Biotransformation of 2-benzoxazolinone and 2-hydroxy-1,4-benzoxazin-3-one by endophytic fungi isolated from Aphelandra tetragona

The biotransformation of the phytoanticipins 2-benzoxazolinone (BOA) and 2-hydroxy-1,4-benzoxazin-3-one (HBOA) by four endophytic fungi isolated from Aphelandra tetragona was studied. Using high-performance liquid chromatography-mass spectrometry, several new products of acylation, oxidation, reduction, hydrolysis, and nitration were identified. Fusarium sambucinum detoxified BOA and HBOA to N-(2-hydroxyphenyl)malonamic acid. Plectosporium tabacinum, Gliocladium cibotii, and Chaetosphaeria sp. transformed HBOA to 2-hydroxy-N-(2-hydroxyphenyl)acetamide, N-(2-hydroxyphenyl)acetamide, N-(2-hydroxy-5-nitrophenyl)acetamide, N-(2-hydroxy-3-nitrophenyl)acetamide, 2-amino-3H-phenoxazin-3-one, 2-acetylamino-3H-phenoxazin-3-one, and 2-(N-hydroxy)acetylamino-3H-phenoxazin-3-one. BOA was not degraded by these three fungal isolates. Using 2-hydroxy-N-(2-hydroxyphenyl)[(13)C(2)]acetamide, it was shown that the metabolic pathway for HBOA and BOA degradation leads to o-aminophenol as a key intermediate.     

A polyphasic approach for the characterization of endophytic Alternaria strains isolated from grapevines

A polyphasic approach was set up and applied to characterize 20 fungal endophytes belonging to the genus Alternaria, recovered from grapevine in different Italian regions.Morphological, microscopical, molecular and chemical investigations were performed and the obtained results were combined in a pooled cluster analysis. Following morphological analyses, all strains were grouped according to their three-dimensional sporulation pattern on PCA and to the colony characteristics on different substrates. After DNA extraction, all strains were analyzed by RAPD-PCR and the resulting profiles were subjected to cluster analysis. The metabolites extracted from the 20 Alternaria endophytes were analyzed by a HPLC and the resulting metabolite profiles were subjected to multivariate statistic analyses. In comparison with reference ‘small-spored’ Alternaria species, the 20 strains were segregated into two morphological groups: one belonging to the A. arborescens species-group and a second to the A. tenuissima species-group. RAPD analysis also showed that grapevine endophytes belonged to either the A. arborescens or the A. tenuissima species-group and that they were molecularly distinct from strains belonging to A. alternata. Chemotaxonomy gave the same grouping: the grapevine endophytic strains belong to A. arborescens or A. tenuissima species-groups producing known metabolites typical of these species-groups. Interestingly, the 20 grapevine endophytes were able to produce also a number of unknown metabolites, whose characterization could be useful for a more precise segregation of the two species-groups.The results show how complementary morphological, molecular and chemical data can clarify relationships among endophyte species-groups of low morphological divergence.     

卷柏内生真菌多样性研究

为探究蕨类植物内生真菌的多样性,本研究采用可培养方法分离江西龙虎山和广东丹霞山的岩壁常见蕨类植物——卷柏的内生真菌,结合形态特征和ITS序列进行菌株鉴定、研究多样性。结果表明：从1 080个组织块中,共分离出234株菌株,卷柏内生真菌的总定殖率为18.98%,总分离率为21.67%。根据分离频率,毛壳菌属Chaetomium（26.50%）、青霉属Penicillium（18.38%）、棒束孢属Isaria（11.97%）、曲霉属Aspergillus（9.42%）为优势属。不同居群地春季卷柏的内生真菌多样性存在差异,龙虎山的分离率为34.72%,而丹霞山仅为6.39%,其优势属也不尽相同。对龙虎山不同季节的卷柏内生真菌多样性研究表明,春季的分离率为34.72%,高于冬季的23.89%,春、冬两季之间的真菌类群相似性系数仅为0.39,差异显著。研究结果将有助于进一步揭示丹霞地貌地区蕨类植物内生真菌物种组成及其影响因素。     

Degradation of keratin substrates by fungi isolated from sewage sludge

Four fungal species including two dermatophytes and two saprophytes were isolated from sewage sludge samples at Basrah (Iraq) they were tested for their degradative ability towards three types of keratin substrates (human hair, chicken feathers and wool). The rate of keratin degradation was expressed as weight loss over three weeks of incubation using a liquid culture medium. Human hair had the highest degradation rate by colonization of Chrysosporium pannicola and Microsporum gypseum at a rate of 62% and 4% respectively. Chicken feathers were highly degraded by Aspergillus flavus (32%) while wool degradation was highest by C. pannicola (45.5%) and Trichophyton mentagrophytes var. erinacei (38%). There was a significant difference (p < 0.00l) in keratin substrate degradation rates by the examined fungi. Keratinase activity was highest for C. pannicola and M. gypseum in the culture medium baited with human hair. Aspergillus flavus revealed the highest activity of this enzyme in cultures amended with chicken feathers while T. mentagrophytes var. erinacei showed highest keratinase activity in cultures with wool substrate. The amount of protein released into the culture medium varied among the tested fungi. The medium's alkalinity increased over incubation time from 6.5 to 7.8. Microscopic examination showed maceration of the keratin substrates by the fungi. This revised version was published online in June 2006 with corrections to the Cover Date.     

Biological activities of extracts from endophytic fungi isolated from Garcinia plants

Sixty-five crude extracts from 51 selected endophytic fungi isolated from Garcinia species were tested for various bioactivities. Eighty per cent of the fungal extracts from fermentation broths and mycelia displayed bioactivities: antimycobacterial (76.9%), antimalarial (14.1%), antiviral (16.7%), antioxidant (22.2%), antiproliferation (11.1% against NCI-H187 and 12.7% against KB cells), and cytotoxicity to Vero cells (40.0%). Based on internal transcribed spacer rRNA sequence analysis, 15 bioactive isolates were identified as Aspergillus, Botryosphaeria, Curvularia, Fusicoccum, Guignardia, Muscodor, Penicillium, Pestalotiopsis, and Phomopsis spp. One isolate (N24) was matched with an unidentified fungal endophyte. These results indicate that endophytic fungi isolated from Garcinia plants in Thailand are potential sources of various bioactive natural products.     

Raw starch-degrading enzyme from newly isolated strains of endophytic fungi

Four newly isolated strains of endophytic fungi namely Gibberella pulicaris, Acremonium sp., `Synnematous' sp. and Nodilusporium sp. were compared for their degradative activity on raw and gelatinized starches, substrate specificity and optimum pH. Results showed that the raw starch-degrading enzyme from Acremonium sp. had a broad activity towards both small and large granule size of raw starches while the enzyme from other strains showed high activity toward starches of smaller granule size. Analysis of the end product by TLC showed that enzyme from Gibberella pulicaris, Acremonium and Nodilusporium sp. hydrolysed raw sago starch to produce solely glucose but the enzyme of `Synnematous' sp. produced glucose and maltose. This revised version was published online in July 2006 with corrections to the Cover Date.     

Two new alkaloid metabolites produced by endophytic fungus Stagonosporopsis oculihominis isolated from Dendrobium huoshanense

Two new alkaloids (1–2), together with six known compounds (3–8), were isolated from cultures of the endophytic fungus Stagonosporopsis oculihominis. Their structures were elucidated through extensive spectroscopic methods including 2D NMR and HRMS analyses. The absolute configurations of 1 was determined by the comparison of experimental and theoretical electronic circular dichroism spectra. The new compounds were investigated for their cytotoxicity against five human cancer cell lines.     

罗汉果内生真菌多样性研究

该文采用传统形态学方法结合r DNA-ITS序列分析,对我国重要药用植物罗汉果中的内生真菌进行了鉴定并研究其多样性。结果表明:采用组织培养法从罗汉果健康植株中共分离得到150株内生真菌,包括罗汉果中雌株的内生真菌96株、雄株的内生真菌54株。122株内生真菌经形态学结合r DNA-ITS序列分析鉴定为9个属,均归属为子囊菌门,包含座囊菌纲(Dothideomycetes)和子囊菌纲(Sordariomycetes)。其中,座囊菌纲(Dothideomycetes)真菌包含3科、3属;子囊菌纲(Sordariomycetes)真菌包含6科、6属。优势属为刺盘孢属(Colletotrichum)和镰刀菌属(Fusarium)。罗汉果雌、雄植株不同组织中内生真菌的定殖率及分离率的变化规律均不相同,雌株中以根中内生真菌的定殖率和分离率最高,叶片中的最低;在雄株中以叶片中的定殖率和分离率最高,根中的最低。不同菌株在雌、雄两种植株的不同组织中的分布均不同,结合内生真菌群落组成的相似性比较结果,表明部分内生真菌对罗汉果雌株和雄株,以及同一植株中的不同组织均具有偏好性。不同组织中内生真菌的多样性指数在...     

Chemical constituents from the aerial parts of Ajania fruticulosa

The isolation and identification of sixteen compounds extracted from the aerial parts of A. fruticulosa have been reported in the present study, including eight flavonoids (1, 2, 3, 4, 5, 6, 7 and 8), three terpenoids (9, 10 and 11), two sterols (12 and 13), one lignan (14), one fatty acid (15) and one fatty acid ethyl ester (16), wherein six compounds (2, 3, 4, 5, 10 and 14) have been isolated from A. fruticulosa for the first time. Furthermore, among the identified compounds, three compounds (6, 7 and 11) have also been reported for the first time in the genus Ajanin and three compounds (8, 15 and 16) have not been isolated and reported from other plants of the family Asteraceae. In addition, the chemotaxonomic significance of these compounds was discussed.     

喜树内生真菌抗水稻纹枯病菌活性的研究

从喜树健康组织叶、茎以及果实中经过分离、纯化共得到26株内生菌株。利用生长速率法测定这26株菌株对植物病原菌——水稻纹枯病病原菌的抑制作用。抑菌试验结果显示：26株菌株中有22株喜树内生真菌的发酵液对植物病原菌菌丝生长均有不同程度的抑制作用;其中,菌株XSY10的发酵液抑菌效果最强,达到了74.97%。水稻盆栽试验结果表明：喷施菌株XSY10次生代谢产物7d后,对水稻纹枯病的防治效果为34.77%。以上试验结果说明喜树内生真菌XSY10对植物病害的防治具有一定的功效。     

Antimicrobial activity in cultures of endophytic fungi isolated from Garcinia species

The aim of the present study was to screen for antimicrobial activity in endophytic fungi isolated from surface sterilized leaves and branches of five Garcinia plants, G. atroviridis, G. dulcis, G. mangostana, G. nigrolineata and G. scortechinii, found in southern Thailand. Fermentation broths from 377 isolated fungi were tested for antimicrobial activity by the agar diffusion method. Minimum inhibitory concentrations (MICs) were obtained for crude ethyl acetate extracts. Seventy isolates (18.6%) displayed antimicrobial activity against at least one pathogenic microorganism, such as Staphylococcus aureus, a clinical isolate of methicillin-resistant S. aureus, Candida albicans and Cryptococcus neoformans. The results revealed that 6-10%, 1-2% and 18% of the crude ethyl acetate extracts inhibited both strains of S. aureus (MIC 32-512 microg mL(-1)), Ca. albicans and Cr. neoformans (MIC 64-200 microg mL(-1)), and Microsporum gypseum (MIC 2-64 microg mL(-1)), respectively. Isolates D15 and M76 displayed the strongest antibacterial activity against both strains of S. aureus. Isolates M76 and N24 displayed strong antifungal activity against M. gypseum. Fungal molecular identification based on internal transcribed spacer rRNA gene sequence analysis demonstrated that isolates D15 (DQ480353), M76 (DQ480360) and N24 (DQ480361) represented Phomopsis sp., Botryosphaeria sp. and an unidentified fungal endophyte, respectively. These results indicate that some endophytic fungi from Garcinia plants are a potential source of antimicrobial agents.     

Chemical constituents from the fresh flower buds of Musa nana and their chemotaxonomic significance

Phytochemical study on the fresh flower of Musa nana Lour. provided seventeen known compounds including two alkaloids, 3-(hydroxyacetyl)-indole (1), bi-indol-3-yl (2), two terpenoids, 5-[(1R)-1-hydroxy-2,6,6-trimethyl-4-oxo-2-cyclohexen-1-yl]-3-methyl-, (2Z, 4E) −2, 4-pentadienoic acid (Valdes), 5, 6(S), 7, 7a(R)-tetrahydro-6-hydroxy-4,4-dimethyl-2(4H)-benzofuranone (4), seven phenols (5–11), three phenylphenalenones, 2-hydroxy-4-(4-methoxyphenyl)-1H-phenalen-1-one (12), 2-methoxy-9-phenyl-1H-phenalen-1-one (13), 2-methoxy-9-(4-methoxyphenyl)-1H-phenalen-1-one (14), and three lipids (15–17). In the present study, all the compounds were isolated for the first time from the species M. nana. Ten compounds including 1-8 and 15-16 have never been previously encountered in the Musaceae family. Furthermore, the chemotaxonomic significance of these isolates was also discussed.     

茶树内生真菌混合培养增强对植物病原真菌的拮抗作用

采用分离自健康茶树叶片组织中的2株内生真菌,长枝木霉CSN-18和曲霉CSN-3,研究了单独培养和混合培养条件下培养原液与培养原液乙酸乙酯萃取液对植物病原真菌的抑制作用。与2菌株单独培养相比,混合培养对稻瘟病菌Magnaporthe grisea、茶花褐斑病菌Phyllosticta camelliaecola和茶云纹叶枯病菌Guignardia camellia等8种植物病原真菌的抑制作用明显增强。混合培养原液的乙酸乙酯萃取液对茶花褐斑病菌、稻瘟病菌和茶云纹叶枯病菌的抑制率分别为79.48%±1.46%、76.99%±0.91%和71.51%±4.93%;对茶花褐斑病菌、茶云纹叶枯病菌和稻瘟病菌孢子萌发的抑制率分别为100.00%、90.90%±2.59%和84.00%±5.29%。混合培养原液对茶花褐斑病菌菌丝生长的抑制作用也比两菌株的单独培养液有明显增强;在低浓度下,混合培养原液对茶花褐斑病菌的孢子萌发有促进作用;但浓度超过40%,则明显抑制茶花褐斑病菌孢子的萌发。     

Effects of endophytic fungi on the phenotypic plasticity of Lolium perenne (Poaceae)

The effects of high vs. low levels of endophytic fungi on the phenotypic plasticity of cloned genotypes were examined in perennial ryegrass (Lolium perenne L.). The objectives were to determine whether endophytic fungi influence plastic responses of host genotypes to variable soil nutrients and whether or not endophyte infection and host genotype interact to determine the extent of this plasticity. Twelve infected genotypes were cloned into ramets: half the ramets were treated with the systemic fungicide Benomyl to reduce or eliminate the endophyte, while the other half were untreated. Ramets of each genotype were subjected to high, medium, or low levels of nutrients in the greenhouse for 11 wk. Tiller number, leaf area, and leaf mass were determined after 11 and 25 wk. The fungicide significantly reduced the level of endophyte infection. Responses to nutrient conditions in relation to fungicide treatment were genotype specific: for some genotypes, high levels of endophytic fungi appeared to reduce plasticity, while for other genotypes the endophyte had no effect. The potential for microscopic symbionts to affect phenotypic plasticity in genetically variable populations has not often been recognized. However, the clandestine effects of symbionts on the plasticity of host genotypes could impact microevolutionary processes occurring within plant populations that occupy heterogeneous environments.     

In vitro antioxidant activities of endophytic fungi isolated from the liverwort Scapania verrucosa

We investigated in vitro antioxidant activities of 49 endophytic fungi isolated from the liverwort Scapania verrucosa. Based on morphological and molecular identification, the endophytic fungi isolated were classified into seven genera (Hypocrea, Penicillium, Tolypocladium, Chaetomium, Xylaria, Nemania, and Creosphaeria), all belonging to one family (Xylariaceae). By screening with the 2,2'-azino-di(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) decolorization assay, the ethyl acetate extracts of five endophytic fungi (T7, T21, T24, T32, and T38 strains), which exhibited remarkable Trolox equivalent (TE) antioxidant capacity (ranging from 997.06 to 1248.10 μmol TE/g extract), were selected and their antioxidant capacity was further evaluated by assays for 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, hydroxyl radical scavenging, reducing power, and ferrous ion chelating. The ethyl acetate extracts of two endophytic fungi (T24 and T38) were found to have comparable scavenging abilities on both DPPH-free radicals (93.9 and 88.7%, respectively, at 50 μg/mL) and hydroxyl radicals (97.1 and 89.4%, respectively, at 2 mg/mL) when compared with those of the positive controls (ascorbic acid and butylated hydroxytoluene, respectively). Although their reducing powers were similar to that of butylated hydroxytoluene, as indicated by absorbance (0.35 and 0.30 at 50 μg/mL, respectively), only the T38 strain's ethyl acetate extract showed ferrous ion chelating ability (92.9% at 1 mg/mL) comparable to that of the EDTA-2Na control. These endophytic fungi in S. verrucosa are a potential novel source of natural antioxidants.     

海藻内生真菌三个中国新记录种

报道了分离自山东沿岸潮间带海藻的3个中国新记录种：石生束格孢霉Sarcinomyces petricola、鼠灰帚霉Scopulariopsis murina和球孢帚霉Scopulariopsis sphaerospora。研究标本保存于中国海洋大学海洋生物标本室（OUCMB）。     

Hydroxylated 2-amino-3H-phenoxazin-3-one derivatives as products of 2-hydroxy-1,4-benzoxazin-3-one (HBOA) biotransformation by Chaetosphaeria sp., an endophytic fungus from Aphelandra tetragona

The biotransformation of the phytoanticipin HBOA and its major degradation metabolites 2-hydroxy-N-(2-hydroxyphenyl)acetamide (7) and N-(2-hydroxyphenyl)acetamide (8) by Chaetosphaeria sp., an endophytic fungus isolated from Aphelandra tetragona, was studied. Three new metabolites could be identified as 2-amino-7-hydroxy-3H-phenoxazin-3-one (12), 2-acetylamino-7-hydroxy-3H-phenoxazin-3-one (13) and 7-hydroxy-2-(2-hydroxyacetyl)-amino-3H-phenoxazin-3-one (14). Structure elucidation of 12 and 13 was performed by MS, 1H, 13C NMR and 2D NMR techniques and confirmed by chemical transformation.     

In vivo nuclear-magnetic-resonance analysis of polyamine and alkaloid metabolism in transformed root cultures of Datura stramonium L.: evidence for the involvement of putrescine in phytohormone-induced de-differentiation

The utilisation and accumulation of ¹⁵N-labeled metabolites by a ¹⁵N-labeled transformed root culture of Daturastramonium L. was investigated by in vivo ¹⁵N-nuclear-magnetic-resonance (NMR) spectroscopy. After resuspension in spent growth medium, the pools of [¹⁵N]glutamate and [¹⁵N]glutamine were rapidly depleted and there was an increase in the ¹⁵N-NMR signals from conjugated putrescines and hyoscyamine. The signal from the conjugated putrescines passed through a maximum 2 d after the roots were resuspended, and it was concluded that putrescine could be stored as putrescine conjugates prior to its utilisation in other pathways. The transient accumulation of ¹⁵N-label in the hydroxy-cinnamoylputrescines was reduced when the de-differentiation of the root cultures into a suspension culture was initiated by exposure to a medium containing α-napthaleneacetic acid and kinetin. This led to the hypothesis that phytohormone-induced de-differentiation of the root cultures required the presence of free polyamines, and this was tested using two potent inhibitors of putrescine biosynthesis, dl-α-difluoromethylarginine and dl-α-difluoromethylornithine. In-vivo ¹⁵N-NMR spectra of roots grown in ¹⁵N-enriched medium supplemented with these inhibitors showed that the ¹⁵N-labelling of the conjugated polyamines and hyoscyamine was markedly reduced. dl-α-difluoromethylarginine also prevented the phytohormone induced de-differentiation of the root cultures, and this effect could be reversed by the supply of exogenous putrescine. Thus the supply of putrescine appears to play a crucial role in mediating the phytohormone induced de-differentiation of the root culture. Received: 13 September 1997 / Accepted: 12 November 1997