应用反向线点杂交技术鉴定临床常见曲霉属和毛霉目真菌

目的应用反向线点杂交技术(reverse line blot hybridization,RLB)快速鉴定临床常见的曲霉属和毛霉目真菌。方法收集我院真菌和真菌病研究中心保存的5种曲霉菌(烟曲霉、黄曲霉、黑曲霉、土曲霉、构巢曲霉)和7种毛霉目真菌(冻土毛霉菌、总状毛霉菌、卷枝毛霉菌、少根根霉、小孢根霉、微小根毛霉、伞状犁头霉),共计98株菌株。利用真菌通用引物ITS1和ITS4对菌株进行PCR扩增,用12个真菌种特异性探针与扩增后产物进行反向线点杂交。将RLB结果与真菌传统形态学鉴定结果、ITS区DNA测序结果进行比较。结果 RLB可以正确鉴定98株实验菌株,与形态学方法和ITS区测序方法鉴定结果100%一致,种特异性探针之间未见交叉杂交,显示出该方法的高度敏感性和特异性。8株阴性对照菌株(白念珠菌、茄病镰刀菌、尖端赛多孢、马尔尼菲青霉、疣状瓶霉、棒曲霉、日本曲霉以及雅致小克银汉霉),使用RLB方法无法鉴定。通过烟曲霉基因组DNA浓度10倍倍比稀释法验证RLB的敏感性为1.8×10-3 ng/μL。结论 RLB技术为实验室早期快速诊断、鉴定临床常见的曲霉属和毛霉目真菌提供参考。     

小麦矮腥黑穗菌差异片段筛选与分子检测体系的建立

采取随机扩增DNA多态性(Random amplified polymorphic DNA,RAPD)引物介导的半特异PCR技术(RAPD primer mediated hemi-specific PCR,RM-PCR),在从不同地域征集的18个小麦矮腥黑穗菌(Tilletia controversa Kühn,TCK)菌株和29个小麦网腥黑穗病菌(Tilletia caries(DC)Tul,TCT)菌株的总基因组DNA中筛选鉴定出TCK独有的大小为1322bp差异基因组片段。根据该片段序列设计筛选出2对特异性引物CQUTCK2/CQUTCK3和CQUTCK4/CQUTCK5,均可以从18个TCK菌株的菌丝体和冬孢子DNA中稳定地扩增出747bp和200bp的单一靶带DNA,而在29个TCT菌株的菌丝体或冬孢子DNA均无任何扩增产物。以腥黑穗菌属通用引物对CQUK6/CQUK7为内置对照,可以确定被检样品是否含PCR抑制物质进而判断检测体系是否正确,同时有效地排除样品检测结果的假阳性和假阴性。采用建立的TCK特异PCR检测技术体系,实现简单而快速地鉴定小麦矮腥黑穗菌冬孢子或罹病小麦组织中侵染菌丝体的目的。     

PCR快速鉴定鼠疫耶尔森氏菌

建立一种简便、快速、特异的PCR检测方法,用于鼠疫耶尔森氏菌的快速鉴定。针对鼠疫耶尔森氏菌特异的一段染色体序列3a设计引物,扩增-276bp片段的鼠疫标识序列。应用该PCR反应体系,对我国17个生态型及1个待定的生态型共计275株鼠疫耶尔森氏菌及48株相关菌株的PCR扩增结果表明,实验菌株均扩增出预期的276bp片段产物带,48株相关菌株均阴性,其检测灵敏度为100pg DNA。说明该方法用于鼠疫耶尔森氏菌的检测鉴定简便、快捷,具有很高的特异性和敏感性。     

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)。     

云南八角莲内生真菌分离、鉴定的初步探索

通过对云南八角莲(D.aurantiocaulis)内生真菌的分离、鉴定,获得87株内生真菌,并对87株进行分离、鉴定发现内生真菌在宿主植物中具有明显的多样性,无孢类群(22株)是优势菌,其次为镰孢菌属(10株)毛壳菌属(10株)、青霉属(9株)、小齿梗霉属(7株)、丛梗孢属(4株),首次报道云南八角莲内生真菌的类群和区系特点.     

食线虫真菌28S rDNA PCR扩增片段的RFLPs分析*

本文对16个食线虫真菌(节丛孢属、隔指孢属和单顶孢属)菌株和3个其他相关丝孢菌(顶辐孢属和单端孢属)菌株的28S rDNA 片段进行了扩增, 并用限制性内切酶Sau3A I, Msp I, Rsa I and Hae III对PCR产物进行了消化。采用UPGMA法对 PCR 产物的限制性图谱特征进行了聚类分析。结果表明,捕食性真菌的聚类群与捕食器官类型相对应,顶辐孢属和单端孢属与捕食性真菌的遗传距离较远。该结果与rDNA 的ITS区间、5.8S和18S的序列分析结果一致。     

食线虫真菌28S rDNA PCR扩增片段的RFLPs分析

本文对16个食线虫真菌(节丛孢属、隔指孢属和单顶孢属)菌株和3个其他相关丝孢菌(顶辐孢属和单端孢属)菌株的28S rDNA 片段进行了扩增, 并用限制性内切酶Sau3A I, Msp I, Rsa I and Hae III对PCR产物进行了消化。采用UPGMA法对 PCR 产物的限制性图谱特征进行了聚类分析。结果表明,捕食性真菌的聚类群与捕食器官类型相对应,顶辐孢属和单端孢属与捕食性真菌的遗传距离较远。该结果与rDNA 的ITS区间、5.8S和18S的序列分析结果一致。     

食线虫真菌28S rDNA PCR扩增片段的RFLPs分析

本文对16个食线虫真菌（节丛孢属,隔指孢属和单顶孢属）菌株和3个其他相关丝孢菌（顶辐孢属和单端孢属）菌株的28S rDNA片段进行了扩增,并用限制性内切酶Sau3 A,I,Msp I,RsaIand HaeⅢ对PCR产物进行了消化,采用UPGMA法对PCR产物的限制性图谱特征进行了聚类分析。结果表明,捕食性真菌的聚类群与捕食器官类型相对应,顶辐孢属和单端孢属与捕食性真菌的遗传距离较远。该结果与rDNA的ITS区间,5．8S和18S的序列分析结果一致。     

肺炎链球菌自溶素和溶血素基因的PCR法鉴定

肺炎链球菌是一种致病率和致死率很高的病原菌 ,若无丰富临床检测经验从临床标准中分离鉴定此菌较困难 ,本实验以寡核苷酸引物YH1 YH2、YH7 YH8分别扩增肺炎链球菌自溶素和溶血素基因的 35 4bp和 30 7bp的DNA片段 ,通过改变各种反应条件 ,建立了这两种病原因子基因的PCR检测方法。用此方法对 2 0株肺炎链球菌标准菌株及 7株对照菌株进行了鉴定 ;其扩增产物分别经限制性内切酶TthHB81和和AccI进行酶切以确认扩增产物是否正确 ;用酚 氯仿抽提纯化的全细胞DNA对PCR方法的检测灵敏度进行了测定 ;并利用此方法对 2 8份临床标本分离物进行了鉴定。结果　所有 2 0株肺炎链球菌均可分别用引物YH1 YH2、YH7 YH8扩增出 35 4bp和 30 7bp的DNA片段 ,而对照菌株均呈阴性 ;自溶素及溶血素基因的扩增产物分别经限制性内切酶TthHB81和AccI消化后产生的片段和预期的完全一致 ;两对产物均可从 10fg的全细胞DNA中扩增出目的DNA片段。所建立的两套PCR系统对 2 8份临床标本分离物进行鉴定 ,其中PCR阳性的 15份分离物经生化学特性检查被鉴定为肺炎链球菌。本试验所建立的两套PCR检测系统具有特异性强 ,灵敏度高及操作简单等优点 ,均可用于肺炎链球菌的鉴定。     

PCR法快速鉴定眼源性蜡样芽胞杆菌

目的建立一种快速、灵敏、特异的眼源性蜡样芽胞杆菌PCR检测方法,为蜡样芽胞杆菌性眼内炎患者的快速诊断提供依据。方法选择编码蜡样芽胞杆菌细胞毒素的cytK为靶基因设计引物,建立检测眼源性蜡样芽胞杆菌PCR;PCR产物用琼脂糖凝胶电泳鉴定,基因序列与GenBank比对验证扩增产物;将计数过的5株蜡样芽胞杆菌菌悬液,梯度稀释后分别提取DNA进行PCR扩增,确定检测方法的灵敏度;分别用眼部常见感染菌金黄色葡萄球菌、表皮葡萄球菌、甲型溶血性链球菌、化脓性链球菌、藤黄微球菌、铜绿假单胞菌、大肠埃希菌、普通变形杆菌和白假丝酵母菌以及枯草芽胞杆菌DNA进行特异性试验;进一步将该方法应用到人工污染致病蜡样芽胞杆菌的房水标本中,并分析其灵敏度。结果5株分离自眼内炎患者标本中的蜡样芽胞杆菌均扩增出360bp左右的DNA片段,测序结果与GenBank比对一致;该法检测在5h内完成,方法灵敏度达7．5～15．0CFU／mL;其他菌株检测未出现非特异性扩增;对模拟感染房水标本的PCR鉴定结果与分离培养对比,二者符合率为100％,模拟标本的检测灵敏度与纯菌结果一致。结论cytK基因为靶基因的PCR用于眼源性蜡样芽胞杆菌的快速检测,具有简便、快速、敏感、特异等特点,为眼内炎患者的快速诊断提供依据,在实际检验工作中有良好的应用前景。     

Assessment of diversity,distribution and antibacterial activity of endophytic fungi isolated from a medicinal plant <Emphasis Type="Italic">Adenocalymma alliaceum</Emphasis> Miers

A study was conducted for isolation, identification and antibacterial potential of fungal endophytes of Adenocalymma alliaceum Miers., (Bignoniaceae), a medicinal shrub vine plant which has long history for its usages in curing various disorders. A total of 149 isolates of endophytic fungi representing 17 fungal taxa were obtained from 270 segments (90 from each stem, leaf and petiole) of this plant. Hyphomycetes (77.85%) were the most prevalent, followed by Ascomycetes (8.05%) and Coelomycetes (4.03%) respectively. A considerable amount of fungal isolates was kept under (10.07%) Mycelia-Sterilia (MS). Leaf harboured maximum colonization of endophytic fungi (72.22%) which was greater than stem (67.78%) and petiole (25.54%). The Jc similarity index was maximum (0.619) between stem vs leaf followed by leaf vs petiole (0.571) and stem vs petiole (0.428). The dominant endophytic fungi were Alternaria alternata, Aspergillus niger, Stenella agalis, Fusarium oxysporum, Curvularia lunata and Fusarium roseum. Among twelve endophytic fungi tested for antibacterial activity, crude extracts of nine endophytic fungi (75%), showed antibacterial potential against one or more clinical human pathogens. Alternaria alternata, Curvularia lunata, Penicillium sp. and Chaetomium globosum exhibited significant antibacterial activity against 4 of 5 tested pathogens, showing broad spectrum activity. This investigation explains the value of sampling from different tissues of a host plant for the greater species diversity, and additionally, the antibacterial screening of some endophytic fungi from this specific medicinal plant may represent a unique source for many of the useful antibacterial compounds.     

Studies on seed-borne fungi of wheat in seed health testing programme

Seed mycoflora associated with wheat was studied on different media with a particular reference to Blotter and potato dextrose agar (PDA) procedures of ISTA. Seed-borne fungi, viz. Fusarium moniliforme, Rhizopus spp., Mucor spp., Alternaria alternata, Aspergillus niger, Aspergillus flavus, Curvularia lunata, Drechslera spp, Alternaria spp. and Penicillium spp., were isolated from the variety HD264. Blotter method was found to be the best media for the isolation of mycoflora whether borne externally or internally. Total number and frequency of occurrence of fungi were recorded. The effect of seed treatment with different chemicals and eco-friendly botanicals was analysed on germination, and growth, better percentage of seed germination and reduction in fungal pathogen were due to biochemical seed treatment.     

Mycolytic effect of fluorescent Pseudomonas in biocontrolling of fungal phytopathogenic Curvularia lunata,Fusarium oxysporum,Alternaria padwickii and Rhizoctonia solani

About 50 bacterial strains, each of Pseudomonas fluorescens, from different rhizospheric soil of different plants were screened for antagonistic activity against Curvularia lunata, Fusarium oxysporum, Alternaria padwickii, Rhizoctonia solani causing black kernel, kernel spotting, root rots, stackburn and sheath blight diseases of rice (Oryza sativa L.). Out of the 50 isolates, 15 isolates were found to be effective in lysing the cell wall of the above-mentioned putative pathogens tested in vitro. These Pseudomonas isolates produced mycolytic enzymes, viz. β-1,3-glucanases, β-1,4-glucanases and lipases. P. fluorescens PAK1 and PAK12 among the strains were more effective for the production of these enzymes while PAK12 produce good level of β-1,3-glucanases, β-1,4-glucanases and lipases against tested fungal pathogens. These findings demonstrate a mechanism of antagonism by P. fluorescens against different fungal plant pathogens.     

Exploring antifungal activities of acetone extract of selected Indian medicinal plants against human dermal fungal pathogens

A broad spectrum of medicinal plants was used as traditional remedies for various infectious diseases. Fungal infectious diseases have a significant impact on public health. Fungi cause more prevalent infections in immunocompromised individuals mainly patients undergoing transplantation related therapies, and malignant cancer treatments. The present study aimed to investigate the in vitro antifungal effects of the traditional medicinal plants used in India against the fungal pathogens associated with dermal infections. Indian medicinal plants (Acalypha indica, Lawsonia inermis Allium sativum and Citrus limon) extract (acetone/crude) were tested for their antifungal effects against five fungal species isolated from skin scrapings of fungal infected patients were identified as including Alternaria spp., Curvularia spp., Fusarium spp., Trichophyton spp. and Geotrichum spp. using well diffusion test and the broth micro dilution method. All plant extracts have shown to have antifungal efficacy against dermal pathogens. Particularly, Allium sativum extract revealed a strong antifungal effect against all fungal isolates with the minimum fungicidal concentration (MFC) of 50–100 μg/mL. Strong antifungal activity against Curvularia spp., Trichophyton spp., and Geotrichum spp. was also observed for the extracts of Acalypha indica, and Lawsonia inermis with MFCs of 50–800 μg/mL respectively. The extracts of Citrus limon showed an effective antifungal activity against most of the fungal strains tested with the MFCs of 50–800 μg/mL. Our research demonstrated the strong evidence of conventional plants extracts against clinical fungal pathogens with the most promising option of employing natural-drugs for the treatment of skin infections. Furthermore, in-depth analysis of identifying the compounds responsible for the antifungal activity that could offer alternatives way to develop new natural antifungal therapeutics for combating resistant recurrent infections.     

Season and Tissue Type Affect Fungal Endophyte Communities of the Indian Medicinal Plant Tinospora cordifolia More Strongly than Geographic Location

A total of 1,151 endophytic fungal isolates representing 29 taxa were isolated from symptom-less, surface-sterilized segments of stem, leaf, petiole, and root of Tinospora cordifolia which had been collected at three locations differing in air pollution in India (Ramnagar, Banaras Hindu University, Maruadih) during three seasons (summer, monsoon, winter). Endophytes were most abundant in leaf tissues (29.38% of all isolates), followed by stem (18.16%), petiole (10.11%), and root segments (6.27%). The frequency of colonization (CF) varied more strongly among tissue type and season than location. CF was maximal during monsoon followed by winter and minimal during summer. A species each of Guignardia and Acremonium could only be isolated from leaves, whereas all other species occurred in at least two tissue types. Penicillium spp. were dominant (12.62% of all isolates), followed by Colletotrichum spp. (11.8%), Cladosporium spp. (8.9%), Chaetomium globosum (8.1%), Curvularia spp. (7.6%), and Alternaria alternata (6.8%). Species richness, evenness, and the Shannon-Wiener diversity index followed the same pattern as the CF with the tissue type and the season having the greatest effect on these indices, suggesting that tissue type and season are more influential than geography. Dissimilarity of endophyte communities in regards to species composition was highest among seasons. Colletotrichum linicola occurred almost exclusively in winter, Fusarium oxysporum only in winter and summer but never during monsoon and Curvularia lunata only in winter and during monsoon but never in summer. Emissions of NO(2), SO(2), and suspended particulate matter were negatively correlated with the CF. Ozone did not have any effect. The frequency of most species declined with increasing pollution, but some showed an opposite trend (e.g., Aspergillus flavus). Five unnamed taxa (sterile mycelia) were identified as Aspergillus tubingensis, Colletotrichum crassipes, Botryosphaeria rhodina, Aspergillus sydowii, and Pseudofusicoccum violaceum, using molecular tools. Fifteen of the 29 endophyte taxa exhibited antibacterial activity. B. rhodina (JQ031157) and C. globosum showed activity against all bacterial human pathogens tested, with the former showing higher activity than the latter.     

Pests and Diseases of Betelvine Piper Betle and Their Natural Enemies in India

Betelvine (Piper betle) is an important cash crop in India. The cultivation of this crop is seriously threatened by several pest insects, for example the homopterans Aleurocanthus rugosa, Dialeuroides pallida, Aphis gossypii and Tricentrus gibbosulus and the thysanopterans Membrothrips indicus, Zaniothrips ricini, Mymarothrips garuda and Aroidothrips longistylus and by the plant pathogenic fungi Curvularia lunata, Phytophthora parasitica, Colletotrichum piperis and Fusarium moniliforme. The pest and pathogen species are attacked by natural enemies belonging to the Coleoptera, Diptera, Heteroptera, Hymenoptera, Aranea and Acari. While occurrence of non-acarine enemies is confined to certain seasons, the acarine enemies are associated with the insect pests and fungal pathogens throughout the year. The predatory mites Walzia indiana and Proctolaelaps pygmaeus and the mycophagous mites Unguizetes clavatus and Acarus sp. seem to have potential to control the insect pests and fungal pathogens, respectively.     

Biodegradation of chemical constituents in tasar silk cocoons after infection with fungi

Cocoons can become heavily contaminated by a large number of fungi during storage. Infection of tasar cocoons by six fungi isolated from naturally contaminated cocoons, viz. Fusarium moniliforme, Aspergillus flavus, Penicillium citrinum, Curvularia lunata, Drechslera spiciferum and Botryodiplodia theobromae exhibited a considerable loss in total, buffer-soluble protein and phenol content of the cocoons at different intervals. In all cases, maximum reduction of proteins and phenol was observed due to F. moniliforme at the end of incubation period.     

Relative inhibition of insect phenoloxidase by cyclic fungal metabolites from insect and plant pathogens

The fungal metabolite kojic acid, which is produced by Aspergillus and Penicillium species fungi that may be pathogens of both insects and plants, was a significant inhibitor of phenoloxidase of different representative beetle and caterpillar insect species. Fusaric acid and picolinic acid, produced by Fusarium spp., were also significant inhibitors of phenoloxidase, while dipicolinic acid and beauvericin were ineffective at concentrations tested. Previous reports of the ability of kojic and fusaric acid to inhibit defensive enzymes of plants suggest that these compounds may be important in allowing the producing fungi to be pathogens of both insects and plants.     

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.