A novel endophytic Huperzine A–producing fungus,Shiraia sp. Slf14, isolated from Huperzia serrata

Aims: To characterize and identify a novel Huperzine A (HupA)‐producing fungal strain Slf14 isolated from Huperzia serrata (Thunb. ex Murray) Trev. in China. Methods and Results: The isolation, identification and characterization of a novel endophytic fungus producing HupA specifically and consistently from the leaves of H. serrata were investigated. The fungus was identified as Shiraia sp. Slf14 by molecular and morphological methods. The HupA produced by this endophytic fungus was shown to be identical to authentic HupA analysed by thin layer chromatographic, High‐performance liquid chromatography (HPLC), LC‐MS, ¹H NMR and acetylcholinesterase (AChE) inhibition activity in vitro. The amount of HupA produced by Shiraia sp. Slf14 was quantified to be 327·8 μg l^?1 by HPLC, which was far higher than that of the reported endophytic fungi, Acremonium sp., Blastomyces sp. and Botrytis sp. Conclusions: The production of HupA by endophyte Shiraia sp. Slf14 is an enigmatic observation. It would be interesting to further study the HupA production and regulation by the cultured endophyte in H. serrata and in axenic cultures. Significance and Impact of the Study: Although the current accumulation of HupA by the endophyte is not very high, it could provide a promising alterative approach for large‐scale production of HupA. However, further strain improvement and the fermentation process optimization are required to result in the consistent and dependable production.     

Endophytic fungus <Emphasis Type="Italic">Cladosporium cladosporioides</Emphasis> LF70 from <Emphasis Type="Italic">Huperzia serrata</Emphasis> produces Huperzine A

A strain LF70 endophytic fungus was isolated from the leaves of Huperzia serrata. The fungus was identified as Cladosporium cladosporioides LF70 according to its morphological characteristics and nuclear ribosomal DNA ITS sequence analysis. The strain could produce Huperzine A (HupA) identified through thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) with authentic HupA. The amount of HupA produced by this endophytic fungus was quantified to be 56.84 μg/L by HPLC, which was higher than that of other reported endophytic fungi, Acremonium sp., Blastomyces sp., and Botrytis sp. Acetylcholinesterase inhibition activity of HupA produced by strain LF70 was also similar to authentic HupA in vitro. Isolation of such a fungus may provide a promising alternative approach to producing HupA, which is used in treating Alzheimer’s disease and preventing further memory degeneration.     

Efficient strategy for maintaining and enhancing the huperzine A production of Shiraia sp. Slf14 through inducer elicitation

Huperzine A (HupA), a naturally occurring lycopodium alkaloid, is a potent, highly specific and reversible inhibitor of acetylcholinesterase and is a potential treatment for Alzheimer’s disease. However, isolating HupA from Huperziaceae plants is inefficient; thus, extracting this compound from endophytic fungi may be more controllable and sustainable. However, the large-scale production of this chemical from endophytes is limited by the innate instability of endophytic fungi. In this study, we maintained the stability and viability of the HupA-producing endophytic fungus Shiraia sp. Slf14 and enhanced the HupA titers during fermentation by adding Huperzia serrata extracts (HSE), l-lysine, and acetic acid into the culture as inducers. Adding trace amounts of HupA clearly improved the HupA production of Shiraia sp. Slf14, reaching a maximum content of approximately 40 μg g^?1. Moreover, the addition of HSE and l-lysine promoted HupA production in the flask fermentation. The aforementioned bioprocessing strategy may be potentially applied to other endophytic fungal culture systems for the efficient production of plant secondary metabolites.     

Isolation and characterization of endophytic huperzine A-producing fungi from Huperzia serrata

Huperzia serrata is a producer of huperzine A (HupA), a cholinesterase inhibitor (ChEI). Over 120 endophytic fungi were recovered from this plant and screened for Hup-A and nine were found. These nine represented seven different fungal genera with the most significant producer being Shiraia sp. A total of 127 endophytic fungi isolates obtained from the root, stem, and leaf segments of H. serrata were grouped into 19 genera based on their morphological traits and sequence analysis of the internal transcribed spacers (ITS1-5.8S-ITS2), indicating endophytic fungi in H. serrata are diverse and abundant. Aspergillus, Podospora, Penicillium, Colletotrichum, and Acremonium were the frequent genera, whereas the remaining genera were infrequent groups. Overall, 39 endophytic fungi isolates showed acetylcholinesterase (AChE) inhibition in vitro. Nine endophytic fungi isolates from seven distinct genera were capable of producing HupA verified by thin-layer chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). Among the HupA-producing fungi, the yield of HupA produced by the Shiraia sp. Slf14 was 327.8 μg/l in potato dextrose broth, and the fungal HupA was further validated by mass spectrometry (ESI-MS). The present study demonstrated that H. serrata was a fascinating fungal reservoir for producing HupA and other ChEIs.     

Ethanol and Methanol Can Improve Huperzine A Production from Endophytic Colletotrichum gloeosporioides ES026

Huperzine A (HupA) is a plant alkaloid that is of great interest as a therapeutic candidate for the treatment of Alzheimer''s disease. However, the current production of HupA from plants in large quantity is unsustainable because the plant resource is scarce and the content of HupA in plants is extremely low. Surprisingly, this compound was recently found to be produced by various endophytic fungi, which are much more controllable than the plants due to simpler genetics and ease of manipulation. However, it might be due to the innate properties of endophytic symbiosis, that production of this chemical in large quantity from endophytes has not yet been put into practice. Endophytic Colletotrichum gloeosporioides ES026 was previously isolated from a HupA producing plant and the fungi also proved to produce HupA. In this study, various fermentation conditions were tried to optimize the production of HupA from C. gloeosporioides ES026. Optimization of these parameters resulted in a 25.58% increase in HupA yield. Potato extracts supplemented with glucose or sucrose but not maltose facilitated HupA producing from the fungi. A final concentration of 0.5–2% ethanol stimulated the growth of fungi while methanol with the same treatment slightly inhibited the growth. However, both methanol and ethanol greatly increased the HupA production with the highest yield of HupA (51.89% increment) coming from ethanol treatment. Further analysis showed that both ethanol and methanol were strong inducers of HupA production, while ethanol was partially used as a carbon source during fermentation. It was noticed that the color of that ethanol treated mycelia gradually became dark while methanol treated ones stayed grey during fermentation. The present study sheds light on the importance of optimizing the fermentation process, which, combined with effective inducers, maximizes production of chemicals of important economic interest from endophytic fungi.     

组蛋白甲基化与去乙酰化对蛇足石杉内生真菌盘长孢状刺盘孢合成石杉碱甲的影响

以蛇足石杉Huperzia serrata内生真菌盘长孢状刺盘孢Cg01菌株为研究对象,利用PEG介导的同源重组转化体系,对Cg01组蛋白甲基化酶基因（histone methyltransferases,HMT）CgClr4和组蛋白去乙酰化酶基因（histone deacetylase,HDAC）CgClr3、CgSir2进行基因敲除与回补,并通过实时荧光定量PCR（RT-qPCR）检测了回补株中对应基因表达量以及高效液相色谱HPLC检测突变体菌株中石杉碱甲huperzine A（HupA）产量。结果显示3个基因敲除突变体菌株ΔCgClr4、ΔCgClr3、ΔCgSir2的HupA产量分别为255μg/L、270μg/L、244μg/L,与野生型菌株相比分别下降了21.3%、16.6%、24.7%。在基因回补突变体菌株ΔCgClr4/CgClr4、ΔCgClr3/CgClr3、ΔCgSir2/CgSir2中,相应回补基因表达均与野生型无显著性差异,其HupA产量分别为351.9μg/L、334.7μg/L、331μg/L,回补菌株的HupA产量回复到野生型水平。结果表明这3个基因均具有调控内生真菌盘长孢状刺盘孢Cg01合成HupA的作用,为研究蛇足石杉内生真菌中石杉碱甲的合成调控机制提供了理论基础和新的思路。     

Identification of a novel endophytic fungus from Huperzia serrata which produces huperzine A

Huperzine A is isolated from Huperzia serrata and is used for treatment of Alzheimer’s disease, due to its low toxicity and long effective period. The decrease in H. serrata sources means that natural huperzine A cannot meet the needs of clinical therapy. In this study, >200 endophytic fungal strains were isolated from H. serrata, and screened using high-performance liquid chromatography. Strain ES026 produced huperzine A. Production was identified and quantified by liquid chromatography–tandem mass spectrometry, and the yield of huperzine A was 1 μg/g dried mycelium. ES026 strain was identified as Colletotrichum gloeosporioides by morphology, polymerase chain reaction with species-specific primers and rDNA internal transcribed spacer sequence. ES026 contributes to the breeding of cultivated strains with high yield of huperzine A. Meanwhile, the strain was suitable for the study of biosynthesis of huperzine A.     

De Novo RNA Sequencing and Transcriptome Analysis of Colletotrichum gloeosporioides ES026 Reveal Genes Related to Biosynthesis of Huperzine A

Huperzine A is important in the treatment of Alzheimer’s disease. There are major challenges for the mass production of huperzine A from plants due to the limited number of huperzine-A-producing plants, as well as the low content of huperzine A in these plants. Various endophytic fungi produce huperzine A. Colletotrichum gloeosporioides ES026 was previously isolated from a huperzine-A-producing plant Huperzia serrata, and this fungus also produces huperzine A. In this study, de novo RNA sequencing of C. gloeosporioides ES026 was carried out with an Illumina HiSeq2000. A total of 4,324,299,051 bp from 50,442,617 high-quality sequence reads of ES026 were obtained. These raw data were assembled into 24,998 unigenes, 40,536,684 residues and 19,790 genes. The majority of the unique sequences were assigned to corresponding putative functions based on BLAST searches of public databases. The molecular functions, biological processes and biochemical pathways of these unique sequences were determined using gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) assignments. A gene encoding copper amine oxidase (CAO) (unigene 9322) was annotated for the conversion of cadaverine to 5-aminopentanal in the biosynthesis of huperzine A. This gene was also detected in the root, stem and leaf of H. serrata. Furthermore, a close relationship was observed between expression of the CAO gene (unigene 9322) and quantity of crude huperzine A extracted from ES026. Therefore, CAO might be involved in the biosynthesis of huperzine A and it most likely plays a key role in regulating the content of huperzine A in ES026.     

An Endophytic Sanguinarine-Producing Fungus from Macleaya cordata,Fusarium proliferatum BLH51

Fermentation processes using sanguinarine-producing fungi other than Macleaya cordata may be an alternative way to produce sanguinarine (SA), which is a quaternary benzo[c]phenanthridine alkaloid possessing antibacterial, anthelmintic, and anti-inflammatory properties. In this study, a SA-producing endophytic fungus strain BLH51 was isolated from the leaves of M. cordata grown in the Dabie Mountain, China. Strain BLH51 produced SA when grown in potato dextrose liquid medium. The amount of SA produced by this endophytic fungus was quantified to be 178 μg/L by HPLC, substantially lower than that produced by the host tissue. The fungal SA—which was analyzed by thin layer chromatography and high-performance liquid chromatography—was shown to be identical to authentic SA. Strain BLH51 was identified as Fusarium proliferatum based on the morphological characteristics and nuclear ribosomal DNA ITS sequence analysis. To the best of our knowledge, this is the first report concerning the isolation and identification of endophytic SA-producing fungi from the host plant, which further proved that endophytic fungi are valuable reservoirs of bioactive compounds.     

蛇足石杉中的新内生真菌及其代谢产物的分离与鉴定

从蛇足石杉内生菌的次级代谢产物中寻找活性成分,为进一步开发利用蛇足石杉药用植物资源提供了新途径,但至今其内生菌代谢产物的系统性研究较为少见。种类丰富的内生真菌普遍存在于各种植物中,但蕨类植物中内生真菌的研究较少。为了寻找蛇足石杉内生菌中的细胞毒活性成分,该研究从蛇足石杉根部分离得到一株球毛壳属(Chaetomium sp.)真菌M336,对其化学成分进行了研究。对蛇足石杉内生真菌M336采用PDA固体培养基扩大发酵,发酵物经提取及乙酸乙酯萃取后,通过正相硅胶柱色谱法、Sephadex LH-20凝胶柱色谱法、薄层制备、高效液相色谱等色谱手段对其发酵物中的化学成分进行分离纯化,利用理化性质、质谱、核磁等波谱分析技术,并结合相关文献数据鉴定化合物的结构。结果表明：从内生真菌M336发酵提取物的乙酸乙酯萃取部分分离并鉴定出8个化合物,分别为chaetoviridines F、chaetoviridines E、5′-epichaetoviridin A、5′-epichaetoviridin A、xanthoquinodins Al、xanthoquinodins A2、xanthoquinodins B1和毛壳菌素。从M336中分离得到8个化合物,化合物3有一定的抑菌作用,其余化合物有一定的细胞毒活性。该研究结果丰富了蛇足石杉内生真菌球毛壳属中的天然细胞毒活性的化合物。     

Variation in huperzine A and B in Australasian Huperzia species

Huperzine A (HupA) and huperzine B (HupB) are alkaloids with acetylcholine esterase inhibitory activity and potential applications in the treatment of Alzheimer's disease. Both alkaloids were isolated in the 1980s from the Chinese lycopod Huperzia serrata (Thunb. ex Murray) Trevis., which has been used since the Tang dynasty as a traditional Chinese medicine. Most of the HupA currently used in herbal supplements and medicines worldwide is sourced from H. serrata which on average contains only 0.18 mg g⁻¹ dry wt HupA and is experiencing a rapid decline in China due to over-harvesting. Eleven Australasian Huperzia species were surveyed and nine species were found to contain both alkaloids, with a significant positive relationship observed between HupA and the lower abundance HupB. An Australian Huperzia carinata plant had one of the highest HupA concentrations recorded for a plant (1.03 mg g⁻¹ dry wt) and an Huperzia phlegmaria plant had the highest HupB value of all species surveyed (0.23 mg g⁻¹ dry wt). Intra-specific variation in huperzine concentration was examined for H. phlegmaria and Huperzia phlegmarioides and both HupA and HupB varied substantially within each species, but this variation was unrelated to foliar nitrogen levels.     

一株产槲皮素类色素的银杏内生真菌的研究

从银杏树根中分离出70余株内生真菌.将它们置于马铃薯葡萄糖(PD)液体培养基中培养,发现其中一株(Gh01)能产生橙黄色色素.经过化学反应及HPLC检测证明该橙黄色色素为槲皮素类糖苷.这是关于内生真菌产槲皮素类糖苷的首次报道.本文深入探讨了碳源,氮源,金属离子,初始PH及培养温度对色素产量的影响.PD液体培养基的最适培养温度和初始PH分别为28℃和7.0.正交设计结果显示:最适碳源和氮源分别为20g/L葡萄糖和5g/L蛋白胨.增加1g/L的氯化锌可提高色素产量.在最适培养条件下连续培养120h色素的产量可达到27.515g/L.     

Spatial Segregation and Aggregation of Ectomycorrhizal and Root-Endophytic Fungi in the Seedlings of Two Quercus Species

Diverse clades of mycorrhizal and endophytic fungi are potentially involved in competitive or facilitative interactions within host-plant roots. We investigated the potential consequences of these ecological interactions on the assembly process of root-associated fungi by examining the co-occurrence of pairs of fungi in host-plant individuals. Based on massively-parallel pyrosequencing, we analyzed the root-associated fungal community composition for each of the 249 Quercus serrata and 188 Quercus glauca seedlings sampled in a warm-temperate secondary forest in Japan. Pairs of fungi that co-occurred more or less often than expected by chance were identified based on randomization tests. The pyrosequencing analysis revealed that not only ectomycorrhizal fungi but also endophytic fungi were common in the root-associated fungal community. Intriguingly, specific pairs of these ectomycorrhizal and endophytic fungi showed spatially aggregated patterns, suggesting the existence of facilitative interactions between fungi in different functional groups. Due to the large number of fungal pairs examined, many of the observed aggregated/segregated patterns with very low P values (e.g., < 0.005) turned non-significant after the application of a multiple comparison method. However, our overall results imply that the community structures of ectomycorrhizal and endophytic fungi could influence each other through interspecific competitive/facilitative interactions in root. To test the potential of host-plants'' control of fungus–fungus ecological interactions in roots, we further examined whether the aggregated/segregated patterns could vary depending on the identity of host plant species. Potentially due to the physiological properties shared between the congeneric host plant species, the sign of hosts'' control was not detected in the present study. The pyrosequencing-based randomization analyses shown in this study provide a platform of the high-throughput investigation of fungus–fungus interactions in plant root systems.     

Raffinose production using α-galactosidase from Paraphaeosphaeria sp.

An α-galactosidase for raffinose synthesis by a reverse hydrolysis reaction was searched from intracellular of endophytic fungi. About 500 strains were screened, one strain was selected and identified as Paraphaeosphaeria sp. Partially purified α-galactosidase (1 U/ml in the reaction mixture), 100 g/L galactose, and 500 g/L sucrose at 60 °C for 48 h resulted in synthesis of 13.3 g/L raffinose and 4.6 g/L planteose at a ratio of about 3:1. The data suggest that α-galactosidase was able to synthesize raffinose at a ratio higher than that of α-galactosidases derived from other fungi.     

A comparison of fungal endophytic community diversity in tree leaves of rural and urban temperate forests of Kanto district,eastern Japan

To clarify the effects of forest fragmentation and a change in tree species composition following urbanization on endophytic fungal communities, we isolated fungal endophytes from the foliage of nine tree species in suburban (Kashiwa City, Chiba) and rural (Mt. Wagakuni, Ibaraki; Mt. Takao, Tokyo) forests and compared the fungal communities between sites and host tree species. Host specificity was evaluated using the index of host specificity (Si), and the number of isolated species, total isolation frequency, and the diversity index were calculated. From just one to several host-specific species were recognized in all host tree species at all sites. The total isolation frequency of all fungal species on Quercus myrsinaefolia, Quercus serrata, and Chamaecyparis obtusa and the total isolation frequency of host-specific species on Q. myrsinaefolia, Q. serrata, and Eurya japonica were significantly lower in Kashiwa than in the rural forests. The similarity indices (nonmetric multidimensional scaling (NMS) and C_MH) of endophytic communities among different tree species were higher in Kashiwa, as many tree species shared the same fungal species in the suburban forest. Endophytic fungi with a broad host range were grouped into four clusters suggesting their preference for conifer/broadleaves and evergreen/deciduous trees. Forest fragmentation and isolation by urbanization have been shown to cause the decline of host-specific fungal species and a decrease in β diversity of endophytic communities, i.e., endophytic communities associated with tree leaves in suburban forests were found to be depauperate.     

一株产紫杉醇罗汉松内生真菌的分离和鉴定

[目的]紫杉醇是重要的抗癌药物,主要从罗汉松等植物中提取,为了保护罗汉松等种质资源,本文从罗汉松植株中分离产紫杉醇内生真菌,并对内生真菌所产紫杉醇的抗肿瘤活性进行了分析.[方法]采用组织块法自罗汉松的根、茎、叶等组织中分离内生真菌;通过四唑蓝(Methyl ThiazolylTetrazolium,MTT)比色法筛选有抗肿瘤活性的内生真菌菌株,通过薄层层析(Thin Layer Chro-matography,TLC)和高效液相色谱(High Performance Liquid Chromatography,HPLC)对内生真菌所产活性物质进行鉴定;采用抽提法抽提内生真菌所产紫杉醇,应用Vero细胞对抽提的紫杉醇的活性进行了分析.[结果]从罗汉松属(Podocrapus)植物中分离到155株内生真菌,其中28株内生真菌具有较高的抑癌活性.将其中一株菌株A2命名为EPTP-1,经形态学和分子分类学分析鉴定为烟曲霉(Aspergillus fumigatus).菌株EPTP-1中抽提的紫杉醇5.553μg/L～555.3 μg/L作用24h表现出明显的致细胞凋亡作用.菌株EPTP-1发酵5天时紫杉醇的产率为0.5578±0.0294 mg/L.[结论]从罗汉松中分离到了一株产紫杉醇内生真菌EPTP-1,可作为紫杉醇类药物工业化生产的候选菌株.     

云南元江干热河谷五种优势植物的内生真菌多样性

从蔓草虫豆（Atylosia scarabaeoides）、余甘子（Phyllanthus emblica）和黄花稔（Sida acuta）等5种云南元江干热河谷植物的525个组织块中,共分离得到内生真菌371株,内生真菌的分离频率在0.61～0.92之间,且所有植物叶内生真菌的分离频率都明显高于茎（P<0.05）。经形态学鉴定,内生真菌分属于拟茎点霉属（Phomopsis sp.）、离蠕孢属（Bipolaris sp.）和交链孢属（Alternaria sp.）等32个分类单元。拟茎点霉属为干热河谷植物优势内生真菌属,从所有被调查植物的茎叶中都分离得到该属真菌,且相对分离频率高达12.90%～50.54%。内生真菌群落组成的多样性和相似性分析结果表明,云南元江干热河谷植物内生真菌多样性偏低、宿主专一性较小。     

Effects of pH,water stress and temperature on in vitro cultures of ectomycorrhizal fungi from Mediterranean forests

A comparative study of the growth of eight species of ectomycorrhizal fungi [Hebeloma edurum Métrod ex Bon, Lactarius deliciosus (L.: Fr.) S.F. Gray., Rhizopogon roseolus (Corda) Th.M.Fr., Suillus collinitus (Fr.) Kuntze, Suillus granulatus (L.: Fr.) Roussel, Suillus luteus (L.: Fr.) Roussel, Tricholoma focale (Fr.) Ricken, Tricholoma striatum (Schaeff.) Sacc.] in pure culture at different pH values, water potentials and temperatures was carried out. The biomass production and mycelial growth of each strain were assessed and characterised morphologically. H. edurum and L. deliciosus showed a clear affinity for neutral or slightly basic media. Most species had the lowest colony diameters and biomass production at pH 2.5. Only H. edurum, R. roseolus and S. collinitus grew at the highest water stress levels (−13.85 and −15.45 bar). All species grew at the lowest values of temperature whereas only R. roseolus and S. collinitus did at 30 °C.     

Natural products of Alternaria sp., an endophytic fungus isolated from Salvadora persica from Saudi Arabia

This study is to evaluate the potential of endophytic fungi of Salvadora persica for the production of bioactive compounds against pathogenic bacteria and fungi. Forty-two fungal isolates were obtained from 135 young and old stem and 125 root segments. Those 42 isolates representing ten fungi include: Trichoderma sp. (the most common), two species of Alternaria, Rhizopus arrhizus and 6 sterile mycelia. The ten fungi were grown in liquid culture and their crude extracts were tested against pathogenic bacteria and fungi. Nine crude extracts gave positive reactions against pathogenic bacteria of which Alternaria sp. (A8) was chosen further study. The fungal isolate was growing as sterile mycelium and was identified by phylogenetic analyses based on LSU rDNA sequence data and it might represent undescribed species of Alternaria. Sixty-two bioactive chemical compounds were identified from the ethyl acetate crude extracts of Alternaria sp., of which the following were recorded as major compounds in the active sub-fractions. These compounds showed strong antibacterial activity in combination.     

云南会泽铅锌矿废弃矿渣堆常见植物内生真菌多样性

从云南会泽铅锌矿废弃矿渣堆上的常见植物硬毛南芥(Arabis hirsuta)、毛萼香茶菜(Rabbosia eriocalyx)和倒挂刺(Rosa longicuspis)等6种植物的690个组织块中共分离得到内生真菌495株,内生真菌的分离频率在0.42—0.93之间,平均为0.72,所有植物茎内生真菌的分离频率都明显高于叶(P<0.05)。经形态学鉴定,内生真菌分属于茎点霉属(Phoma)、交链孢属(Alternaria)和派伦霉属(Peyronellaea)等20个分类单元,其中茎点霉属和派伦霉属为该废弃矿渣堆上常见植物的优势内生真菌属。6种植物内生真菌的多样性指数在1.05—2.29之间,与其它非重金属污染环境植物内生真菌的多样性指数相似,说明在重金属污染地区仍然存在多种重金属耐受的内生真菌种类。6种植物内生真菌的相似性系数(0.455—0.833)表明,会泽铅锌矿区植物内生真菌的宿主专一性较小。