Phillyrin produced by Colletotrichum gloeosporioides, an endophytic fungus isolated from Forsythia suspensa

To investigate the phillyrin-producing endophytic fungi from the medicinal plant Forsythia suspensa, a total of 24 strains of endophytic fungi were isolated from the healthy stems, leaves and fruits of the plant, 9 from stems, 5 from leaves, and 10 from fruits respectively. All fungal isolates were fermented in liquid PDA medium and their extracts were preliminary analyzed by TLC. One isolated strain G10, which was from the fruit of F. suspensa, had the same R_f value as authentic phillyrin. The potential phillyrin-producing fungus G10 was further analyzed by HPLC and HPLC-MS, and the results showed that the isolate G10 possessed of a retention time and ion peaks identical with the authentic compound phillyrin. The isolate G10 was identified as Colletotrichum gloeosporioides based on the morphological characteristics and ITS sequence analysis. The current research indicates that the numerous endophytic fungi inside the medicinal plants are precious resource for the pharmaceutical natural products that are originally from the plants.     

Structure identification for compound I separated and purified fromtaxoids-produced endophytic fungi （Alternaria. alternata var. taxi 1011）

Endophytic fungi are widely found in almost all kinds of plants. Many endophytic fungi can produce some physio-logical active compounds, which are same to or analog to those isolated from their hosts. Producing physiological active compounds through microbial fermentation can give a new way to resolve resource limitation and to find out alternative source.Through the methods of organic solvent extraction, thin layer chromatography (TLC) and column chromatography, compound I was isolated, purified from the liquid fermentation metabolites of the taxoids-produced endophytic fungi (Altemaria. altemata var.taxi 1011 Y. Xiang et LU An-guo) that was screened from the bark of Taxus. cuspidata Sieb.et Zucc.. Compound I was identified as one kind of taxoids type Ⅲ, based on the analyzing results by using the methods of ultraviolet spectroscopy (UV), infrared spectroscopy (IR), mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR). This study provides a completed method for separation and purification of the endophytic fungi as well as structure identification of its fermentation metabolite     

Diversity and bioactivity of bacterial endophyte community of Cupressaceae

The plant family Cupressaceae (Coniferales) harbours diverse endophytic fungi with antifungal, antibacterial and antiproliferative activities. Here, endophytic association of a broad bacterial community with the healthy foliar tissues of Cupressus arizonica, Cupressus sempervirens, Juniperus communis and Thuja orientalis (Cupressaceae) is shown. We isolated over 69 endophytic bacterial strains of Proteobacteria, Bacilli and Actinobacteria from Cupressaceae. The initial screening for antifungal activity against Pyricularia oryzae identified eleven superior bacterial strains which were identified as Brevundimonas diminuta CAE₂₄ (G^?, Caulobacteraceae, Alphaproteobacteria); Stenotrophomonas maltophilia CAE₂₃, CSE₄, CSE₁₂, CSE₄₂, CSE₄₉ and CSE₆₂ (G^?, Xanthomonadaceae, Gammaproteobacteria); Bacillus pumilus CSE₆₆ and Bacillus subtilis POE₂₆ (G⁺, Bacillaceae, Bacilli); and Microbacterium resistens CSE₁₉ (G⁺, Microbacteriaceae, Actinobacteria). The dominant isolated bacterial species was S. maltophilia. This species represented 63.6% of the superior strains which was ubiquitous and also non‐host specific. In addition, the superior bacterial strains produced bioactive secondary metabolites and volatile compounds (VOCs) with antifungal activity against the fungal pathogens of Cupressaceae, that is Diplodia seriata, Phaeobotryon cupressi and Spencermartinsia viticola. The antagonistic activity of the endophytic bacteria on the target fungi was also confirmed in vitro. To our knowledge, this is the first to document such an endophytic bacterial community in Cupressaceae and its bioactivity. These findings may find application in organic agroforestry for plant disease biocontrol and in biopharmacy for lead molecule discovery.     

Temporal variations in endophytic fungal assemblages of Ginkgo biloba L.

We isolated endophytic fungi from living healthy leaves, petioles, and current-year twigs of Ginkgo biloba L. from April to November 2004 with the objective of identifying the dominant endophytic fungal taxa, and monitoring their occurrence and frequency. A total of 9 fungal taxa were identified to the genus level. Diversity measures inferred from the Shannon–Wiener, Morisita–Horn, and S?rensen indices indicated that leaves and petioles harbored more diverse endophytic fungal assemblages than twigs, and that fungal taxa involved in twigs shared less with those in leaves and petioles. Among the organs, the occurrence pattern of overall endophytic fungi differed significantly, and two taxa, Phomopsis sp. and Phyllosticta sp., were the most frequently isolated and thus regarded as the dominant endophytic fungi. Phomopsis sp. was isolated frequently from twigs (84.8%) but rather few from leaves (16.1%) and petioles (24.3%). Phyllosticta sp. was isolated frequently from leaves (72.9%) and petioles (65.7%) but was never isolated from twigs. Temporal changes in relative frequency of total endophytic fungi tended to differ among sampling dates for all three organs. The occurrence of Phyllosticta sp. in both leaves and petioles was first detected in August and peaked in October. Phomopsis sp. was detected in twigs throughout the growing season. These results suggest that the distribution of the two dominant endophytic fungi was organ-specific and differed within seasons.     

Endohyphal bacteria from fungal endophytes of the Mediterranean cypress (Cupressus sempervirens) exhibit in vitro bioactivity

Endohyphal organisms of endophytic fungi can induce, or promote, beneficial effects of their respective host fungi to the host plant that harbours them. The Cupressaceae plant family (Coniferales) hosts highly bioactive endophytic fungi. Here, we show that a fraction of such endophytic fungi harbours bioactive endohyphal bacteria with a non‐obligatory symbiotic lifestyle. Indeed, 5 of 16 (31.25%) endophytic fungi of Cupressus sempervirens harboured endofungal bacterial strains of Bacillaceae (G⁺, Bacilli) and Sphingomonadaceae (G^?, α‐Proteobacteria), that is Bacillus pumilus (from the fungi Leptosphaeria CSE₂₁₁ and Pyrenochaeta CSE₁₃₄), Bacillus subtilis (from the fungi Leptosphaeria CSE₂₁₂ and Ascorhizoctonia CSE₁₄₈) and Sphingomonas paucimobilis (from the fungus Ascorhizoctonia CSE₁₉₅). Notably, each endophytic fungal species contained only one endofungal bacterial species that was stably maintained in symbiosis over several rounds of subculturing. Moreover, we investigated whether cypress endofungal bacteria (CEB) could benefit their host fungus competing with other fungi and bacteria or the host plant against the invading microorganisms. In vitro assays indicated that CEB possessed antagonistic activity against cypress endophytic and pathogenic microbiome. Also, CEB metabolites and volatile compounds (VOCs) exhibited antifungal and antibacterial activity against the target microbiome. Bioactivity of CEB was less than that of the endophytic microbiome of Cupressaceae, on which we reported earlier. In conclusion, our work is the first to document endohyphal bacteria of fungal endophytes of C. sempervirens and the bioactivity of such endohyphal symbionts. These findings implicate a complicated interrelationship among host plant, endophytic microbiome and endofungal bacteria, which might be of high importance for evolutionary, as well as environmental and agricultural studies. Eventually, endohyphal bacteria may be introduced as a novel source for lead molecule discovery.     

Identification of Taxus cuspidata Sieb. et Zucc. endophytic fungi-new species, species known and their metabolite

A total of 94 isolates of endophytic fungi were isolated from the bark of 200-yr.-old Taxus cuspidata Sieb. et Zucc.in the primeval forest of the Changbai Mountain Natural Reserve, and 19 species of endophytic fungi were identified, including 10 new recorded-genus-species, 2 new species (Phomopsis Iongiscoleosporu Y. Xiang et Lu An Guo Wu Wen Fang, Coniothyrium macrospoum Y. Xiang J.X. et Lu An Guo Wu Wen Fang), 1 new varied species (Alternaria alternata (Fr.) Keissler var. taxi Y.Xiang et Lu An Guo) and 6 known species of China (Eurotium amstelodomi Mgngin, Eurotium repens de Bary, Botrytis sp.,Penicillium citrinum Thom, Epicoccum nigrium LinK, Fusarium sp.). Through thin layer chromatography (TLC), liquid fermentation metabolite of the strains was determined, and four strains (Alternaria alternata (Fr.) Keissler var. taxi Y. Xiang et Lu An Guo Wu Wen Fang, Botrytis sp., Eurotium amsteloodomi Mgngin, Eurotium repens de Bary) were screened out, whose metabolites reacted positively with the vanillic aldehyde that was one special taxoid developer. Among the four strains, Alternaria alternata (Fr.) Keissler var. taxi Y. Xiang et Lu An Guo, produced one compound largely, which positively reacted with one alkaloids developer-Bismuth potassium iodide. The compound is identified as taxoids type through spectrum analysis. This demonstrates that Alternaria alternata (Fr.) Keissler var. taxiY. Xiang et Lu An Guo can highly produce taxoids largely.     

盘龙参内生真菌分布特征的研究

从盘龙参[Spiranthes sinensis(Pers)Ames]的根、茎、叶中共分离得到49株内生真菌,经形态观察,鉴定为3目、4科、9属。其中曲霉属、镰刀孢属、丝核菌属为优势种群,分别占已分离菌株数的16.3%、14.3%和14.3%。不同组织部位所分离得到的内生真菌在种群及数量上都存在差异:根中的优势属为镰刀孢属,占到了根中分离菌种数的23.5%;叶中的优势属是链格孢属,占到叶中分离菌种数的26.3%;茎中的优势属为长蠕孢属,占到叶中分离菌种数的23.1%。表明盘龙参内生真菌的分布具有一定的组织专一性。     

杨树内生真菌的分离和拮抗生防菌的筛选

从健康杨树植株的叶(芽)、皮、枝中分离获得154株内生真菌,经形态观察分类鉴定归属于3个目、5个科、13个属中.利用两点对峙培养拮抗试验、孢子萌发试验和人工接种试验,从中筛选杨树烂皮病病原菌的拮抗菌.试验结果表明所获内生真菌(4属)菌株对病原菌菌丝生长和孢子萌发均存在不同程度的抑制作用,其中,青霉属真菌(Penicillium sp.)的菌株拮抗效果表现最明显.青霉菌可迅速对病原菌菌落包围、覆盖,从而使植物病原菌生长受到抑制,孢子萌发试验中,3 d的培养滤液就可对病原菌孢子萌发产生抑制作用,表现出一定的生防潜能.     

紫杉链格孢1011菌株产紫杉烷类物质的分离纯化和结构鉴定(英文)

植物体内普遍存在内生真菌,它们可以产生与宿主相同或相似的生理活性物质。通过微生物发酵产生生理活性物质可以为解决能源短缺和寻找替代能源开辟一条新途径。作者初步探讨了分离和纯化高产菌株以及对其发酵代谢产物的结构鉴定的方法。采用溶剂提取法、薄层色谱法和柱色普法,对从东北红豆杉(T.cuspidataSieb.etZucc.)上分离筛选出的高产紫杉烷类物质紫杉链格孢(Alternariaalternatavar.taxi1011Y.XiangetLUAn-guo)1011菌株的发酵产物进行分离纯化,提取了一个化合物Ⅰ。经紫外扫描、红外扫描、质谱、核磁共振等方法鉴定,确定该化合物为紫杉烷类二萜Ⅲ型化合物。图1表2参12。     

Nursery inoculation of Eucalyptus seedlings in Western Australia and Southern China using spores and mycelial inoculum of diverse ectomycorrhizal fungi from different climatic regions

A large-scale ectomycorrhizal (ECM) fungus inoculation program was conducted in plantation forest tree nurseries in Australia and China over a period of 2 years. These experiments used a wide diversity of fungi (90 isolates belonging to 23 genera), most of which were obtained from Australian eucalyptus plantations or forests. These trials compared the effectiveness of mycelial slurries (homogenised liquid fungal cultures) and spore suspensions (ground dried fruit bodies) as inoculum forms in nurseries with widely differing management regimes. The success rate of inoculation was moderate, regardless of nursery management regime (38% overall). However, there were substantial differences in overall performance between fungal genera, with agaricoid genera, such as Descolea and Laccaria, and hypogeous (truffle-like) fungi performing better than sequestrate genera, such as Pisolithus and Scleroderma. Comparison of congeneric fungal isolates from different climatic regions did not show significant differences in the performance of fungi from regions where climatic conditions were most similar to nursery conditions relative to those from disparate climatic regions (tropical, temperate, or mediterranean). There was a higher overall success rate for inoculation with spore suspensions (49%) than for mycelial slurries obtained from liquid cultures (35%). Spore-based inoculum was also easier to use and much less expensive to produce than mycelial slurries. It is recommended that future studies investigate the use of mixtures of fungi and attempt to optimise spore germination to increase the reliability of eucalypt seedling nursery inoculation.     

东北红豆杉内生真菌的鉴定-----新种和已知种及其代谢产物

从长白山自然保护区原始森林200多年生的东北红豆杉(TasxuscuspidateSiebetZucc.)的树皮中分离出94株内生真菌,鉴定出19种。其中,除10个新记录属种外,还包括2个新种:长线孢拟茎点(PhomopsislongiscoleosporuY.XiangetLuAnGuo,WuWenFang)和大孢盾壳霉(ConiothyriummacrospoumY.Xiang,LuAnGou,WuWenFang);1个新变种为紫杉链格孢[Alternariaalternata(Fr.)Keisslervar.taxiY.XiangetLuAnGuo];还有6个国内已知种:阿姆斯特丹散囊菌(EurotiumamstelodomiMgngin),匐散囊菌原变(EurotiumrepensdeBary),葡萄孢(Botrytissp.),桔青霉(PenicilliumcitrinumThom),黑附球孢(EpicoccumnigriumLinK),镰刀孢(Fusariumsp.)。用薄层层析技术,测定了各菌种发酵代谢产物,经筛选发现紫杉链格孢[Alternaria(Fr.)Keisslervar.taxiY.XiangetJ.X.Cui],葡萄孢(Botrytissp.),阿玛姆斯丹散囊菌(EurotiumamsteloodomiMgngin),匍匐散囊菌原变种(EurotiumrepensdeBary)4种内生真菌的代谢产物可与紫杉烷类物质显色剂——香草试液发生较明显的颜色反应,其中紫杉链格孢[Alternariaalternata(Fr.)Keisslervar.taxiY.XiangetJ.X.Cui]菌种的代谢产物中有一种含量较大的组分,还可与稀碘化铋钾生物碱试剂发生反应,该组分化合物经过光谱分析,为紫     

Characterization of endophytic bacteria with plant growth promotion and biological control potential isolated from walnut trees

In this study, a total of 68 endophytic bacteria were isolated from different tissues of walnut trees. About 55% and 22% of the strains had the ability to produce indole acetic acid and gibberellic acid, respectively. The capability of isolates to solubilize phosphate, growth on N‐free medium, siderophore, protease and lipase production was varied. Based on phenotypic grouping and plant growth promotion properties, twelve isolates were selected and 16S rRNA gene‐based phylogenetic analysis revealed that strains showed 99%–100% similarity to Pseudomonas, Bacillus, Arthrobacter, Roseomonas and Streptomyces genera. Amongst the selected strains, PS12, KS54, JS66 and KS74 showed root and shoot growth enhancement of poplar cutting. NS70, KS54 and PL36 strains showed antagonistic activity against Pseudomonas syringae pv. syringae;RR47, KS74 and NR69 strains had inhibition effects against Brenneria nigrifluens; and JS66 and RR26 strains had antagonistic activity against both phytopathogens under in vitro conditions. This is the first reported study to elucidate the endophytic bacterial diversity associated with walnut trees with beneficial attributes.     

Two Burkholderia strains from nodules of Dalbergia odorifera T. Chen in Hainan Island, southern China

Two Burkholderia strains 8111 and 8201 were isolated from root nodules of Dalbergia odorifera, an endemic woody legume in southern China. Phylogenetic analysis of 16S rRNA gene and 16S-23S gene intergenic spacer (ITS) showed that these two bacterial strains were closely related to Burkholderia cepacia and they were also similar in carbon source utilization using Biolog GN2 plate tests. The DNA G+C content of strains 8111 and 8201 were 65.8 and 65.5?mol%. Inoculation tests demonstrated that both strains 8111 and 8201 formed functional root nodules in their original host D. odorifera, and significantly enhanced plant growth (as measured by plant biomass and nitrogen content), compared to the no-inoculated control plants. Sequencing of 16S rRNA gene in nodules of D. odorifera seedlings inoculated with strains 8111 and 8201 confirmed their identity. However, these two strains did not induce root nodulation in Acacia auriculiformis and Erythrophleum fordii. This implies that the nodulation capacity between Burkholderia strains 8111 or 8201 and their legume hosts may be specific. Our results show that both Burkholderia strains 8111 and 8201 are able to form functional nodules on D. odorifera and are potentially beneficial inoculants for seedling propagation to be used in large scale D. odorifera plantations.     

狭叶柴胡内生真菌分布特征的研究

从狭叶柴胡(Bupleurum scorzonerifolium Willd.)中共分离得到13株内生真菌,经菌落形态观察及显微观察,鉴定为3目、4科、7属,并未见明显的优势菌群,且没有一个属能够同时从根、茎、叶三种组织中分离得到,表明不同菌属在宿主体内分布存在一定的组织特异性。研究为狭叶柴胡内生真菌的进一步开发奠定了理论与实践基础。     

红树属植物内生真菌多样性及其代谢产物研究进展

红树林是生长在热带和亚热带潮间带河口地带的耐盐植物群落,分布在南纬30°与北纬30°之间,具有盐胁迫、高矿物组成、强还原性、频繁的潮汐等特征,也使植物对营养、空间的竞争异常激烈,特殊生境使红树林植物内生真菌多样性丰富,必然会造成不同于其他生态系统的良好生物活性、独特化学结构的活性功能分子。红树属是红树林植物中分布较广泛的一个属,包括8个种,分别是红海榄、红树、红茄苳、美洲红树、Rhizophora harrisonii、R.racemosa、树冬红树和萨摩红树。迄今为止,从红树属分离出内生真菌类群已达23目41科64属,其中对拟盘多毛孢属、青霉属和毛霉属的内生真菌代谢产物研究的最为广泛;从红树属内生真菌中分离鉴定出195种天然产物,包括生物碱类、萜类化合物、香豆素类、色酮类化合物、醌类化合物、杂氧蒽醌类化合物、肽类、酚酸类化合物、内酯类等化合物,从红树属植物中发现许多结构新颖的次生代谢产物,从中国南海红树林红茄苳内生拟盘多毛孢真菌中发现一系列罕见的具有亲脂性取代基的新色酮类化合物pestalotiopsones A-F (107—112),从拟盘多毛孢属JCM2A4中发现2个具有柔性结构的补身烷型倍半萜-环青霉醛酸的新骨架化合物pestalotiopens A和B (83, 84)。此外,许多化合物显示出良好的抗肿瘤、抗菌和抗氧化等生物活性,如化合物pestalpolyol 1(119)具有很强的抑制作用,对小鼠淋巴瘤L5178Y细胞的半抑制浓度(half maximal inhibitory concentration,IC₅₀)值为4.10μmol·L^-1。从内生真菌毛霉QEN-189中分离到的吲哚生物碱rhizovarins A,B和F(46, 47, 50)对肺癌细胞A549抑制作用强,IC₅₀值分别为11.5、6.3和9.2μmol·L^-1,对白血病细胞HL-60有强抑制作用,IC₅₀值为9.6、5和7μmol·L^-1。     

东北红豆杉内生真菌的鉴定-中国新记录属种及其代谢产物

从长白山自然保护区原始森林200多年生东北红豆杉(TasxuscuspidataSiebetZucc.)树皮中分离94株内生真菌,鉴定出19种。其中,除2个新种、1个新变种和6个国内已知种(已详细描述并发表在《林业研究》2003,14(4):290-294),本文还对包括9个新记录属种的形态特征进行了详细描述:坚孢葡萄单孢(StaphylotrichumcoccosporumMeyeretNicot)、砖红葡萄痕孢(BotryodemalateritiumPapendoorf&Upadhyay)、球头孢[Oedocephalumglomerulosum(RulliardSacc.)、球头三型孢(TrichosporonoidesoedocephalisHaskins&Spencer)、扁圆球孢[Beniowskiasphaeroidea(KalChbrenneretCaeke)Mason]、疣顶孢(ThermomycesverrucosusPugh,Blakeman&Morgan-jones)、腐殖厚壁孔孢(GilmaniellahumicolaBrron)、球乳突孢[Papulariasphaerosperma(Pers.:Fr.)vonHohnel]、褐钝孢[Ambrosiellabrunnea(Verrall)Batra]和1个新记录种:假线壳囊孢(CytosporinanothaDied.)。对中国新记录属种内生真菌,提供了形态学特征描述与图解及其生境、寄主的记载。用薄层层析技术,测定了各菌种发酵代谢产物可与紫杉烷类物质显色剂棗香草试液和稀碘化铋钾试液不发生明显的颜色反应。图10参23。     

Isolation and identification of endophytic bacteria with plant growth promoting and biocontrol potential from oak trees

Samples of roots, leaves and stems of healthy oak trees were collected from various locations in the Baneh and Marivan regions, Iran. In total, 63 endophytic bacteria were isolated and grouped according to phenotypic properties. Seven selected isolates were further identified by partial sequencing of the 16S rRNA gene. Isolates Pp54, Pp88, Pp95 and Pp177 were Pseudomonas spp., isolates Sm59 and Sm79 were Stenotrophomonas spp., and isolate Bf 172 was a Bacillus sp. The ability of these isolates for plant hormone production such as auxin and gibberellin was evaluated, along with siderophore production, phosphate solubilization, atmospheric nitrogen fixation, protease and hydrogen cyanide production. All strains produced auxin and gibberellin in different amounts. Atmospheric nitrogen fixation ability was positive for strains Sm79, Pp54, Pp88 and Pp95. All strains except Sm79 solubilized phosphate. Strains Sm59, SM79, Pp88, Pp177 and Bf172 produced protease. Pp88, Pp95, Bf172 and Pp177 were able to produce siderophore. Strains Sm79 and Pp95 released low concentrations of hydrogen cyanide. Amongst the strains tested, Pp95, Pp88, Pp177 and Sm79 had different inhibitory effects on the bacterial plant pathogen, Pseudomonas syringae pv. syringae under in vitro conditions. This is the first reported case of endophytic Bacillus, Pseudomonas and Stenotrophomonas strains from oak trees in Iran.     

Effects of salicylic acid, 1‐aminocyclopropan‐1‐carboxylic acid and methyl jasmonate on the frequencies of endophytic fungi in Quercus serrata leaves

To examine the effects of salicylic acid (SA), the ethylene precursor 1‐aminocyclopropan‐1‐carboxylic acid (ACC) and methyl jasmonate (MJ), which induce defence responses against pathogen in plants, on foliar endophytic fungi, we performed exogenous treatments of Quercus serrata leaves with these chemicals and subsequently isolated endophytic fungi. SA and ACC significantly reduced the isolation frequency of the most dominant fungus, a Xylaria species as identified by sequence analysis of the internal transcribed spacer (ITS) region of the ribosomal DNA. In contrast, the frequency of the second most frequent fungus increased by SA and ACC. A few other fungi were also affected positively or negatively. Shannon diversity indices decreased by all treatments because of the uneven isolation frequencies of the endophytes. Our results imply that host defence responses induced by the chemical treatments reduced the number of infections of some endophytes while the frequency of some other endophytes increased benefiting from the reduction of competitors.     

In vitro TAXOL production,by Pestalotiopsis breviseta — A first report

Coelomycetous fungi were screened for the production of TAXOL. TAXOL production of Pestalotiopsis breviseta fungi is confirmed by Ultra Violet (UV) spectroscopic analysis, Infra Red (IR) analysis, high performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR) and LC–MASS spectroscopy. TAXOL isolated from the P. breviseta fungus was identical with authentic TAXOL and produces 0.064mg/L (0.128% dry weight of fungal mat).