both upregulated and downregulated flora in diseased fruit. Five compounds, including two new (5,6-dimethoxy-[1',1:4,1″-terphenyl]-2-ol [compound 1] and 5-(methoxycarbonyl)-2-methylbenzo[d][1,3]dioxole-2-carboxylic acid [compound 2]) and three known compounds (3,7-dihydroxy-1,9-dimethyldibenzofuran [compound 3], methyl 3-hydroxybenzoate [compound 4], and uracil [compound 5]), were isolated and identified for the first time from the endophytic fungus Medicopsis romeroi. In general, the diversity of fungal communities on diseased fruit was lower than that on healthy fruits, while the antibacterial activity of artificially cultured endophytic fungi on diseased fruits was generally stronger than that on healthy fruits, suggesting excellent promise for the development of secondary metabolites from active strains on diseased fruit as antibacterial agents. IMPORTANCE Powdery fruit disease is a notorious disease of Cinnamomum burmannii that causes severe loss in fruit production. Studies on the function of endophytic fungal communities in healthy plant tissues are not new, while little is known about the functional changes of fungal communities in disease-causing plant tissues. Our results demonstrate that fungal communities in diseased fruits differ from those in healthy fruits at the level of phylum, class, order, or genus, with significant differences in the species and relative abundance of dominant groups. Endophytic fungi in diseased fruits appeared to produce secondary metabolites with stronger antibacterial properties, although the community diversity was not as varied as that in healthy fruits. In addition, secondary metabolites of the Medicopsis romeroi strain from diseased fruits were identified for the first time. These results have important implications for understanding the functional variation of bioactivity in fungal communities and for developing a broader resource of bioactivity.

Pseudocryphonectriaelaeocarpicola gen. et sp. nov. (Cryphonectriaceae, Diaporthales) causing stem blight of Elaeocarpus spp. in China.

Cryphonectriaceae is a diaporthalean family containing important plant pathogens of which Cryphonectriaparasitica is the most notorious one. An emerging stem blight disease on Elaeocarpusapiculatus (Elaeocarpaceae) and E.hainanensis was observed in Guangdong Province of China recently. Typical Cryphonectria blight-like symptoms including cankers on tree barks with obvious orange conidial tendrils were observed. Forty-eight isolates were obtained from diseased tissues and conidiomata formed on the hosts E.apiculatus and E.hainanensis. These isolates were further identified based on both morphology and molecular methods using the combined sequence data of the internal transcribed spacer (ITS) region, large subunit of the nrDNA (LSU), the translation elongation factor 1-alpha (tef1) and DNA-directed RNA polymerase II second largest subunit (rpb2) genes. As a result, the fungus represents an undescribed genus and species within the family Cryphonectriaceae. Hence, Pseudocryphonectriaelaeocarpicola gen. et sp. nov. is proposed herein to represent these isolates from diseased barks of E.apiculatus and E.hainanensis. Pseudocryphonectria differs from the other genera of Cryphonectriaceae in having dimorphic conidia. Further inoculation results showed that P.elaeocarpicola is the causal agent of this emerging blight disease in China, which can quickly infect and kill the hosts E.apiculatus and E.hainanensis.

Diversity and antibacterial activity of fungal endophytes from Eucalyptus exserta.

BACKGROUND: Eucalyptus bacterial wilt caused by Ralstonia solanacearum is an important eucalyptus disease. Endophytic fungi, an important source of natural active substances, provide a new breakthrough for the control of plant diseases. RESULTS: In the present study, 80 endophytic fungal isolates were obtained from the healthy branches and fruits of Eucalyptus exserta. Fifteen distinct isolates (MK120854-MK120868) were selected for further taxonomic identification through morphological trait assessments and internal transcribed spacer (ITS) region-rRNA gene sequence analysis. Thirteen genera, namely, Phyllosticta, Penicillium, Eutypella, Purpureocillium, Talaromyces, Lophiostoma, Cladosporium, Pestalotiopsis, Chaetomium, Fusarium, Gongronella, Scedosporium and Pseudallescheria, were identified on the basis of their morphological characteristics. Members of the genus Phyllosticta were the primary isolates, with a colonization frequency (CF) of 27.5 %. Most of the fungal isolates displayed antibacterial activity. The crude extracts obtained from Lophiostoma sp. Eef-7, Pestalotiopsis sp. Eef-9 and Chaetomium sp. Eef-10 exhibited strong inhibition on the test bacteria, and Lophiostoma sp. Eef-7 was further cultured on a large scale. Three known compounds, scorpinone (1), 5-deoxybostrycoidin (2) and 4-methyl-5,6-dihydro-2 H-pyran-2-one (3), were isolated from the endophytic fungus Lophiostoma sp. Eef-7 associated with E. exserta. The structures of these compounds were elucidated by analysis of 1D and 2D NMR and HR-ESI-MS spectra and a comparison of their spectral data with published values. Compounds 1 and 2 showed weak antimicrobial activity against Ralstonia solanacearum. CONCLUSIONS: Endophytic fungi from Eucalyptus exserta may represent alternative sources of antimicrobial agents. Lophiostoma sp. Eef-7 can produce 2-azaanthraquinone derivatives and shows weak antibacterial activity against Ralstonia solanacearum.

Rapid Screening of Essential Oils as Substances Which Enhance Antibiotic Activity Using a Modified Well Diffusion Method.

Antimicrobial resistance is recognized as one of the major global health challenges of the 21st century. Synergistic combinations for antimicrobial therapies can be a good strategy for the treatment of multidrug resistant infections. We examined the ability of a group of 29 plant essential oils as substances which enhance the antibiotic activity. We used a modified well diffusion method to establish a high-throughput screening method for easy and rapid identification of high-level enhancement combinations against bacteria. We found that 25 essential oils possessed antibacterial activity against Escherichia Coli ATCC 25922 and methicillin-resistant Staphylococcus aureus (MRSA) 43300 with MICs that ranged from 0.01% to 2.5% v/v. We examined 319 (11 × 29) combinations in a checkerboard assay with E. Coli ATCC 25922 and MRSA 43300, and the result showed that high-level enhancement combinations were 48 and 44, low-level enhancement combinations were 214 and 211, and no effects combinations were 57 and 64, respectively. For further verification we randomly chose six combinations that included orange and Petitgrain essential oils in a standard time-killing assay. The results are in great agreement with those of the well diffusion assays. Therefore, the modified diffusion method was a rapid and effective method to screen high-level enhancement combinations of antibiotics and essential oils.

Duclauxin Derivatives From Fungi and Their Biological Activities.

Duclauxin is a heptacyclic oligophenalenone dimer consisting of an isocoumarin and a dihydroisocoumarin unit. These two tricyclic moieties are joined by a cyclopentane ring to form a unique hinge or castanets-like structure. Duclauxin is effective against numerous tumor cell lines because it prevents adenosine triphosphate (ATP) synthesis by inhibiting mitochondrial respiration. There are about 36 reported natural duclauxin analogs mainly produced by 9 Penicillium and Talaromyces species (T. duclauxii, T. aculeatus, T. stipitatus, T. bacillisporus, T. verruculosus, T. macrosporus, P. herquei, P. manginii, and Talaromyces sp.). These metabolites exhibit remarkable biological activities, including antitumor, enzyme inhibition, and antimicrobial, showing tremendous potential in agricultural and medical applications. This review highlights the chemical structures and biological activities of fungal duclauxins, together with biosynthesis, absolute configuration, and mode of action for important duclauxins. Furthermore, phylogenetic analysis and correct names of Penicillium and Talaromyces species producing duclauxins are presented in this review.

New nitrogen-containing metabolites from cultures of rice false smut pathogen Villosiclava virens.

Two new nitrogen-containing metabolites methyl N-acetyl-O-(4-acetylphenyl)-L-homoserinate (1), dimethyl (1H-indole-3-carbonyl)-D-glutamate (2), and two new natural products, 1,2-O-isopropylidene-D-mannitol (3), N-acetyl-ß-methyl-L-phenylalanine (4), along with five known compounds (5-9) were isolated from the rice false smut pathogen Villosiclava virens UV-8b cultured in the solid rice medium. The structures were elucidated by spectroscopic analysis and by comparison of their physical and spectroscopic data with the literature. These metabolites were evaluated for their antibacterial and phytotoxic activities. Compounds 5-7 showed weak inhibition against the tested bacteria, while compounds 4-6 and 9 displayed inhibitory activity against the radicle elongation of rice seeds.

A new p-terphenyl derivative from the insect-derived fungus Aspergillus candidus Bdf-2 and the synergistic effects of terphenyllin.

A new p-terphenyl derivative 4″-deoxy-2'-methoxyterphenyllin (1), along with six known p-terphenyl derivatives (2-7), a known flavonoid derivative dechlorochlorflavonin (8) and a known fellutanine A (9), were isolated from the insect-derived strain of the fungus Aspergillus candidus Bdf-2, associated with Blaptica dubia. The structure of 1 was established by the analysis of the 1D and 2D NMR and HR-ESI-MS spectra. Compounds 1-9 were evaluated for antibacterial activities against Staphylococcus aureus ATCC29213, Escherichia coli ATCC25922 and Ralstonia solanacearum, and for antioxidant activities. Synergistic effects of compound 2 with the other compounds were also investigated. As a result, compound 6 displayed the best antibacterial activities in all single compound with MIC value of 32 µg/mL against S. aureus ATCC29213 and R. solanacearum, respectively. However, no antibacterial effect against E. coli ATCC25922 was detected from any single compound. The combination of 2 + 6 exhibited obvious synergistic effect against S. aureus ATCC29213 and the MIC value was 4 µg/mL. Compound 6 also showed the best antioxidant activity as a single compound with an IC50 value of 17.62 µg/mL. Combinations of 5 + 6, 2 + 4 + 5 and 2 + 4 + 5 + 6 displayed synergistic effect and their antioxidant activities were better than that of any single compound.

Mollicellins O⁻R, Four New Depsidones Isolated from the Endophytic Fungus Chaetomium sp. Eef-10.

Four new depsidones, mollicellins O⁻R (compounds 1⁻4), along with three known compounds 5⁻7, were isolated from cultures of the fungus Chaetomium sp. Eef-10, an endophyte isolated from Eucalyptus exserta. The structures of the new compounds were elucidated by analysis of the 1D and 2D NMR and HR-ESI-MS spectra. The known compounds were identified by comparison of their spectral data with published values. Compounds 1⁻7 were evaluated for antibacterial activities against Staphylococcus aureus (sensitive and resistant strains), Escherichia coli, Agrobacterium tumefaciens, Salmonella typhimurium, Pseudomonas lachrymans, Ralstonia solanacearum, Xanthomonas vesicatoria and cytotoxic activities against two human cancer cell lines (HepG2 and Hela). Mollicellin H (6) displayed best antibacterial activity, with IC50 values of 5.14 µg/mL against S. aureus ATCC29213 and 6.21 µg/mL against S. aureus N50, MRSA, respectively. Mollicellin O (1) and mollicellin I (7) also exhibited antibacterial activities against S. aureus ATCC29213 and S. aureus N50. Mollicellin G (5) was active against both two human cancer cell lines, with IC50 values of 19.64 and 13.97 µg/mL while compounds 6 and 7 only showed cytotoxic activity against one cell line. In addition, mollicellin O (1) showed antioxidant activity based on DPPH radical scavenging, with an IC50 value of 71.92 µg/mL.

Bioactive spirobisnaphthalenes from the endophytic fungus Berkleasmium sp.

Nine new spirobisnaphthalenes, palmarumycins B1-B9 (1-9), along with 13 known compounds (10-22), were isolated from cultures of the fungus Berkleasmium sp., an endophyte isolated from the medicinal plant Dioscorea zingiberensis C. H. Wright. The structures of the new compounds were elucidated by analysis of the 1D and 2D NMR and HRESIMS spectra and by comparison with known compounds. Compounds 7-9 contain an uncommon 2,3-dihydro-1H-inden-1-one unit. All isolated compounds were evaluated for their antibacterial activities against Bacillus subtilis, Staphylococcus hemolyticus, Agrobacterium tumefaciens, Pseudomonas lachrymans, Ralstonia solanacearum, and Xanthomonas vesicatoria and for their antifungal effects against the spore germination of Magnaporthe oryzae. Palmarumycin C8 (22) exhibited the best antibacterial and antifungal effects. In addition, diepoxin δ (11) and palmarumycin C8 (22) showed pronounced cytotoxic activities against five human cancer cell lines (HCT-8, Bel-7402, BGC-823, A 549, A 2780) with IC50 values of 1.28-5.83 µM.

Preparative separation of spirobisnaphthalenes from endophytic fungus Berkleasmium sp. Dzf12 by high-speed counter-current chromatography.

High-speed counter-current chromatography (HSCCC) was applied for the first time for the preparative separation of spirobisnaphthalenes from a crude extract of the endophytic fungus Berkleasmium sp. Dzf12, associated with the medicinal plant Dioscorea zingiberensis. Six spirobisnaphthalenes were successfully separated by HSCCC with a two-phase solvent system composed of n-hexane-chloroform-methanol-water (1.5:3.0:2.5:2.0, v/v). About 18.0 mg of diepoxin κ (1), 245.7 mg of palmarumycin C13 (2), 42.4 mg of palmarumycin C16 (3), 42.2 mg of palmarumycin C15 (4), 32.6 mg of diepoxin δ (5), and 22.3 mg of diepoxin γ (6) with purities of 56.82, 71.39, 76.57, 75.86, 91.01 and 82.48%, respectively, as determined by high-performance liquid chromatography (HPLC), were obtained from 500 mg of the crude extract in a one-step elution within 7 h of separation procedure by HSCCC. The purified spirobisnaphthalenes were further structurally characterized by means of physicochemical and spectrometric analysis.

Purification of ustiloxins A and B from rice false smut balls by macroporous resins.

Ustiloxins are cyclopeptide mycotoxins produced by Villosiclava virens, the pathogenic fungus of rice false smut disease. Both resins SP207 and SP700 were screened to show the best adsorption and desorption properties for ustiloxins A and B among 20 commercial macroporous resins. Dynamic adsorption and desorption tests were carried out to optimize the process parameters. The optimal conditions for adsorption of resin SP207 were a processing volume as 32 bed volumes (BV), pH value of 4, and flow rate of 2 BV/h; and those for desorption of resin SP207 were a 40:60 (v/v) ratio of ethanol to water, an eluent volume of 4 BV, pH value of 4 and a flow rate of 3 BV/h. The optimal conditions for adsorption of resin SP700 were a processing volume of 26 BV, pH value as 4, flow rate of 2 BV/h; and those for desorption of resin SP700 were a 30:70 (v/v) ratio of ethanol to water solution as eluent, volume of 4 BV, pH value as 4 and flow rate of 2 BV/h. Under the optimal conditions; the purities of ustiloxins A and B obtained with resin SP207 increased 23.06-fold and 19.78-fold, respectively; and their recoveries were 96.67% and 81.25%; respectively. Similarly; the purities of ustiloxins A and B obtained with resin SP700 increased 14.75-fold and 15.33-fold and their recoveries were 93.65% and 88.64%; respectively. The results show that adsorption and desorption on SP207 and SP700 resins are effective strategies for purifying ustiloxins A and B. The developed methods are beneficial for large-scale preparation and purification of ustiloxins A and B from rice false smut balls.

Enhancement of Palmarumycins C(12) and C(13) production in liquid culture of endophytic fungus Berkleasmium sp. Dzf12 after treatments with metal ions.

The influences of eight metal ions (i.e., Na+, Ca2+, Ag+, Co2+, Cu2+, Al3+, Zn2+, and Mn4+) on mycelia growth and palmarumycins C(12) and C(13) production in liquid culture of the endophytic fungus Berkleasmium sp. Dzf12 were investigated. Three metal ions, Ca2+, Cu2+ and Al3+ were exhibited as the most effective to enhance mycelia growth and palmarumycin production. When calcium ion (Ca2+) was applied to the medium at 10.0 mmol/L on day 3, copper ion (Cu2+) to the medium at 1.0 mmol/L on day 3, aluminum ion (Al3+) to the medium at 2.0 mmol/L on day 6, the maximal yields of palmarumycins C(12) plus C(13) were obtained as 137.57 mg/L, 146.28 mg/L and 156.77 mg/L, which were 3.94-fold, 4.19-fold and 4.49-fold in comparison with that (34.91 mg/L) of the control, respectively. Al3+ favored palmarumycin C(12) production when its concentration was higher than 4 mmol/L. Ca2+ had an improving effect on mycelia growth of Berkleasmium sp. Dzf12. The combination effects of Ca2+, Cu2+ and Al3+ on palmarumycin C(13) production were further studied by employing a statistical method based on the central composite design (CCD) and response surface methodology (RSM). By solving the quadratic regression equation between palmarumycin C(13) and three metal ions, the optimal concentrations of Ca2+, Cu2+ and Al3+ in medium for palmarumycin C(13) production were determined as 7.58, 1.36 and 2.05 mmol/L, respectively. Under the optimum conditions, the predicted maximum palmarumycin C(13) yield reached 208.49 mg/L. By optimizing the combination of Ca2+, Cu2+ and Al3+ in medium, palmarumycin C(13) yield was increased to 203.85 mg/L, which was 6.00-fold in comparison with that (33.98 mg/L) in the original basal medium. The results indicate that appropriate metal ions (i.e., Ca2+, Cu2+ and Al3+) could enhance palmarumycin production. Application of the metal ions should be an effective strategy for palmarumycin production in liquid culture of the endophytic fungus Berkleasmium sp. Dzf12.

Determination and analysis of ustiloxins A and B by LC-ESI-MS and HPLC in false smut balls of rice.

Ustiloxins are cyclopeptide mycotoxins produced by the pathogenic fungus Villosiclava virens of rice false smut. Ustiloxins A and B as two main mycotoxins were determined conveniently by LC-ESI-MS in the water extract from rice false smut balls which were mostly composed of the chlamydospores and mycelia of the pathogen. Both ustiloxins A and B in the water extract were also quantitatively analyzed by HPLC. This is the first report on the determination and analysis of ustiloxins A and B simultaneously by LC-ESI-MS and HPLC in false smut balls of rice.

Medium optimization for exopolysaccharide production in liquid culture of endophytic fungus Berkleasmium sp. Dzf12.

Berkleasmium sp. Dzf12, an endophytic fungus from Dioscorea zingiberensis, is a high producer of spirobisnaphthalenes with various bioactivities. The exopolysaccharide (EPS) produced by this fungus also shows excellent antioxidant activity. In this study, the experimental designs based on statistics were employed to evaluate and optimize the medium for EPS production in liquid culture of Berkleasmium sp. Dzf12. For increasing EPS yield, the concentrations of glucose, peptone, KH(2)PO(4), MgSO(4)·7H(2)O and FeSO(4)·7H(2)O in medium were optimized using response surface methodology (RSM). Both the fractional factorial design (FFD) and central composite design (CCD) were applied to optimize the main factors which significantly affected EPS production. The concentrations of glucose, peptone and MgSO(4)·7H(2)O were found to be the main effective factors for EPS production by FFD experimental analysis. Based on the further CCD optimization and RSM analysis, a quadratic polynomial regression equation was derived from the EPS yield and three variables. Statistical analysis showed the polynomial regression model was in good agreement with the experimental results with the determination coefficient (adj-R(2)) as 0.9434. By solving the quadratic regression equation, the optimal concentrations of glucose, peptone and MgSO(4)·7H(2)O for EPS production were determined as 63.80, 20.76 and 2.74 g/L, respectively. Under the optimum conditions, the predicted EPS yield reached the maximum (13.22 g/L). Verification experiment confirmed the validity with the actual EPS yield as 13.97 g/L, which was 6.29-fold in comparison with that (2.22 g/L) in the original basal medium. The results provide the support data for EPS production in large scale and also speed up the application of Berkleasmium sp. Dzf12.

Extraction optimization of water-extracted mycelial polysaccharide from endophytic fungus Fusarium oxysporum Dzf17 by response surface methodology.

Water-extracted mycelial polysaccharide (WPS) from the endophytic fungus Fusarium oxysporum Dzf17 isolated from Dioscorea zingiberensis was found to be an efficient elicitor to enhance diosgenin accumulation in D. zingigerensis cultures, and also demonstrated antioxidant activity. In this study, response surface methodology (RSM) was employed to optimize the extraction process of WPS from F. oxysporum Dzf17 using Box-Behnken design (BBD). The ranges of the factors investigated were 1-3 h for extraction time (X(1)), 80-100 °C for extraction temperature (X(2)), and 20-40 (v/w) for ratio of water volume (mL) to raw material weight (g) (X(3)). The experimental data obtained were fitted to a second-order polynomial equation using multiple regression analysis. Statistical analysis showed that the polynomial regression model was in good agreement with the experimental results with the determination coefficient (R(2)) of 0.9978. By solving the regression equation and analyzing the response surface contour plots, the extraction parameters were optimized as 1.7 h for extraction time, 95 °C for extraction temperature, 39 (v/w) for ratio of water volume (mL) to raw material weight (g), and with 2 extractions. The maximum value (10.862%) of WPS yield was obtained when the WPS extraction process was conducted under the optimal conditions.

Preparative separation of helvolic acid from the endophytic fungus Pichia guilliermondii Ppf9 by high-speed counter-current chromatography.

High-speed counter-current chromatography (HSCCC) was applied for preparative separation of helvolic acid from the crude extract of the endophytic fungus Pichia guilliermondii Ppf9, associated with the medicinal plant Paris polyphylla var. yunnanensis for the first time. The two-phase solvent system consisted of n-hexane-ethyl acetate-methanol-water (4.5:4.5:5.0:5.0, v/v) appending with phosphoric acid (0.2%, v/v) was employed. The revolution speed of the separation column, flow rate of the mobile phase and separation temperature of the apparatus were 800 rpm, 3 ml min(-1) and 25°C, respectively. About 6.8 mg of helvolic acid was successfully obtained from 450 mg of the crude extract by HSCCC within 4 h separation procedure, and its purity reached to 93.2% according to the HPLC analysis. The product was further characterized by MS, (1)H-NMR and (13)C-NMR spectra.

Enhancement of diepoxin ζ production in liquid culture of endophytic fungus Berkleasmium sp. Dzf12 by polysaccharides from its host plant Dioscorea zingiberensis.

This study is the first report of the enhancement of diepoxin ζ production in liquid culture of the endophytic fungus Berkleasmium sp. Dzf12 by the polysaccharides from its host plant Dioscorea zingiberensis which serve as elicitors. Three polysaccharides, namely water-extracted polysaccharide (WEP), sodium hydroxide-extracted polysaccharide and acid-extracted polysaccharide were sequentially prepared from the rhizomes of D. zingiberensis. Among them, WEP was found to be the most effective elicitor to enhance diepoxin ζ production. When WEP was added to the medium at 400 mg l(-1) on day 3 of culture, the maximal diepoxin ζ yield (intracellular diepoxin ζ in mycelia plus extracellular diepoxin ζ in medium) of 350.76 mg l(-1) on day 15 was achieved, which was about 2.69-fold in comparison with that (130.43 mg l(-1)) of the control.

Enhancement of palmarumycin C12 and C13 production in liquid culture of the endophytic fungus Berkleasmium sp. Dzf12 by oligosaccharides from its host plant Dioscorea zingiberensis.

Three crude oligosaccharides were respectively prepared by acid hydrolysis of three polysaccharides, which were water-extracted polysaccharide (WEP), sodium hydroxide-extracted polysaccharide (SEP) and acid-extracted polysaccharide (AEP) from the rhizomes of Dioscorea zingiberensis. Among the three oligosaccharides, the crude oligosaccharide prepared by acid hydrolysis of WEP was found to be the most efficient elicitor to enhance the production of palmarumycins C(12) and C(13) in liquid culture of endophytic fungus Berkleasmium sp. Dzf12. When OW was applied to the medium at 300 mg/L on day 3 of culture, the maximal yields of palmarumycin C(12) (87.96 mg/L) and palmarumycin C(13) (422.28 mg/L) were achieved on day 15 of culture, which were 9.83 and 3.24-fold in comparison with those (8.95 and 130.43 mg/L) of control, respectively. The results indicate that addition of the oligosaccharides from the host plant D. zingiberensis should be an effective strategy for enhancing production of palmarumycins C(12) and C(13) in liquid culture of endophytic fungus Berkleasmium sp. Dzf12.