刺五加内生真菌分离鉴定及抗菌活性研究

为开发利用刺五加内生真菌资源,寻找新型抗菌物质,从刺五加根茎叶中分离纯化得到内生真菌71株,经菌落形态和显微形态去重后进行ITS序列分子鉴定为18个种,来自于4目、8科、11属,其中链格孢属与镰孢霉属为优势属。根、茎、叶中内生真菌多样性指数分别为1. 112 31、1. 523 658、0. 264 1。18种刺五加内生真菌中,16种内生真菌至少对一种指示菌有抗菌活性。CWJ-5(镰孢霉属Fusarium oxysporum)对金黄色葡萄球菌抑菌效果显著,抑菌直径27. 17±0. 07 mm; CWJ-6(链格孢属Alternaria alternata)对大肠埃希菌抑菌效果显著,抑菌直径18. 07±0. 01 mm。结果表明刺五加内生真菌资源丰富,有很大的开发利用空间,部分菌种抑菌活性显著,值得进一步研究。     

北细辛内生真菌的分离鉴定及代谢产物的生物活性

采用表面消毒法分别从3种北细辛中分离获得10株形态特征不同的优势内生真菌,经形态学和18S rDNA ITS分子分类学分析鉴定为小丛壳属(Glomerella sp.)、叶点霉属(Phyllosticta sp.)、柄孢壳菌属(Po-dospora sp.)、刺盘孢属(Colletotrichum sp.)和镰孢属(Fusarium sp.)。对北细辛优势内生真菌的发酵产物进行体外抗肿瘤和抗菌活性检测,结果表明:除菌株E3、E4和E10外,其余菌株均有不同程度的抗肿瘤和抗菌活性;镰孢属(Fusarium sp.)菌株E9对A549、MDA-MB-231和PANC-1肿瘤细胞抑制率达75%以上;小丛壳属(Glomerella sp.)菌株E1和叶点霉属(Phyllosticta sp.)菌株E2靶向FabI的抗菌活性较强,抑制率达59%。     

盾叶薯蓣内生真菌及其对宿主培养物生长和皂苷元生产的影响

从盾叶薯蓣根状茎中分离并鉴定了9株内生真菌,经悬浮培养14d,分别制备灭活菌丝和菌液浓缩物。其中,内生尖孢镰刀菌Dzf17能有效地提高盾叶薯蓣无菌苗和培养细胞薯蓣皂苷元的含量和产率,且灭活菌丝的诱导效果要强于菌液浓缩物。Dzf17灭活菌丝处理无菌苗,薯蓣皂苷元的产率为78.697mg/L,是对照(27.471mg/L)的2.865倍;用Dzf17菌液浓缩物处理无菌苗,皂苷元产率为41.822mg/L,是对照的1.522倍。Dzf17灭活菌丝处理培养细胞,薯蓣皂苷元的产率为1.391mg/L,是对照(0.691mg/L)的2.013倍;用Dzf17菌液浓缩物处理培养细胞,皂苷元产率为1.214mg/L,是对照的1.757倍。结果表明,在盾叶薯蓣无菌苗或细胞培养中添加一定量的内生真菌灭活菌丝或菌液浓缩物对于提高薯蓣皂苷元含量和产量将是一种有效的方法。     

1株产纤维素酶木霉菌种的鉴定

对自行筛选分离的1株木霉菌进行形态学及分子生物学鉴定。采用CTAB法抽提其基因组总DNA,利用真菌通用引物ITS1和ITS4扩增菌株rDNA ITS区序列,扩增产物纯化后进行测序。测序结果在GenBank中进行同源性搜索,并下载部分具有代表性种的ITS序列,利用软件MEGA4构建分子系统发育树,通过序列分析,并结合形态学鉴定该菌属于半知菌亚门,丝孢纲,丛梗孢目,木霉属,康宁木霉(Trichoderma koningii)。     

千层塔内生真菌的分离与鉴定

目的：为从千层塔中分离具有药用价值的内生真菌奠定基础。方法：新鲜千层塔茎段,经酒精和升汞消毒后,接种于PDA平板培养基上进行内生真菌的分离、纯化;根据菌落形态和孢子等形态特征,结合核糖体基因居间序列（ITS序列）进行菌株鉴定。结果：从千层塔的茎中分离出4株内生真菌。内生真菌I菌落形态和孢子特征与枝状枝孢霉属的特征相符合,ITS序列与GenBank中多条属于枝状枝孢霉的ITS序列相似,鉴定该菌株属于枝状枝孢霉;内生真菌II菌落形态和孢子特征与黄青霉的特征相符合,鉴定该菌株属于黄青霉;内生真菌III菌落形态和孢子特征与尖孢镰刀菌的特征相符合,ITS序列与GenBank中多条属于尖孢镰刀菌的ITS序列相似程度高,鉴定该菌株属于尖孢镰刀菌;内生真菌Ⅳ菌落形态与盾壳霉相似,ITS序列与GenBank中6条属于盾壳霉的ITS序列具有较高的相似性,鉴定该菌株属于盾壳霉。结论：从千层塔中分离和鉴定出4株内生真菌,分别属于枝状枝孢霉、黄青霉、尖孢镰刀茵和盾壳霉。     

刺五加内生青霉鲨烯合酶基因的克隆与序列分析

【目的】克隆刺五加内生青霉Penicillium minioluteum P116-1a的鲨烯合酶(Squalene synthase,SS)基因。【方法】采用cDNA 5末端快速扩增(Rapid Amplification ofcDNA 5 Ends,5 RACE)技术扩增P.minioluteum P116-1a SS基因的全长cDNA序列和DNA序列;运用生物信息学方法对该基因进行分析,预测其编码蛋白的结构与功能;并通过RT-PCR法和SDS-PAGE法检测SS的表达情况。【结果】P.minioluteum P116-1a的SS基因含有4个外显子和3个内含子,开放阅读框长1 416 bp,编码471个氨基酸,预测蛋白含67.73%的α螺旋,5.31%的延伸链,2.97%的β折叠,23.99%的无规则卷曲,含有鲨烯合酶和八氢番茄红素合成酶的特异性识别区域,定位于内质网膜。与P.marneffei和Talaromyces stipitatus中SS蛋白的氨基酸同源性达90%以上。不同温度下SS的表达情况不同。【结论】首次在刺五加内生青霉P.minioluteum P116-1a中克隆到SS基因,为进一步研究P.minioluteum P116-1a提高刺五加皂苷含量的机制奠定基础。     

一株产银杏黄酮内生真菌的初步筛选

从银杏(Ginkgo biloba L.)的根、茎、叶中共分离得到18株内生真菌,用显色反应、薄层层析(TLC)及紫外-可见分光光度法对发酵提取液进行分析并筛选得到一株产黄酮类物质的内生真菌(编号ROOT3.1),经形态学和分子生物学初步鉴定其为镰孢属黄色镰孢菌(Fusarium culmorum).对此菌株发酵液中的黄酮含量进行了测定,其含量为0.017 2 mg·mL-1.     

虎杖内生真菌产白藜芦醇苷菌株的分离鉴定

利用组织培养法对秦巴山区虎杖进行内生真菌分离,通过内生真菌的液体发酵,对发酵液和菌丝的乙酸乙酯萃取液进行HPLC分析检测,筛选出一株产白藜芦醇苷的菌株M-56,其产量达1.029 mg/L。根据该菌株的形态特征及菌丝ITS序列分析,将该株菌确定为无性型真菌丝孢纲链格孢菌Alternaria alternaria。     

产黄酮成分的绶草内生真菌的鉴定

利用薄层层析法进行的定性分析表明,分离自绶草Spiranthes sinensis的3株内生真菌产生了黄酮类化合物,在形态学鉴定的同时,扩增、测序了3株内生真菌的rDNA的ITS序列,利用MEGA4.0软件和邻接法（Neighbour-joining methods）进行了聚类分析,菌株S-1鉴定为不等弯孢Curvularia inaequalis,S-2和S-3鉴定为嗜松青霉Penicillium pinophilum。     

山苍子叶内生真菌的纯化与鉴定

本研究选用PDA培养基,通过组织块分离法从山苍子叶中分离得到两株内生真菌。对照真菌鉴定手册,根据菌株和菌丝体形态学特征,并给合ITS区段的碱基序列分析,鉴定两株内生分别属于顶孢霉属和芒果球座菌属。     

刺五加鲨烯合酶基因cDNA的克隆与序列分析

采用改良的异硫氰酸胍法提取刺五加总RNA,逆转录为cDNA,根据已报道的人参鲨烯合酶基因(squalene synthase gene,SS) cDNA序列设计引物,利用RT-PCR法克隆刺五加SS基因的cDNA序列.克隆得到长度为1258 bp的刺五加SS基因cDNA序列,开放阅读框全长1248 bp,编码415个氨基酸残基,GenBank登录号为HQ456918,与人参的SS1、SS2和SS3氨基酸序列一致性分别为91.73％、97.59％和96.63％.首次分离并报道了刺五加SS基因cDNA序列,为刺五加苷生物合成中关键酶的表达分析及调控机理研究提供参考.     

The photolyase gene from the plant pathogen Fusarium oxysporum f. sp. lycopersici is induced by visible light and alpha-tomatine from tomato plant

Survival of irradiated spores from Fusarium oxysporum with ultraviolet radiation (UV) was increased following exposition to visible light, indicating that this phytopathogenic fungus has a mechanism of photoreactivation able to counteract the lethal effects of UV. A genomic sequence containing the complete photolyase gene (phr1) from F. oxysporum was isolated by heterologous hybridisation with the Neurospora crassa photolyase gene. The F. oxysporum phr1 cDNA was isolated and expressed in a photolyase deficient Escherichia coli strain. The complementation of the photoreactivation deficiency of this E. coli mutant by phr1 cDNA demonstrated that the photolyase gene from F. oxysporum encodes a functional protein. The F. oxysporum PHR1 protein has a domain characteristic of photolyases from fungi (Trichoderma harziaium, N. crassa, Magnaporthe grisea, Saccharomyces cerevisiae) to bacteria (E. coli), and clusters in the photolyases phylogenetic tree with fungal photolyases. The F. oxysporum phr1 gene was inducible by visible light. The phr1 expression was also detected in presence of alpha-tomatine, a glycoalkaloid from tomato damaging cell membranes, suggesting that phr1 is induced by this cellular stress.     

Isolation of Fusarium oxysporum with Potential for Biocontrol of the Witchweed (Striga hermonthica) in the Nigerian Savanna

A fungus isolated from wilted Striga hermonthica plants was identified as Fusarium oxysporum. A comparison of this isolate (PSM-197) with four other Fusarium spp. for control of S. hermonthica using conidial foliar sprays, showed that it was the most pathogenic and virulent. The isolate, grown on sorghum grain and incorporated into soil, completely inhibited the emergence of S. hermonthica compared with other substrates.     

The chromatographic co-elution of dihydrodehydrodiconiferyl alcohol monopyranose with eleutheroside E in Eleutherococcus senticosus: implications for eleutheroside E assays

During a quantification assay of the constituents of Eleutherococcus senticosus by reverse-phase HPLC using acetonitrile:water gradient elution, it was observed that a recently reported component, dihydrodehydrodiconiferyl alcohol monopyranose, co-eluted with eleutheroside E. The implications of this finding for researchers and the herbal medicine industry are discussed. C     

六种藜科植物提取物对植物病原菌的抑制活性

研究了角果藜（ Ceratocarpus arenarius）、盐穗木（ Halostachys caspica）、里海盐爪爪（ Kalidium caspicum） 、叉毛蓬（ Petrosimonia sibirica ）、盐角草（ Salicornia europaea ）和小叶碱蓬 （ Suaeda microphylla ）等六种新疆藜科植物提取物及其不同极性萃取部分对根癌土壤杆菌（Agrobacterium tumefaciens ）、黄瓜角斑病菌（P Pseudomonas lachrymarts）、番茄疮痂病菌（ Xanthomonas vesicatoria ） 等植物病原细菌以及杨树溃疡病菌（ Botryosphaeria dothidea ）、棉花枯萎病菌（Fusarium oxysporum f. sp. vasinfectum ）、稻纹枯病菌（Rhizoctonia solani）等病原真菌的抑制活性。结果显示角果藜、叉毛蓬和盐角草乙醇粗提物表现出一定抗细菌活性,其中以叉毛蓬和盐角草提取物对黄瓜角斑病菌的抑制活性最强。多数植物提取物及其不同极性萃取部分对杨树溃疡病菌表现出强的抑制活性。抗真菌活性成分主要存在于供试植物的石油醚、氯仿和正丁醇萃取部分中,提示活性成分为极性中等的化合物。角果藜和盐角草乙醇粗提物及其不同极性萃取部分对供试真菌有较好的抑制活性。     

Epicoccum nigrum for biocontrol agents in vitro of plant fungal pathogens

Epicoccum nigrum strains were evaluated in vitro as potential biological agents for control of the growth of Fusarium avenaceum, F. graminearum, F. oxysporum and Botrytis cinerea. Individual biotic effects of five strains of E. nigrum on the various fungi were determined using the biotic series method elaborated for fungi, on potato dextrose agar (PDA - Difco) medium. Our results show that E. nigrum strains limited the growth of all isolates of Fusarium spp., but not of those of Botrytis cinerea.     

Volatile organic compounds: a potential direct long-distance mechanism for antagonistic action of Fusarium oxysporum strain MSA 35

Fusarium oxysporum MSA 35 [wild-type (WT) strain] is an antagonistic Fusarium that lives in association with a consortium of bacteria belonging to the genera Serratia, Achromobacter , Bacillus and Stenotrophomonas in an Italian soil suppressive to Fusarium wilt. Typing experiments and virulence tests provided evidence that the F. oxysporum isolate when cured of the bacterial symbionts [the cured (CU) form], is pathogenic, causing wilt symptoms identical to those caused by F. oxysporum f. sp. lactucae . Here, we demonstrate that small volatile organic compounds (VOCs) emitted from the WT strain negatively influence the mycelial growth of different formae speciales of F. oxysporum. Furthermore, these VOCs repress gene expression of two putative virulence genes in F. oxysporum lactucae strain Fuslat10, a fungus against which the WT strain MSA 35 has antagonistic activity. The VOC profile of the WT and CU fungus shows different compositions. Sesquiterpenes, mainly caryophyllene, were present in the headspace only of WT MSA 35. No sesquiterpenes were found in the volatiles of ectosymbiotic Serratia sp. strain DM1 and Achromobacte r sp. strain MM1. Bacterial volatiles had no effects on the growth of the different ff. spp. of F. oxysporum examined. Hyphae grown with VOC from WT F. oxysporum f. sp. lactucae strain MSA 35 were hydrophobic whereas those grown without VOCs were not, suggesting a correlation between the presence of volatiles in the atmosphere and the phenotype of the mycelium. This is the first report of VOC production by antagonistic F. oxysporum MSA 35 and their effects on pathogenic F. oxysporum. The results obtained in this work led us to propose a new potential direct long-distance mechanism for antagonism by F. oxysporum MSA 35 mediated by VOCs . Antagonism could be the consequence of both reduction of pathogen mycelial growth and inhibition of pathogen virulence gene expression.     

Phylogeny and pathogenicity of Fusarium oxysporum isolates from cottonseed imported from Australia into California for dairy cattle feed

A unique biotype of the Fusarium wilt pathogen, Fusarium oxysporum Schlecht. f.sp. vasinfectum (Atk) Sny. & Hans., found in Australia in 1993 is favored by neutral or alkaline heavy soils and does not require plant parasitic nematodes to cause disease. This makes it a threat to 4-6 million acres of USA Upland cotton ( Gossypium hirsutum L.) that is grown on heavy alkaline soil and currently is not affected by Fusarium wilt. In 2001-2002, several shiploads of live cottonseed were imported into California for dairy cattle feed. Thirteen F. oxysporum f.sp. vasinfectum isolates and four isolates of a Fusarium spp. that resembled F. oxysporum were isolated from the imported cottonseed. The isolates, designated by an AuSeed prefix, formed four vegetative compatibility groups (VCG) all of which were incompatible with tester isolates for 18 VCGs found in the USA. Isolate AuSeed14 was vegetatively compatible with the four reference isolates of Australian biotype VCG01111. Phylogenetic analyses based on EF-1α, PHO, BT, Mat1-1, and Mat1-2 gene sequences separated the 17 seed isolates into three lineages (race A, race 3, and Fusarium spp.) with AuSeed14 clustering into race 3 lineage or race A lineage depending on the genes analyzed. Indel analysis of the EF-1α gene sequences revealed a close evolutionary relationship among AuSeed14, Australian biotype reference isolates, and the four Fusarium spp. isolates. The Australian seed isolates and the four Australian biotype reference isolates caused disease with root-dip inoculation, but not with stem-puncture inoculation. Thus, they were a vascular incompetent pathotype. In contrast, USA race A lineage isolates readily colonized vascular tissue and formed a vascular competent pathotype when introduced directly into xylem vessels. The AuSeed14 isolate was as pathogenic as the Australian biotype, and it or related isolates could cause a severe Fusarium wilt problem in USA cotton fields if they become established.