一株高抗氧化活性银杏内生真菌SG0016的鉴定及其培养条件优化

对从银杏叶片中分离的1株具有高抗氧化活性的内生真菌SG0016,并对SG0016菌株进行形态学和分子生物学鉴定,以发酵液DPPH自由基清除率为指标,采用单因素和正交试验对SG0016菌株的接种量、培养温度、装液量、培养时间和碳源、氮源等培养条件进行优化,以提高其发酵液抗氧化活性。结果显示,SG0016菌为球毛壳菌(Chaetomium globosum);其发酵液抗氧化活性最佳培养条件为:接种量10%、培养温度23℃、装液量100 mL(250mL的三角瓶)、培养时间7d,葡萄糖为碳源,酵母膏为氮源。在此条件下,发酵液的DPPH自由基清除率最高,为96.17%,比优化前提高了23.6%。银杏内生真菌球毛壳菌SG0016代谢产物具有较好的抗氧化活性,为天然抗氧化剂的开发提供了新的来源。     

杜仲内生球毛壳菌的抗氧化活性研究

本文研究了分离自杜仲叶片的内生真菌球毛壳菌菌株No.173发酵液提取物的抗氧化活性,采用铁氰化钾还原力测定法、β-胡萝卜素/亚油酸模型和光照核黄素体系评价了发酵液提取物的抗氧化作用,并采用Folin-Ciocalteu法测定杜仲内生球毛壳菌发酵液提取物总多酚含量。结果表明,其抗氧化能力与Vc基本相当;清除超氧阴离子的能力优于芦丁;多酚含量为255.53±1.38mg/g,是其主要的抗氧化活性成分。     

桦褐孔菌发酵及其提取物清除自由基活性的研究

用1,1-diphenyl-2-picryhydrazyl(DPPH)酶标仪法,对桦褐孔菌发酵液的甲醇提取物的自由基清除率进行了测定,发现该提取物具有较强的清除自由基的活性;进一步用DPPH薄层试验,结果发现该菌的CHCL3提取物中主要含有两个具清除自由基活性的组分.在此基础上,以清除自由基活性为指标,对桦褐孔菌液体发酵条件进行优化,发现当培养条件为:葡萄糖20g/L、甘油6mL/L、蛋白胨15g/L、CuSO4 0.005g/L、酪氨酸0.05g/L;种龄为7d、装液量为50mL/250mL、转速为180r/min、接种量为10%时,桦褐孔菌发酵液提取物的清除自由基活性最强.     

黄花倒水莲内生真菌生物活性评价及HNLF-44菌株鉴定

为充分开发黄花倒水莲(Polygala fallax)的内生真菌资源，获得具有抗植物病原真菌、抗氧化活性的内生真菌，该文以黄花倒水莲内生真菌为研究对象，使用平板对峙法检测内生真菌对6种植物病原真菌的抑菌活性，测定内生真菌发酵液的DPPH清除自由基能力和总还原能力，评价内生真菌的抗氧化活性，并对具有强抑菌活性和抗氧化活性的菌株进行形态和ITS鉴定。结果表明：(1)黄花倒水莲内生真菌中有2株内生真菌对香蕉专化尖孢镰刀菌、柑橘树脂病菌、叶点霉菌、香蕉具条叶斑病菌、茄病镰刀菌、三七根腐病菌具有明显的抑菌活性，抑菌率在50.3%～91.4%之间，其中HNLF-5对柑橘树脂病菌的抑菌率为73.2%,HNLF-44对香蕉专化尖孢镰刀菌抑菌率为91.4%。(2)内生真菌发酵液具有良好的抗氧化活性，DPPH清除率均在80%以上，总还原能力吸光值范围为0.279 2～0.748 8。(3)HNLF-44菌株为链格孢属真菌。该研究表明，药用植物黄花倒水莲内生真菌具有较好的生物活性，为后续从黄花倒水莲内生真菌中挖掘潜在新型抑菌活性和抗氧化活性物质奠定了基础。     

疏花水柏枝内生真菌QY-1的抗氧化活性分析

以从疏花水柏枝(Myricaria laxiflora)中分离的1株内生真菌QY-1为研究对象,为分析其体内及体外的抗氧化活性,对其发酵液及其提取物的活性进行了综合评价。对nr DNA内转录间隔区进行测序鉴定。分别采用总抗氧化力试剂盒、自由基清除试剂盒和还原力测定试剂盒评价发酵液及粗提物的体外抗氧化能力活性;对发酵液提取物及其主成分进行了大肠埃希菌、人神经母瘤细胞SH-SY5Y的体内抗氧化损伤保护作用分析。结果表明QY-1是1株球毛壳菌(Chaetomium globosum),其粗提物具有很高的抗氧化活性,总抗氧化活力、自由基清除率及总还原力均接近维生素C的50%,远高于国际报道水平。且其发酵液提取物有促进细胞增殖和抗氧化保护作用,显著提高大肠埃希菌细胞和神经细胞在H2O2胁迫下的存活率。其粗提物可保护神经细胞膜的完整性,有效减低乳酸脱氢酶(LDH)的渗漏率。从粗提物中分离到一种主成分SF2,可显著抑制凋亡蛋白Caspase 3和Caspase 9的活性。结果表明内生真菌球毛壳菌QY-1不管在体内还是体外都具备较强的抗氧化能力,是一种具有潜在开发价值的天然抗氧化剂资源。     

柱孢绿僵菌产抗氧化活性成分培养条件的研究

通过液体摇瓶培养,以自由基清除率为指标,对一株柱孢绿僵菌(Metarhizium cylindrosporae)在不同液体培养条件下的抗氧化活性进行了研究,确定了其产抗氧化活性物质的最佳培养条件。结果表明最佳培养条件为:碳源为葡萄糖和麦芽糖组成的组合碳源;氮源为蚕蛹粉;无机盐及生长因子为MgSO4.7H2O、柠檬酸铵和VB;起始pH值为6.5;装液量为40mL/100mL;接种量为5%~7.5%;摇床转速为160~180r/min;培养时间为8d。正交实验结果表明最佳培养基配方为麦芽糖10g/L、蚕蛹粉10g/L、MgSO4.7H2O0.1g/L、柠檬酸铵0.4g/L、VB0.3g/L时,其次生代谢产物的抗氧化活性最强,可达82.16%。     

林下参内生球毛壳菌FS-01对人参病原真菌的抑制作用

为探究林下参内生真菌球毛壳菌(Chaetomium globosum)FS-01菌株对人参病原菌的抑菌作用,该研究在实验室条件下,测定了FS-01菌株菌丝、发酵液和孢子悬浮液对人参黑斑病菌(Alternaria panax)、人参菌核病菌(Sclerotinia schinseng)、人参灰霉病菌(Botrytis cinerea)、人参立枯病菌(Rhizoctonia solani)、人参根腐病菌(Fusarium solani)5种人参病原菌的抑制作用。结果表明:内生真菌球毛壳菌FS-01对5种病原菌均有抑制作用,其中,对人参黑斑病菌的抑制作用最高,为30.80%,其次是人参立枯病菌、人参菌核病菌、人参根腐病菌和人参灰霉病菌; 发酵液抑菌实验结果表明,在加入内生真菌球毛壳菌FS-01菌株发酵液的PDA培养基上,对人参灰霉病菌的抑制作用最高,为82.09%,其次是人参菌核病菌、人参黑斑病菌、人参立枯病菌和人参根腐病菌; 孢子抑菌实验结果表明,在加入内生真菌球毛壳菌FS-01菌株孢子悬浮液的PDA培养基上,对人参黑斑病菌的抑制作用最高,为83.72%,其次是人参灰霉病菌、人参立枯病菌、人参菌核病菌和人参根腐病菌。综上结果认为,内生真菌球毛壳菌FS-01菌株对人参病原菌均有很高的抑菌作用,可作为人参病原菌的生防菌株资源。     

牡蛎形拟层孔菌生物学特性、驯化栽培及抗氧化活性

对采自江西省萍乡市的野生牡蛎形拟层孔菌Fomitopsis ostreiformis子实体分离的菌株进行生物学特性的单因素及正交试验,在对其菌丝体最适培养条件研究结果的基础上,对其进行驯化栽培研究。通过水提醇沉法对获得的牡蛎形拟层孔菌栽培子实体进行粗多糖提取,并通过测定其粗多糖对DPPH自由基和羟自由基的清除率,讨论该菌体外抗氧化活性。结果显示：牡蛎形拟层孔菌的最适生长碳源与氮源分别为葡萄糖和酵母浸粉,最适生长pH为5.0-6.0,最适生长温度为35℃,温度因素对其菌丝体生长的影响最大;栽培种接种后,60d左右可形成成熟的子实体;其栽培子实体粗多糖的提取率为10.88%,提取粗多糖中总糖含量为23.87%;粗多糖对DPPH自由基和羟自由基均具有较强的清除率,且均存在一定的量效关系,表明该菌具有较高的抗氧化应用潜力。     

蒺藜内生真菌抗氧化活性菌株的筛选

为研究21株蒺藜内生真菌的抗氧化活性及筛选出一株抗氧化活性较好的菌株,本实验首先以总抗氧化能力为指标评价PDB培养基和察氏培养基发酵条件下所有菌株的抗氧化活性,选出总抗氧化能力都较强的前9株菌株测定其对DPPH·自由基、羟自由基和超氧阴离子自由基的清除作用,发现JL13、JL14和JL17菌株发酵产物的清除效果最为明显,因此对三株菌株发酵产物乙酸乙酯萃取部位清除自由基的活性进一步评价,结果显示:JL13菌株发酵产物清除DPPH·自由基、羟自由基和超氧阴离子自由基的能力都是最强的,ECs0依次为149.67、439.91和514.77μg/mL.该结果表明,蒺藜内生真菌具有较好的抗氧化潜力,具有一定的开发利用价值,尤其是JL13菌株,可以作为进一步实验研究的对象.     

核桃内生真菌抗氧化菌株的筛选及其代谢产物活性研究

本试验以29株核桃内生真菌为研究对象,采用总还原力(FRAP法)及DPPH·清除试验评价其代谢产物的抗氧化能力,从中筛选出高活性菌株,并对该菌株发酵液进行浓缩萃取,研究各萃取物的抗氧化活性。结果表明,供试29株菌的代谢产物都表现出一定的抗氧化活性,其中菌株LTS-6-6(Pyrenochaeta)为高活性菌株。该菌株发酵液乙酸乙酯萃取物(EAE)显示了较强的抗氧化活性,对DPPH·的EC_(50)为7μg/mL,总还原力为527.59±10.66 mg V_C/g,总酚和总黄酮含量分别达到641.14±7.50 mg没食子酸/g、100.30±8.44 mg芦丁/g。该结果表明,核桃内生真菌代谢产物具有一定的抗氧化活性,是寻找天然抗氧化活性物质的良好资源。     

Tracking down the "head blob": comparative analysis of wingless expression in the developing insect procephalon reveals progressive reduction of embryonic visual system patterning in higher insects

The evolution of larval head morphology in holometabolous insects is characterized by reduction of antennal appendages and the visual system components. Little insight has been gained into molecular developmental changes underlying this morphological diversification. Here we compare the expression of the segment polarity gene wingless (wg) in the pregnathal head of fruit fly, flour beetle and grasshopper embryos. We provide evidence that wg activity contributes to segment border formation, and, subsequently, the separation of the visual system and protocerebrum anlagen in the anterior procephalon. In directly developing insects like grasshopper, seven expression domains are formed during this process. The activation of four of these, which correspond to polar expression pairs in the optic lobe anlagen and the protocerebral ectoderm, has shifted to postembryonic stages in flour beetle and Drosophila. The remaining three domains map to the protocerebral neuroectoderm, and form by disintegration of a large precursor domain in flour beetle and grasshopper. In Drosophila, the precursor domain remains intact, constituting the previously described “head blob”. These data document major changes in the expression of an early patterning gene correlated with the dramatic evolution of embryonic visual system development in the Holometabola.     

Phylogeny of cardinalfishes (Teleostei: Gobiiformes: Apogonidae) and the evolution of visceral bioluminescence

The cardinalfishes (Apogonidae) are a diverse clade of small, mostly reef-dwelling fishes, for which a variety of morphological data have not yielded a consistent phylogeny. We use DNA sequence to hypothesize phylogenetic relationships within Apogonidae and among apogonids and other acanthomorph families, to examine patterns of evolution including the distribution of a visceral bioluminescence system. In conformance with previous studies, Apogonidae is placed in a clade with Pempheridae, Kurtidae, Leiognathidae, and Gobioidei. The apogonid genus Pseudamia is recovered outside the remainder of the family, not as sister to the superficially similar genus Gymnapogon. Species sampled from the Caribbean and Western Atlantic (Phaeoptyx, Astrapogon, and some Apogon species) form a clade, as do the larger-bodied Glossamia and Cheilodipterus. Incidence of visceral bioluminescence is found scattered throughout the phylogeny, independently for each group in which it is present. Examination of the fine structure of the visceral bioluminescence system through histology shows that light organs exhibit a range of morphologies, with some composed of complex masses of tubules (Siphamia, Pempheris, Parapriacanthus) and others lacking tubules but containing chambers formed by folds of the visceral epithelium (Acropoma, Archamia, Jaydia, and Rhabdamia). Light organs in Siphamia, Acropoma, Pempheris and Parapriacanthus are distinct from but connected to the gut; those in Archamia, Jaydia, and Rhabdamia are simply portions of the intestinal tract, and are little differentiated from the surrounding tissues. The presence or absence of symbiotic luminescent bacteria does not correlate with light organ structure; the tubular light organs of Siphamia and chambered tubes of Acropoma house bacteria, those in Pempheridae and the other Apogonidae do not.     

An Evaluation of the Rust FungusGymnoconia nitensas a Potential Biological Control Agent for AlienRubusSpecies in Hawaii

The rust fungusGymnoconia nitensinfects blackberry (Rubus argutus) systemically in regions of the continental United States, producing bright yellow–orange masses of spores on newly developing floricanes during springtime. In tests to determine the suitability of this rust as a biological control agent forR. penetransin Hawaii, a species now thought to be conspecific withR. argutus,rooted cuttings of the Hawaiian plants were grown at North Carolina State University, inoculated, and observed. Other introduced weedyRubusspp. in Hawaii, includingR. ellipticus, R. rosifolius,andR. glaucus,as well as the two endemic speciesR. hawaiensisandR. macraei,also were inoculated. No species ofRubusare of commercial importance in Hawaii, but the protection of the native species, of whichR. macraeiis rare, was of utmost concern. The native Hawaiian species did not survive well in North Carolina in this study, however. Later availability of a plant pathogen containment laboratory in Hawaii enabled similar tests to be conducted at that facility. In addition to the above species,R. spectabilis(salmonberry), a species native to the Pacific Northwest with which the HawaiianRubusspp. are thought to share a common ancestor, was inoculated in Hawaii. Infection withG. nitensunder natural field conditions becomes apparent only when sporulation occurs on floricanes the second year following infection. However, experimental inoculation led to early responses of chlorotic leaf flecking and puckering, leaf and stem contortion, and stem gall formation, indicating the sensitivity ofR. penetrans(=R. argutus),R. hawaiensis,andR. macraeito this rust. Apparent systemic infection also resulted in sporulation on one plant ofR. macraei.Ability to attack the endemic species suggests thatG. nitenswould not be suitable for release in Hawaii as a biological control agent, at least on the islands with populations of the native species.     

Evaluation of the co-agglutination test in diagnosis of experimental microsporidiosis

Microsporidiosis is an emerging and opportunistic infection associated with wide range of clinical syndromes in humans. Confirmation of the presence of microsporidia in different samples is laborious, costly and often difficult. The present study was designed to evaluate the utility of the Co-agglutination test (Co-A test) for detection of urinary, fecal and circulating microsporidial antigens in experimentally infected mice. One hundred and twenty male Swiss albino mice were divided into non infected control and infected experimental groups which were further subdivided into two equal subgroups; immunosuppressed and immunocompetent. Microsporidial spores were isolated from human stools and identified to be Encephalitozoon intestinalis by the molecular methods. They were used to infect each subgroup of mice, then their urine, stools and sera were collected at the 1st, 3rd, 5th, 7th and 9th days post-infection (PI). Co-A test, using prepared hyperimmune serum, was used to detect antigens in all samples collected. The cross reactivity of microsporidial hyperimmune sera with antigens of Cyclospora cyatenensis and Cryptosporidium parvum was investigated by Co-A test. The results showed that Co-A test was effective in detecting microsporidial antigen in stool of immunosuppressed infected mice from the 1st day PI, and in urine and serum from the 3rd day PI till the end of the study. In the immunocompetent subgroup, Co-A test detected microsporidial antigens in stool, serum and urine of mice from the 1st day, 3rd day and the 5th day PI, respectively till the end of the study, without cross reactivity with C. cyatenensis or C. parvum in both subgroups. Co-A test proved to be simple and suitable tool for detecting microsporidial antigen in different specimens and did not need sophisticated equipment. It is very practical under field or rural conditions and in poorly equipped clinical laboratories.     

Molecular phylogeny based on the 16S rRNA gene of elite rhizobial strains used in Brazilian commercial inoculants

Nitrogen is often a limiting nutrient, therefore the sustainability of food crops, forages and green manure legumes is mainly associated with their ability to establish symbiotic associations with stem and root-nodulating N2-fixing rhizobia. The selection, identification and maintenance of elite strains for each host are critical. Decades of research in Brazil resulted in a list of strains officially recommended for several legumes, but their genetic diversity is poorly known. This study aimed at gaining a better understanding of phylogenetic relationships of 68 rhizobial strains recommended for 64 legumes, based on the sequencing of the 16S rRNA genes. The strains were isolated from a wide range of legumes, including all three subfamilies and 17 tribes. Nine main phylogenetic branches were defined, seven of them related to the rhizobial species: Bradyrhizobium japonicum, B. elkanii, Rhizobium tropici, R. leguminosarum, Sinorhizobium meliloti/S. fredii, Mesorhizobium ciceri/M. loti, and Azorhizobium caulinodans. However, some strains differed by up to 35 nucleotides from the type strains, which suggests that they may represent new species. Two other clusters included bacteria showing similarity with the genera Methylobacterium and Burkholderia, and amplification with primers for nifH and/or nodC regions was achieved with these strains. Host specificity of several strains was very low, as they were capable of nodulating legumes of different tribes and subfamilies. Furthermore, host specificity was not related to 16S rRNA, therefore evolution of ribosomal and symbiotic genes may have been diverse. Finally, the great diversity observed in this study emphasizes that tropics are an important reservoir of N2-fixation genes.     

Parasitoid-induced mortality of house fly pupae by Pteromalid wasps in the laboratory

House fly, Musca domestica L., pupae were exposed to six species of pteromalid parasitoids, Muscidifurax zaraptor Kogan and Legner, M. raptor Girault and Sanders, M. raptorellus Kogan and Legner, Pachycrepoideus vindemiae (Rondani), Spalangia nigroaenea Curtis, and Urolepis rufipes Ashmead. Exposures were made for 48 h at six parasitoid-to-host ratios to measure the effect of parasitoid density on parasitoid-induced mortality (PIM) of hosts (excluding mortality as measured by parasitoid emergence). PIM was evident at all parasitoid-to-host ratios for all six species. Fly eclosion declined with a corresponding increase in the parasitoid-to-host ratio; the reverse was generally true for PIM. Parasitoid emergence increased initially with a corresponding increase in the parasitoid-to-host ratio to a point (depending on the parasitoid species), but then declined. The three Muscidifurax spp. and P. vindemiae exhibited similar behavior and generally avoided previously stung hosts until ovipositional restraints broke down at the higher parasitoid-to-host ratios. S. nigroaenea and U. rufipes exhibited little ovipositional restraint, resulting in a high proportion of PIM of hosts. Understanding factors that influence PIM will provide better evaluations of field releases of parasitoids to control flies and will aid in the development of the most economic procedures for large scale rearing of pteromalid parasitoids.     

Molecular phylogeny of Phebalium (Rutaceae: Boronieae) and related genera based on the nrDNA regions ITS 1+2

Parsimony analyses of the internal transcribed spacer regions of nuclear ribosomal DNA (ITS 1 & ITS 2) for 38 taxa sampled from the Phebalium group (Rutaceae: Boronieae) and two outgroups confirm that, with the exception of Phebalium sensu stricto and Rhadinothamnus, six of the currently recognised genera within the group are monophyletic. The data indicate that Phebaliums. str. is paraphyletic with respect to Microcybe, and Rhadinothamnus is paraphyletic with respect to Chorilaena. Rhadinothamnus and Chorilaena together are the sister group to Nematolepis. Drummondita, included as an outgroup taxon, clustered within the ingroup as sister to Muiriantha and related to Asterolasia.The phylogeny suggests that the evolution of major clades within a number of these genera (e.g. Phebalium) relates to vicariance events between eastern and south-western Australia. Leionema is an eastern genus, with the most basal taxon being the morphologically distinct Leionema ellipticum from northern Queensland. Leionema also includes one species from New Zealand, but this species (as with some others) proved difficult to sequence and its phylogenetic position remains unknown. Taxonomic changes at the generic level are recommended.The authors wish to thank Paul G.Wilson, PERTH, for advice and discussion, and Paul Forster, BRI, for collecting and providing material of Leionema ellipticum. The project was supported by a Melbourne University Postgraduate Award (to BM), the Australian Biological Resources Study (ABRS), Australian Systematic Botany Society and Wolf Den (Australia) Investments.     

Removal of wastewater pathogen indicators in a constructed wetland in Leon, Spain

This paper studies the seasonal presence and removal of the pathogenous micro-organisms Escherichia coli, total coliforms (TC), Clostridium perfringens (Cp), faecal streptococci (FS), Giardia cysts, Cryptosporidium oocysts and helminth eggs, in a constructed wetland treatment system. The removal efficiency of this system with respect to the indicator micro-organisms achieved maximum values in spring and autumn at 99.9% for E. coli and TC, respectively, in winter at 97.0% for FS, in summer at 100% for Clostridium and throughout the year, also at 100%, in the case of Giardia cysts, Cryptosporidium oocysts and helminth eggs. In general, very low protozoan and helminth egg counts were found, and the system demonstrated efficient reduction of the wastewater indicator pathogens.     

Phylogeographic patterns in Leccinum sect. Scabra and the status of the arctic-alpine species L. rotundifoliae

We investigated inter- and intraspecific phylogenetic relationships in the ectomycorrhizal fungal genus Leccinum section Scabra. Species of this section are exclusively associated with Betula and occur throughout the Northern Hemisphere. We compared the phylogenetic relationships of arctic, alpine, boreal and temperate accessions of section Scabra based on DNA sequences of the single-copy nuclear gene Gapdh and the multiple-copy nuclear region 5.8S-ITS2. Exclusively arctic lineages were not detected in species that occur both in arctic-alpine or boreal regions, except in L. rotundifoliae that was restricted to cold climates. L. scabrum and L. holopus showed an intercontinental phylogeographic pattern, and L. variicolor showed a pattern unrelated to geographical distribution. Molecular clock estimates indicated that L. rotundifoliae is as old as other species in section Scabra. Individual gene trees suggest that interspecific hybridisation occurred several times in the evolution of section Scabra.     

The phylogenetic placement of the Leucogastrales, including Mycolevis siccigleba (Cribbeaceae), in the Albatrellaceae using morphological and molecular data

The morphology of many hypogeous fungi converges on a homogeneous reduced form, suggesting that disparate lineages are subject to a uniform selection pressure. The primary goal of this study was to evaluate the morphology and infer the phylogeny of the Leucogastrales with Mycolevis siccigleba using a Bayesian methodology. A comprehensive morphological assessment was used for an a priori phylogenetic inference to guide the sequencing effort. All structures except spore ornamentation pointed to the Albatrellaceae as the most likely sister taxon. Polyporoletus sublividus, a close relative of Albatrellus, produces ornamented basidiospores with a similar structure to M. siccigleba basidiospores. The ITS from 30 taxa was used for the molecular phylogenetic analysis. P. sublividus was found sister to Mycolevis. Leucophleps spinispora and L. magnata formed a group sister to the Polyporoletus/Mycolevis group, whereas Leucogaster was polyphyletic with respect to the core of the Leucogastrales and sister to A. caeruleoporus. This relationship was expected as previously undescribed chlamydospores produced by members of Albatrellus had a similar morphology to the basidiospores of L. rubescens.