Screening and identification of a fungal strain affecting hydroxylation of naringin

柚皮苷是中草药枳实、橘红的主要有效成分,用于心血管疾病的防治.为了开发生物法生产柚皮苷的羟基化衍生物,从土壤中分离筛选到一株能转化柚皮苷为3'-羟基柚皮苷和3',5'-二羟基柚皮苷的真菌菌株NT-02.通过对菌株NT-02的形态学观察和ITS序列系统发育分析,构建了系统发育树,NT-02菌株与哈茨木霉(Trichoderma harzianum/Hypocreaum lixii)形成一个类群,且有99％的同源性,初步鉴定真菌NT-02为木霉属(Trichoderma)菌株.     

康宁木霉对龟裂链霉菌MY02诱导活性及其诱导组分的初步分析

利用真菌作为诱导菌株评价其对龟裂链霉菌(Streptomyces rimosus) MY02的活性代谢产物的影响,并对其诱导条件进行优化。当以康宁木霉(Trichoderma koningii)和香菇真菌(Lentinus edodes)作为诱导菌株,黄瓜枯萎病菌(Fusarium oxysporum f sp.cucumarinum)作为指示菌,以菌株MY02作为被诱导菌株时,康宁木霉和香菇真菌对菌株MY02的抗真菌活性均具有明显的正向诱导作用,菌株MY02的抗真菌活性均比对照明显增强。以康宁木霉作为诱导菌株时,其最佳诱导条件为接种量2%,添加时间为发酵36 h。通过初步分析康宁木霉的诱导组分为其发酵产生的多糖类物质。     

粮食上木霉菌的分离鉴定及其生防效果

【背景】粮食在生长和收储期极易受到病原真菌或产毒真菌的污染,造成严重的损失。众多实践证明木霉属(Trichoderma)可以有效防治植物病原真菌。【目的】鉴定和筛选能有效抑制粮食常见危害真菌的木霉生防菌株,开发生防菌剂,保障粮食生产安全。【方法】从粮食上分离筛选出35株木霉,通过多基因系统发育分析和形态学观察方法进行菌种鉴定,利用平板对峙试验筛选出对粮食常见危害真菌有抑制作用的菌株。【结果】35株木霉分属于8个种,分别为非洲哈茨木霉(Trichodermaafroharzianum)、类棘孢木霉(Trichodermaasperelloides)、 Trichoderma amoenum、近深绿木霉(Trichoderma paratroviride)、Trichoderma obovatum、长枝木霉(Trichoderma longibrachiatum)、东方木霉(Trichodermaorientale)和深绿木霉(Trichodermaatroviride)。对峙试验结果表明,这8种木霉对于粮食上分离到的10种危害真菌均具有较好的抑制效果。非洲哈茨木霉(T.afroharzi...     

里氏木霉RutC30常温常压等离子体（ARTP）诱变筛选pyr4基因缺陷型菌株及转化系统的建立

以里氏木霉Trichoderma reesei重要工业生产菌RutC30为出发菌株,通过等离子体（ARTP）诱变筛选,以5-氟乳清酸（5-FOA）和尿苷（Uridine）进行筛选,获得一株pyr4基因缺陷株RutC30ΔU3。用含有野生型里氏木霉pyr4基因的互补质粒转化突变株,可回复野生性状。经测序发现其pyr4基因在核酸序列多个位点发生突变,其中包括两个错义突变和一个移码突变,从而导致乳清酸核苷-5′-磷酸脱羧酶失活。经遗传稳定性研究分析,传代5次后仍保持良好的尿苷依赖性、去葡萄糖阻遏以及高产纤维素酶特性。经实验筛选获得了pyr4基因缺陷菌株可作为基因表达系统的受体菌株,建立了以尿苷营养缺陷为筛选标记的木霉转化系统。     

人肠道产柚苷酶菌株分离、鉴定及其对柚皮苷的转化特性

【目的】从健康人肠道微生物菌群中分离能将柚皮苷高效转化为柚皮素的特定细菌菌株,对分离得到的菌株进行菌种鉴定并研究菌株对柚皮苷转化特性,目的是为柚皮素的高效生物合成提供新细菌资源。【方法】取健康人新鲜粪样,在厌氧工作站内培养24 h后进行梯度稀释并涂板,从板上挑取单菌落与底物柚皮苷进行厌氧培养,用高效液相色谱检测底物被转化情况。根据16S rDNA序列及相关生理生化特性分析,对所分离的柚皮苷转化菌进行菌种鉴定,并测定转化菌株对底物柚皮苷的转化动态和转化能力。【结果】从人肠道菌群中分离得到4株能将柚皮苷转化为柚皮素的细菌菌株,分别命名为AUH-JLD3、AUH-JLD7、AUHJLD104和AUH-JLD109。根据16S rDNA序列分析,结合形态学及相关生理生化特性等,将其分别鉴定为布劳特氏菌(Blautia sp.AUH-JLD3)、肠球菌(Enterococcus sp.AUH-JLD7)、拟杆菌(Bacteroides sp.AUHJLD104)和巴氏链球菌(Streptococcus pasteurianus subsp.AUH-JLD109)。转化动态研究结果表明,所分离的4株细菌菌株均能在12 h内将0.2 mmol/L底物柚皮苷转化为柚皮素;转化能力测定结果显示,菌株AUHJLD3、AUH-JLD7、AUH-JLD104及AUH-JLD109能够高效转化底物柚皮苷的最大浓度分别为0.2 mmol/L(平均转化率66.67%)、0.8 mmol/L(平均转化率86.49%)、0.2 mmol/L(平均转化率73.68%)以及1.6 mmol/L(平均转化率93.20%)。【结论】本研究首次从人肠道菌群中分离得到4株能将柚皮苷转化为柚皮素的细菌菌株,其中巴氏链球菌AUH-JLD109对底物柚皮苷转化能力最强。     

一株抗肿瘤活性的粗榧内生真菌的鉴定及其产物特性初步研究

利用传统分类学方法、电子显微镜观察、18S rDNA和ITS序列的测定以及系统发育分析对一株分离自粗榧抗肿瘤活性内生真菌HCCB1621进行分类鉴定,并对其产物抗肿瘤活性进行了初步研究．各种指标鉴定其为木霉属真菌绿色木霉（Trichoderma viride Pers．ex Fr）．该菌株经发酵培养120h,抗瘤活性达最强;活性在pH4～9范围内稳定：60℃处理1h,活性稳定,这些特性为活性产物的分离纯化和稳定性研究提供了必要的参考．     

柚皮素-7-O-乙酸酯和柚皮素-7-O-丙酸酯的制备及抗血小板聚集活性

为改善柚皮素的水溶性而不降低其抗血小板聚集活性,本文以柚皮苷为原料,经"4'位羟基苄基化-酸水解苷键-酰化-加氢脱苄基"四步反应,合成出柚皮素-7-O-乙酸酯和柚皮素-7-O-丙酸酯。两种衍生物在水中的溶解度分别为637.34±53.23μg/m L和59.74±4.81μg/m L,均高于柚皮素的溶解度。两种衍生物均对二磷酸腺苷诱导的兔体外和大鼠体内血小板聚集有显著的抑制活性,且抑聚率均高于柚皮素。实验结果表明,通过选择酰化柚皮素的7位羟基,引入含1~2个碳的短脂肪烃基链,能显著改善水溶性,提高抗血小板聚集活性。     

木霉属Trichoderma组和Pachybasium组的分子系统学研究

对来源不同的木霉及其有性型Longibrachiatum组,Trichoderma组和Phachybasium组的81个菌株进行了ITS序列测定,并对ITS1序列用PHYLIP程序包中的DNAPARS程序进行系统发育分析。结果表明Trichoderma组和Pachybasium组的所有菌株可分成两个群（A,B）,B群进一步分为4个分支（B1,B2,B3,B4）;A组由Trichoderma组的H.aureoviridis和未鉴定到种的3个Hypocrea菌株构成：B1,B2,B4群均由Pachybasium组菌构成;3群由Pachybasium组的T.hamatum,T.strigosum和Trichoderma组的T.asperellum,T.atroviride,T,koningii,T.viride和Hypocrea菌株T261构成。2个组相互交叉,组间没有明确的区分,进一步证明Pachybasium组是多系的。建议将Trichoderma组中的T.virideaggr.,T.atroviride,T.koningii归并入Pachybasium组,对Trichoderma组重新定义。     

三种水生植物内生真菌多样性及其抗真菌活性

从华凤仙(Impatiens chinensis L.)、问荆(Equisetum arvense L.)和轮叶狐尾藻(Myriophyllum verticillatum L.) 3种植物中共分离到内生真菌155株。经鉴定, 它们分属于26个不同的分类单元。以杨桃炭疽菌(Colletotrichum gloeosporioides)、香蕉疫霉(Phytophthora nicotianae)和腐皮镰刀菌(Fusarium solani)等6种植物病原真菌为指示菌, 对3种水生植物内生真菌代谢产物进行抗菌活性研究。结果表明, 有37株内生真菌(占分离菌株总数的23.9%)显示出对一种或多种病原菌的抑菌活性。来自华凤仙、问荆和轮叶狐尾藻的抗菌活性菌株比例分别为29.2%、13.9%及37.1%, 都远低于已报道的陆生植物内生真菌中活性菌株的比例。抗菌活性菌株主要分布于枝孢属(Cladosporium)、木霉属(Trichoderma)和地霉属(Geotrichum)等5个属中。     

木霉属2中国新记录种

[目的]明确我国南部地区土壤中分离得到的2株木霉菌的分类地位.[方法]采用PDAm培养基分离木霉菌菌株,通过形态学鉴定和内转录间隔区(ITS)序列、翻译延长因子1-alpha (Tef1-α)部分序列相似性分析对菌株进行初步鉴定;选取MEGA 4.0软件用Bootstrap最大简约法、水平3、重复1 000次来构建ITS、Tef1-α序列系统进化树并分析其亲缘关系.[结果]菌株CM01的ITS序列与GenBank基因库中Trichoderma intricatum strain GJS 02-78 ITS序列同源性高达99％,在ITS序列系统进化树中与模式菌株T.intricatum GJS 02-78、Trichoderma atroviride DAOM 179514亲缘关系最近;其Tef1-α序列与GenBank基因库中Hypocrea intricate strain GJS 02-78 Tef1-α序列同源性高达99％,在Tef1-α序列系统进化树中与模式菌株H.intricata GJS 02-78亲缘关系最近;其形态描述和模式菌株一致.菌株SCGA5003的ITS序列与GenBank基因库中Trichoderma stromaticum strain GJS 97-181 ITS序列同源性高达99％,在ITS序列系统进化树中与模式菌株T.stromaticum GJS 97-179、GJS 97-180、GJS 97-181、GJS 97-182、GJS 97-183亲缘关系最近;其Tef1-α序列与GenBank基因库中T.stromatieum strain GJS 97-183 Tef1-α序列同源性高达94％,在Tef1-α序列系统进化树中与模式菌株T.stromaticum CQSQ1032、GXNN7006亲缘关系最近;其形态描述和模式菌株一致.[结论]结合形态学特征与分子鉴定结果,判定菌株CM01为交织木霉(Trichoderma intricatum Samuels&Dodd/Hypocrea intricata Samuels et Dodd)、SCGA5003 为子座木霉(Trichoderma stromaticum Samuels & Pardo-Schulth/Hypocrea stromatica Bezerra,Costa & Bastos),即发现木霉属2个中国新记录种.     

Multilocus phylogenetic structure within the Trichoderma harzianum/Hypocrea lixii complex

Trichoderma harzianum is a ubiquitous species in the environment and is effective in the biological control of plant-pathogenic fungi. T. harzianum has not been linked unequivocally to its sexual state nor has its phylogeny been studied in detail. It has been suggested that T. harzianum is a species complex based on the phenotypic and genotypic variability encountered. On the basis of morphological and cultural characters and DNA sequence data analysis of four genes (ITS rDNA, translation elongation factor 1-alpha, calmodulin, and alpha-actin), Hypocrea lixii was found to be the sexual state of T. harzianum. Both the asexual and sexual states of this species have wide geographic distributions. Phylogenetic analysis of four genes showed that T. harzianum/H. lixii is a cohesive group that is supported by bootstrap values higher than 95%. Principles of genealogical concordance indicated that T. harzianum/H. lixii is a complex of independent monophyletic lineages, but no diagnostic morphological distinctions were identified that justify formal taxonomic recognition for the different lineages.     

Application of DNA bar codes for screening of industrially important fungi: the haplotype of Trichoderma harzianum sensu stricto indicates superior chitinase formation

Selection of suitable strains for biotechnological purposes is frequently a random process supported by high-throughput methods. Using chitinase production by Hypocrea lixii/Trichoderma harzianum as a model, we tested whether fungal strains with superior enzyme formation may be diagnosed by DNA bar codes. We analyzed sequences of two phylogenetic marker loci, internal transcribed spacer 1 (ITS1) and ITS2 of the rRNA-encoding gene cluster and the large intron of the elongation factor 1-alpha gene, tef1, from 50 isolates of H. lixii/T. harzianum, which were also tested to determine their ability to produce chitinases in solid-state fermentation (SSF). Statistically supported superior chitinase production was obtained for strains carrying one of the observed ITS1 and ITS2 and tef1 alleles corresponding to an allele of T. harzianum type strain CBS 226.95. A tef1-based DNA bar code tool, TrichoCHIT, for rapid identification of these strains was developed. The geographic origin of the strains was irrelevant for chitinase production. The improved chitinase production by strains containing this haplotype was not due to better growth on N-acetyl-beta-D-glucosamine or glucosamine. Isoenzyme electrophoresis showed that neither the isoenzyme profile of N-acetyl-beta-glucosaminidases or the endochitinases nor the intensity of staining of individual chitinase bands correlated with total chitinase in the culture filtrate. The superior chitinase producers did not exhibit similarly increased cellulase formation. Biolog Phenotype MicroArray analysis identified lack of N-acetyl-beta-D-mannosamine utilization as a specific trait of strains with the chitinase-overproducing haplotype. This observation was used to develop a plate screening assay for rapid microbiological identification of the strains. The data illustrate that desired industrial properties may be an attribute of certain populations within a species, and screening procedures should thus include a balanced mixture of all genotypes of a given species.     

M13-microsatellite PCR and rDNA sequence markers for identification of Trichoderma (Hypocreaceae) species in Saudi Arabian soil

Seven fungal isolates were identified as pan-global Hypocrea/Trichoderma species, from section Trichoderma, on the basis of their morphology. These species were H. lixii/T. harzianum and H. orientalis/T. longibrachiatum. PCR-based markers with primer M13 (core sequence of phage M13) and internal-transcribed spacer sequences of ribosomal DNA were used to confirm the identity of the two Trichoderma species. Sequence identification was performed using the TrichOKEY version 2.0 barcode program and the multilocus similarity search database TrichoBLAST. Sequences from the ribosomal DNA internal-transcribed spacer regions showed limited variation among the Trichoderma species. This analysis divided the isolates into two main groups. Grouping the isolates based on cluster analysis of their DNA profiles matched the grouping based on morphological taxonomy. Molecular data obtained from analyses of gene sequences are essential to distinguish phonetically cryptic species in this group and to establish phylogenetic relationships.     

Soils of a Mediterranean hot spot of biodiversity and endemism (Sardinia, Tyrrhenian Islands) are inhabited by pan-European, invasive species of Hypocrea/Trichoderma

We have used a Mediterranean hot spot of biodiversity (the Island of Sardinia) to investigate the impact of abiotic factors on the distribution of species of the common soil fungus Trichoderma . To this end, we isolated 482 strains of Hypocrea / Trichoderma from 15 soils comprising undisturbed and disturbed environments (forest, shrub lands and undisturbed or extensively grazed grass steppes respectively). Isolates were identified at the species level by the oligonucleotide BarCode for Hypocrea / Trichoderma ( TrichO KEY), sequence similarity analysis ( Tricho blast ) and phylogenetic inferences. The majority of the isolates were positively identified as pan-European and/or pan-global Hypocrea / Trichoderma species from sections Trichoderma and Pachybasium , comprising H. lixii/T. harzianum , T. gamsii , T. spirale , T. velutinum , T. hamatum , H. koningii/T. koningii , H. virens/T. virens , T. tomentosum , H. semiorbis , H. viridescens/T. viridescens , H. atroviridis/T. atroviride , T. asperellum , H. koningiopsis/T. koningiopsis and Trichoderma sp. Vd2. Only one isolate represented a new, undescribed species belonging to the Harzianum–Catoptron Clade. Internal transcribed spacer sequence analysis revealed only one potentially endemic internal transcribed spacer 1 allele of T. hamatum . All other species exhibited genotypes that were already found in Eurasia or in other continents. Only few cases of correlation of species occurrence with abiotic factors were recorded. The data suggest a strong reduction of native Hypocrea / Trichoderma diversity, which was replaced by extensive invasion of species from Eurasia, Africa and the Pacific Basin.     

Biochemical and metabolic profiles of Trichoderma strains isolated from common bean crops in the Brazilian Cerrado, and potential antagonism against Sclerotinia sclerotiorum

Some species of Trichoderma have successfully been used in the commercial biological control of fungal pathogens, e.g., Sclerotinia sclerotiorum, an economically important pathogen of common beans (Phaseolus vulgaris L.). The objectives of the present study were (1) to provide molecular characterization of Trichoderma strains isolated from the Brazilian Cerrado; (2) to assess the metabolic profile of each strain by means of Biolog FF Microplates; and (3) to evaluate the ability of each strain to antagonize S. sclerotiorum via the production of cell wall-degrading enzymes (CWDEs), volatile antibiotics, and dual-culture tests. Among 21 isolates, we identified 42.86% as Trichoderma asperellum, 33.33% as Trichoderma harzianum, 14.29% as Trichoderma tomentosum, 4.76% as Trichoderma koningiopsis, and 4.76% as Trichoderma erinaceum. Trichoderma asperellum showed the highest CWDE activity. However, no species secreted a specific group of CWDEs. Trichoderma asperellum 364/01, T. asperellum 483/02, and T. asperellum 356/02 exhibited high and medium specific activities for key enzymes in the mycoparasitic process, but a low capacity for antagonism. We observed no significant correlation between CWDE and antagonism, or between metabolic profile and antagonism. The diversity of Trichoderma species, and in particular of T. harzianum, was clearly reflected in their metabolic profiles. Our findings indicate that the selection of Trichoderma candidates for biological control should be based primarily on the environmental fitness of competitive isolates and the target pathogen.     

Phylogeny of the genus trichoderma based on sequence analysis of the internal transcribed spacer region 1 of the rDNA cluster

Sequences of the internal transcribed spacer region 1 (ITS1) of the ribosomal DNA were used to determine the phylogenetic relationships of species of Trichoderma sect. Pachybasium. To this end, 85 strains-including all the available ex-type strains-were analyzed. Parsimony analysis demonstrated that the section is nonmonophyletic, distributing the 85 strains among three main groups that were supported by bootstrap values. Group A comprises two clades (A1 and A2), with A1 including T. polysporum, T. piluliferum, and T. minutisporum, while A2 included T. hamatum, T. pubescens, and T. strigosum in addition to species previously included in sect. Trichoderma (i.e., T. viride, T. atroviride, and T. koningii). The ex-type strain of T. fasciculatum formed a separate branch basal to clade A. Clade B contained the sect. Pachybasium members T. harzianum, T. fertile, T. croceum, T. longipile, T. strictipile, T. tomentosum, T. oblongisporum, T. flavofuscum, T. spirale, and the anamorphs of Hypocrea semiorbis and H. cf. gelatinosa. Sequence differences among clades A1, A2, and B were in the same order of magnitude as between each of them and T. longibrachiatum, which was used as an outgroup in these analyses. Sequence differences within clades A1, A2, and B were considerably smaller: in some cases (i.e., T. virens and T. flavofuscum; T. strictipile and H. cf. gelatinosa), the ITS1-sequences were identical, suggesting conspecifity. In other cases (e.g., T. crassum and T. longipile; T. harzianum, T. inhamatum, T. croceum, T. fertile, and H. semiorbis; T. hamatum and T. pubescens; and T. viride, T. atroviride, and T. koningii) differences were in the range of 1-3 nt only, suggesting a very close phylogenetic relationship. The sequence of a previously described aggressive mushroom competitor group of T. harzianum strains (Th2) was strikingly different from that of the ex-type strain of T. harzianum and closely related species and is likely to be a separate species. Copyright 1998 Academic Press.     

Genetic and metabolic diversity of Trichoderma: a case study on South-East Asian isolates

We have used isolates of Trichoderma spp. collected in South-East Asia, including Taiwan and Western Indonesia, to assess the genetic and metabolic diversity of endemic species of Trichoderma. Ninety-six strains were isolated in total, and identified at the species level by analysis of morphological and biochemical characters (Biolog system), and by sequence analysis of their internal transcribed spacer regions 1 and 2 (ITS1 and 2) of the rDNA cluster, using ex-type strains and taxonomically established isolates of Trichoderma as reference. Seventy-eight isolates were positively identified as Trichoderma harzianum/Trichoderma inhamatum (37 strains) Trichoderma virens (16 strains), Trichoderma spirale (8 strains), Trichoderma koningii (3 strains), Trichoderma atroviride (3 strains), Trichoderma asperellum (4 strains), Hypocrea jecorina (anamorph: Trichoderma reesei; 2 strains), Trichoderma viride (2 strains), Trichoderma hamatum (1 strain), and Trichoderma ghanense (1 strain). Analysis of biochemical characters revealed that T. virens, T. spirale, T. asperellum, T. koningii, H. jecorina, and T. ghanense formed clearly defined clusters, thus exhibiting species-specific metabolic properties. In biochemical character analysis T. atroviride and T. viride formed partially overlapping clusters, indicating that these two species may share overlapping metabolic characteristics. This behavior was even more striking with T. harzianum/T. inhamatum where genotypes defined on the basis of ITS1 and 2 sequences overlapped significantly with adjacent genotypes in the biochemical character analysis, and four strains from the same location (Bali, Indonesia) even clustered with species from section Longibrachiatum. The data indicate that the T. harzianum/T. inhamatum group represents species with high metabolic diversity and partially unique metabolic characteristics. Nineteen strains yielded three different ITS1/2 sequence types which were not alignable with any known species. They were also uniquely characterized by morphological and biochemical characters and therefore represent three new taxa of Trichoderma.     

花青素对神经退行性疾病保护作用的研究进展

对近年来花青素在神经退行性疾病中的研究进展进行综述,并从抑制氧化应激、缓解神经炎症、缓解兴奋性毒性和抑制蛋白异常聚集等方面对花青素的神经保护作用机制进行探讨,为进一步了解和研究花青素类化合物在神经退行性疾病中的预防作用提供科学依据。     

Self-fusion of protoplasts enhances chitinase production and biocontrol activity in Trichoderma harzianum

Protoplasts were isolated from Trichoderma harzianum strain PTh18 using lysing enzymes and self-fusion of T. harzianum protoplasts was carried out using polyethylene glycol in STC buffer. The fused protoplasts of T. harzianum were regenerated and 15 self-fusants were selected to study the chitinase production and biocontrol activity. High chitinase activity was measured in the culture filtrates of most of the self-fusants (87%) than the parent. Among the fusants, the strain SFTh8 produced maximum chitinase with a two-fold increase as compared to the parent strain. All the self-fusants exhibited increased antagonistic activity against Rhizoctonia solani than the parent. The crude chitinase preparation of SFTh8 lysed the mycelia of T. harzianum, Trichoderma viride and Trichoderma reesei and released the protoplasts in higher number than the crude chitinase preparation of parent strain PTh18.     

Scanning electron microscopy observations of the interaction between Trichoderma harzianum and perithecia of Gibberella zeae

Chronological events associated with the interaction between a strain of Trichoderma harzianum, T472, with known biological control activity against perithecial production of G. zeae, were studied with scanning electron microscopy to investigate the mechanisms of control. Large clusters of perithecia consisting of 5-15 perithecia formed on the autoclaved, mulched wheat straw inoculated with G. zeae alone (control) with an average of 157 perithecia per plate. Small clusters consisting of 3-6 and an average of 15 perithecia per plate perithecia formed on straw that was treated with T. harzianum. The mature perithecia from straw treated with T. harzianum produced less pigment and were lighter in color than those from the control plates. Furthermore the cells of the outer wall of these perithecia were abnormal in appearance and unevenly distributed across the surface. Immature perithecia were colonized by T. harzianum approximately 15 d after inoculation (dai) with the biocontrol agent and pathogen. Few perithecia were colonized at later stages. The affected perithecia collapsed 21 dai, compared to the perithecia in the control samples that began to collapse 28 dai. Abundant mycelium of T. harzianum was seen on the perithecia of treated samples. Perithecial structures may be resistant to penetration by the mycelium because direct penetration was not observed. Trichoderma harzianum colonized the substrate quickly and out-competed the pathogen, G. zeae.