| Insects symbiosis is a kind of microorganisms share long-term evolution with insects, which possess a huge diversity of species and metabolic pathways. Some direct evidence showed that insect gut fungi will be an important source of active natural products as well as the important new microorganism resources, which are the important microorganism resources.In this paper, the diversity of fungi associated with the Pantala flavescens larvae were investigated using a culture dependent method, molecular identification based on the internal-transcribed-spacer sequence analysis and morphological analysis. Further research for the symbiotic relationship and internal relation between fungi and bacteria in the gut of P. flavescens larvae were performed by using PCR and Laser Microscope. One fungi was isolated and identified from the gut of Ceriagrion glabrum. Combined with the results of the activity screening, the metabolites of two fungi (QTYC11 and QTYC1) and another fungus (DN02) isolated from the gut of C. glabrum were further studied by using the method of column chromatography (Silica gel chromatography, LH-20 Sephadex column chromatography, C18-column chromatography, HPLC, etc.). All compounds were identified on the basis of extensive spectroscopic (1H-NMR,13C-NMR, HMQC, COSY, HMBC, and ESI-MS, etc.) analysis. A total of 12 compounds were isolated, in which 11 compounds were authenticated, including one new compound. In addition, we further studied the herbicidal, antibacterial and other biological activities of compounds. The details as follows:1> The diversity of fungi associated with the Pantala flavescens larvae, were investigated using a culture dependent method and molecular identification based on the internal-transcrib-ed spacer sequence analysis. In total,48 fungal isolates were obtained, which were included in four classes and 11 different genera, including one new fungal speceis. Fourteen bacterial 16S rDNA sequences, derived from total genomic DNA extractions of fungal mycelia were obtained from the 48 fungi. Most were members of the Proteobacteria (13/14), including one of Bacillaceae (1/14). Leclercia sp., Oceanobacillus oncorhynchi and Methylobacterium extorquens, as the bacterial endosymbiont reside in fungi were first reported. Viable bacteria were observed within living hyphae of symbiotic QTYC45 using Live/Dead stain on the basis of laser microscopy. Biological activities of the fungal culture extracts were tested against pathogenic bacteria Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6633) and Escherichia coli (ATCC 8739) and a relatively high proportion of positive results were obtained. This study is the first report on the diversity and antibacterial activity of symbiotic fungi residing in the gut of P. flavescens larvae. The new strain QTYC44 showed good cellulose enzyme activity, which the cellulose enzyme activity can reach 1654.6 U/mL after 6 d.of fermentation. The present results show that they have rich diversity and could be exploited as a potential source of bioactive compounds.2^ Chemical investigations of the Paraphaeosphaeria sp. QTYC11 isolated from the gut of Pantala flavescens larvae, led to the isolation of 10 metabolites (1-9), which were determined on the basis of extensive spectroscopic analysis and by comparison of the corresponding data reported previously. Among of them lunatoic acid C (1) was first reported. Compounds 1 and 6 showed good activities against the radical growth of Amaranthus retroflexus with the IC50 values of 10.2 and 27.6μg/mL, respectively. The metabolites 1 and 4 had good phytotoxic activities against the radical growth of Echinochloa crusgalli with IC50 values of 5.9 and 16.0μg/mL respectively, and compound 1 showed moderate post-emergence activity for E. crusgalli with 46.0% decrease in dry wight. The ingredient 1 showed extremely potent antifungal activities against the Valsa mali (IC50= 3.3μg/mL), Gibberella sanbinetti (IC50=1.7μg/mL) and Fusarium oxysporum f. sp. cucumerinum (IC50 =4.2μg/mL), which were comparable to those of referenced cycloheximide with IC50 values of 0.3,3.3 and 4.9μg/mL, respectively. The compound 1 also showed good antibacterial activities against Staphylococcus aureus and Bacillus subtilis with the inhibition zone of 17.1 and 28.3 mm, respectively.3、The bioactive metabolites of C. crepinii QTYC-1 was isolated and identified. Potted activity tests showed that the fermented liquid of C. crepinii QTYC-1 could significantly inhibit the growth of Echinochloa crusgalli and Amaranthus retroflexus, with the biggest inhibition rate of 95.0% and 90.1% respectively. Sprayed with the fermented liquid of QTYC-1 in vivo, the victimization rate of weed seedling was 71.1%. The ethyl acetate extract (100μg/mL) exhibited significant phytotoxic activities against the radical growth of E. crusgalli and A. retroflexus with inhibitory rate of 56.8% and 71.2%, respectively, and it also showed good security for some common crops with the inhibition rate of lower than 35%. The ethyl acetate extract (25μg/mL) also showed strong inhibitory effect on Dothiorella gregaria, Altemaria solani, Fusarium. oxysporum f. sp. mornordicae, F. oxysporum f. sp. cucumerinu, Gibberella sanbinetti with the inhibition rate of 69.0%,86.3%,46.6%,74.3% and 71.2%, respectively, which were comparable to the positive control cycloheximide. Compound (11) was isolated from the ethyl acetate extract and showed good activity against E. crusgalli and A. retroflexus with the inhibition rate of 56.8% and 71.2% under the concentration of 100μg/mL, respectively.4、Chemical investigations of Cochliobolus lunatus DN02 were performed. The ethyl acetate extract exhibited significant phytotoxic activities against the radical growth of E. crusgalli and A. retroflexus with IC50 values of 22.9 and 136.7μg/mL, respectively. When sprayed with fermentation broth of DN02, victimization rate, average plant height and fresh weight of E. crusgalli were 66.7%,5.15 cm,13.8 mg, respectively, which were comparable to the positive control 2,4-D (70.0%、4.1 cm、12.4 mg). A main active compound was isolated from the crude extract and was determined as radicinin.In conclusion, the metabolites of the fungi resided in the gut of P. flavescens larvae and Ceriagrion glabrum showed good biological activity, which could be an important resource of natural bioactive molecules. It is worth to further research. |
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