| An endophyte is a bacterial (in cluding actinomycete) or fungal microorganism, which"colonizes symptomlessly the living, internal tissues of host plant, even though the endophyte may, after an incubation or latency period, cause disease."Due to the specialized living niches, little knowledge of these"special organisms"has been accumulated. However, much renewed attention is now being paid to the biodiversity, chemistry and bioactivity of functional metabolites and the related physiological and/or ecological role of these microorganisms. Pyrethrum cinerariifolium Trev, a famous traditional Chinese medicinal herb know to be the producer of Pyrethrum, was found to be a widespread insecticide and have antifungal activity. In this paper, we investigated the distribution, bioactivity, biological and chemical characteristics of the representative fungal endophytes colonized inside the leaves of P. cinerariifolium. The results and some points of renovation are discussed as follows:1. Systemically studied the fungal endophyte distribution and biodiversity of P.cinerariifolium for the first time. A total of 128 fungal were isolated from roots, stems leaves and flowers of P.cinerariifolium and 117 fungal strains were identified after morphology observation.. Among them, these strains were classified into 12 genera: Fusarium, Chaetomium, Phoma, Alternaria, Endophragmiella, Pestalotiopsis, Humicola, Nigrospora, Toruloa, Setodochium, Papularia and Rhizoctonia which showed higher diversity of endophytic fungi in P.cinerariifolium. It was found that the amount, species and distribution of endophytic fungi varied in different parts of P. cinerariifolium.2. The bioassay activities of extracellular metabolic products from 128 strains endophytic fungi were tested. Antagonistic tests showed that the inhibition rates of the 56 strains were more than 75% against at least one pathogen and among them fermentation of Y1, Y2 have better antagonistic action against six pathogens (Botrytis cinerea, Phytophthora capsic, Colletotrichum gloeosporioides, Exserohilum turcicum, Rhizoctonia cerealis and Fusarium graminearum) at the concentration 10 times of fermentation . Among 56 strains, acetone extraction of Y1, Y2 and H2 showed inhibition activities against the above 6 pathogens. Fermentation of strains Y1, Y2, show inhibition rates higher than 80% towards germination of Botrytis cinerea and Apple anthracnose at the concentration 5 times of fermentation. Results of pot culture experiment in greenhouse indicate that strains Y1, Y2, Y7, H2 showed antifungal activities more than 50% against Botrytis cinerea, Phytophthora capsici and Cucumber downy mildew. The results indicated that the strains of Y1, Y2, H2 and Y7 were broad-spectrum and stronger antifungal activity.3. The complete 5.8S rDNA sequence of Y1 and Y2 strain were cloned and sequenced respectively, and phylogenetic tree based on 5.8S rDNA sequences and analyses sequence homology were constructed. The characteristics of morphology showed that strain Y1 was identified as Pestalotiopsis sp., the analysis of DNA sequences showed that the sequence homology of Y1 strain was 100% with Pestalotiopsis microspora, and the 5.8s rDNA sequences number was EU638329 on Gene Bank。The characteristics of morphology showed that the macroconidium was falciform with five or seven diaphragm; microconidium has zero or one diaphragm. The analysis of DNA sequences showed that the sequence homology of Y2 strain was 100% with Fusarium oxysporum. The characteristics of morphology and the analysis of DNA sequences suggest that the strain of Y2 was identified as Fusarium oxysporum, and the 5.8s rDNA sequences number was EU152473 on Gene Bank。4.The biology characteristics of the two strains are preliminary defined. Results show that the effect of environmental and nutritional factors on its growth and reproduction was remarkable. To strain Y1 :ⅰ) Temperature from 10℃to 35℃was suitable for mycelial growth, germination and sporulations. The optimum temperature was 30℃for sporulation, and the lethal temperature was 65℃when treated for 10 minutes.ⅱ) pH value ranged from 6.5 to 8.0 was favorable for mycelial growth, sporulation and spore germination, and the optimum pH was 7.5.ⅲ) The best carbon sources for mycelial growth and sporulations were corn flour and dextrin.ⅳ) As to nitrogen sources, the fungus utilized organic nitrogen very well, while NO3–N (nitrate) inhibited its growth and sporulations. To strain Y2:ⅰ) Temperature from 20℃to 33℃were suitable for mycelial growth, sporulations and germination. The optimum temperature was 30℃for sporulation, and the lethal temperature was 60℃treated for 10 minutes.ⅱ) pH value ranged from 4.0 to 9.5 was favorable for mycelial growth, sporulation and spore germination. The optimum pH was 5.5.ⅲ) The best carbon sources for mycelial growth and sporulations were corn flour or dextrin.ⅳ) As to nitrogen sources, the fungus utilized organic nitrogen very well.5. The fermentation conditions of strains Y1 and Y2 were studied and better components of culture media and conditions were determined. The response surface methodology indicated that basic culture media of strainY1 were D-xylose 19.86 g·L-1, peptone 2.91 g·L-1, MgSO4?7H2O 0.305 g·L-1,FeSO4 0.0061 g·L-1 , K2HPO4 1.219 g·L-1 ,KCl 0.610 g·L-1and optimal incubation conditions of initial pH , medium volume in flask, shaking speed, temperature and inoculation amount were 7.64, 68.81 mL·250 mL-1, 220 r·min-1, 26.7℃and 9.95%, respectively. Under the optimal condition, the biomass weight and antibiotic activity were enhanced by 6.99% and 2.46% respectively compared with that under the initial condition.The single factor experiment indicated that basic culture media of Y2 were maltose 10 g, peptone 6 g, KH2PO4 2 g, KCl 1 g, FeSO4 0.02 g, MgSO4?7H2O 1 g every liter; and optimal incubation conditions of initial pH , medium volume in flask ,shaking speed , temperature and inoculation amount were7.0 , 100 mL·250 mL-1, 180 r·min-1 , 28.0℃and 21% , respectively. Under the optimal condition, the biomass weight and antibiotic activity were 897.5 mg·1000 mL-1 and 96.6%, and were enhanced by 8.5% and 4.7% respectively compared with that under the initial condition.6. In the process of isolation of intracellular metabolic components, bioactivity is tested using Botrytis cinerea to detect antifungal and antibacterial activities, respectively. Thirteen antifungal chemical components are isolated from intracellular metabolic products of Y1 and Y2. Six chemical components are newly isolated from intracellular metabolic products of Y1, the chemical structure of one of them (P2 and P3) is clarified, which is dulcitol and 3-methylbenzene-1,2-diol- dextrose indicant (new chemical component), and the chemical structure of P1 would be Benzoic acid 4,4'-N,N-cyclic propylene ester.The bioassay results indicate that these two compounds inhibit spore of Botrytis cinerea germination, and at 0.25 mg·mL-1, the inhibitory rates of P1, P2 and P3 against bourgeon of the spore of Botrytis cinerea are more than 50%. Seven antifungal chemical components are isolated from metabolic products of Y2, the chemical structure of the four (F1, F2, F3, F5) is clearly explained, which may be 4-methoxybenzonitrile,2-hydroxyethyl tridecanoate,docosane and dulcitol. The bioassay results indicate that the inhibitory rates of F2, F3and F4 against Botrytis cinerea are more than 60% at 1 mg·mL-1 after 7d. Hence, isolation and identification of antifungal active components of Y1 and Y2 need to be studied further.
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