Rumen Bacterial Community Transition During Adaptation to High-grain Diet

Transitional changes of the ruminal bacterial community structure in cows during the switch from roughage to high-grain diet were monitored by PCR amplification and sequencing of 16S rDNA clone libraries. In total, one hundred fifty 16S rDNA sequences of almost full-length (1.4 kb) were analysed from three libraries prepared from the rumen fluid on day 0, 3, and 28 of switch to high-grain diet. In the first library (day 0, hay diet) of 51clones, 90.2% of sequences were belonging to the low G+C Gram-positive bacteria (LGCGPB) phylum, with the minor inclusion of theCytophaga-Flavobacter-Bacteroides (CFB;3.9%), Proteobacteria (3.9%) and high G+C Gram-positive bacteria (HGCGPB;2.0%) phyla-related sequences. Six LGCGPB sequences were clustered with the well-known cellulolytics of the rumen, Ruminococcus flavefaciens and R. albus. In the second library (day 3 of high-grain diet) of 58 clones, the LGCGPB-related sequences still dominated (72.4%), albeit being represented by other species than in the first library. In particular, this library was enriched by representatives of Selenomonas-Succiniclasticum-Megasphaera group IX (17.2%), lactobacilli- (6.9%) and Butyrivibrio fibrisolvens lineage 3-related (8.6%) sequences. Other phyla were represented by CFB (22.4%) and HGCGPB (3.4%). In the third library (day 28 of high-grain diet) of 41 clones, 95% of sequences fell into the LGCGPB phylum. About half of them (46%) were clustered within theSelenomonas-Succiniclasticum-Megasphaera group in Clostridium cluster IX. No HGCGPB-related sequences were detected and CFB was represented by only a single clone. No Streptococcus bovis -related sequences were detected in any of the three clone libraries.     

广西水牛瘤胃中的细菌多样性

[目的]了解广西水牛瘤胃中细菌的组成及其可能的降解纤维素细菌的主要类群。[方法]提取水牛瘤胃内容物和高效降解滤纸的水牛瘤胃内容物的富集培养物的宏基因组DNA,以宏基因组DNA为模板,扩增16S rRNA基因序列,构建该两种样品的细菌的16S rRNA基因文库。通过对16S rRNA基因序列的分析,了解这两种样品的细菌群体种类及数量。 [结果] 水牛瘤胃内容物与其富集培养物中均主要含有LGCGPB (low G+C Gram-Positive Bacteria)、CFB (Cytophaga-Flexibacter-Bacteroides)两大类菌群和少数的螺旋体菌（Spirochaetes）,且LGCGPB所占的比例都是最高的,LGCGPB在水牛瘤胃内容物细菌中的比例为56.66%,而在富集培养物细菌中的比例升高为73.33%。在水牛瘤胃内容物中,丝状杆菌（Fibrobacteres）占3.33%,但在富集培养物中未被检测到。而在富集培养物中占13.33%的变形杆菌（Proteobacteria）,在水牛瘤胃内容物中未被检测到。本研究还发现了分类地位尚未明确的一菌群（R46）。[结论]细菌类群LGCGPB、Proteobacteria可能在水牛瘤胃中的纤维素降解过程中起重要作用。此外,水牛瘤胃中的细菌组成和牦牛、牛、羊瘤胃中的细菌组成较相似但比例有所不同。     

Effect of Plant Age on Endophytic Bacterial Diversity of Balloon Flower (Platycodon grandiflorum) Root and Their Antimicrobial Activities

Balloon flower (Platycodon grandiflorum) is widely cultivated vegetable and used as a remedy for asthma in East Asia. Experiments were conducted to isolate endophytic bacteria from 1-, 3-, and 6-year-old balloon flower roots and to analyze the enzymatic, antifungal, and anti-human pathogenic activities of the potential endophytic biocontrol agents obtained. Total 120 bacterial colonies were isolated from the interior of all balloon flower roots samples. Phylogenetic analysis based on 16S rRNA gene sequences showed that the population of ‘low G + C gram-positive bacteria’ (LGCGPB) gradually increased 60.0–80.0% from 1 to 6 years balloon flower sample. On the other hand, maximum hydrolytic enzyme activity showing endophytic bacteria was under LGCGPB, among the bacterial strains, Bacillus sp. (BF1-1 and BF3-8), Bacillus sp. (BF1-2 and BF3-5), and Bacillus sp. (BF1-3, BF3-6, and BF6-4) showed maximum enzyme activities. Besides, Bacillus licheniformis (BF3-5 and BF6-6) and Bacillus pumilus (BF6-1) showed maximum antifungal activity against Phytophthora capsici, Fusarium oxysporum, Rhizoctonia solani, and Pythium ultimum. Moreover, Bacillus licheniformis was found in 3 and 6 years balloon flower roots, but Bacillus pumilus was found only in 6 years sample. It is presumed that older balloon flower plants invite more potential antifungal endophytes for there protection from plant diseases. In addition, Bacillus sp. (BF1-2 and BF3-5) showed maximum anti-human pathogenic activity. So, plant age is presumed to influence diversity of balloon flower endophytic bacteria.     

天山大峡谷原始森林黏细菌的分离鉴定及其抗菌活性

【背景】黏细菌是一类具有多细胞群体行为特征的高等原核生物,其对植物病原真菌和细菌的捕食特性使其在植物病害防治方面具有重要的应用潜力。【目的】探究乌鲁木齐天山大峡谷原始森林可培养黏细菌的多样性并分析其抗菌活性,为发掘黏细菌生防菌株奠定基础。【方法】以天山大峡谷原始森林采集的土样和腐木为分离材料,采用兔粪诱导法和被捕食菌诱导法从中分离纯化黏细菌菌株,结合形态学观察、生理生化测定和16S rRNA基因序列分析确定其分类地位,并以6种植物病原真菌[大丽轮枝菌(Verticillium dahliae)、尖孢镰刀菌萎蔫专化型(Fusarium oxysporum f. sp. vasinfectum)、拟轮枝链孢霉(Fusarium verticillioides)、立枯丝核菌(Rhizoctonia solani)、黄色镰刀菌(Fusarium culmorum)、细极链格孢菌(Alternaria tenuissima)]和1种植物病原细菌[梨火疫病菌(Erwinia amylovora)]为靶标菌,通过平板对峙法和菌苔捕食法测定其抗菌活性。【结果】从采集的样品中分离出70株菌株,经纯化后获得36株黏细菌纯培养物。经鉴定隶属于4个属,黏球菌属(Myxococcus) 30株、孢囊杆菌属(Cystobacter) 3株、珊瑚球菌属(Corallococcus) 2株和原囊菌属(Archangium) 1株。抗菌活性分析显示,本研究获得的36株黏细菌至少对2种植物病原真菌有抗菌活性,表现出广谱的抗真菌活性,初步筛选出一株菌株NSE37-1兼具广谱和高效抗真菌活性;供试的15株黏细菌对梨火疫病菌均具有捕食活性,初步筛选出一株对梨火疫病菌具有较强捕食能力的黏细菌菌株NSE25。【结论】天山大峡谷可培养黏细菌资源比较丰富,黏球菌属是该地区可培养黏细菌菌群中的优势菌。分离纯化出的黏细菌菌株均表现出广谱的抗植物病原菌活性,具有进一步研究和开发的潜在价值。     

Phylogenetic Diversity of Aerobic Saprotrophic Bacteria Isolated from the Daqing Oil Field

A diverse and active microbial community in the stratal waters of the Daqing oil field (China), which is exploited with the use of water-flooding, was found to contain aerobic chemoheterotrophic bacteria (including hydrocarbon-oxidizing ones) and anaerobic fermentative, sulfate-reducing, and methanogenic bacteria. The aerobic bacteria were most abundant in the near-bottom zones of injection wells. Twenty pure cultures of aerobic saprotrophic bacteria were isolated from the stratal waters. Under laboratory conditions, they grew at temperatures, pH, and salinity values typical of the stratal water from which they were isolated. These isolates were found to be able to utilize crude oil and a wide range of hydrocarbons, fatty acids, and alcohols. Phylogenetic analysis carried out with the use of complete 16S rRNA sequences showed that the isolates could be divided into three major groups: gram-positive bacteria with a high and a low G+C content of DNA and gram-negative bacteria of the -subclass of the Proteobacteria. Gram-positive isolates belonged to the genera Bacillus, Brevibacillus, Rhodococcus, Dietzia, Kocuria, Gordonia, Cellulomonas, and Clavibacter. Gram-negative isolates belonged to the genera Pseudomonas and Acinetobacter. In their 16S rRNA sequences, many isolates were similar to the known microbial species and some probably represented new species.     

Natural Product‐Based Fungicides Discovery: Design,Synthesis and Antifungal Activities of Some Sarisan Analogs Containing 1,3,4‐Oxadiazole Moieties

A series of sarisan analogs containing 1,3,4‐oxadiazole moieties were synthesized by iodine‐mediated oxidative cyclization and screened in vitro for their antifungal activities at 50 μg/mL against five phytopathogenic fungi such as Valsa mali, Curvularia lunata, Alternaria alternate, Fusarium solani and Fusarium graminearum. 1,3,4‐Oxadiazole derivatives 7e , 7p , 7r , 7t and 7u exhibited potent and a broad spectrum of antifungal activities against at least three phytopathogenic fungi at the concentration of 50 μg/mL. Especially, compound 7r displayed more potent antifungal activities against five phytopathogenic fungi than the positive control hymexazol. The EC₅₀ of 7r against V. mali, C. lunata and A. alternate were 12.6, 14.5 and 17.0 μg/mL, respectively. Additionally, some interesting results of structure‐activity relationships (SARs) were also observed.     

Diversity of indigenous endophytic bacteria associated with the roots of Chinese cabbage (<Emphasis Type="Italic">Brassica campestris</Emphasis> L.) cultivars and their antagonism towards pathogens

The study aimed to reveal the diversity of endophytic bacteria in the roots of Chinese cabbage (CC) cultivated in two areas in Korea, namely, Seosang-gun (SS) and Haenam-gun (HN), and also in a transgenic plant (TP) from the laboratory. A total of 653 colonies were isolated from the interior of CC roots, comprising 118, 302, and 233 isolates from SS, HN, and TP samples, respectively. Based on 16S rRNA gene sequence analysis, the isolates belonged to four major phylogenetic groups: high-G+C Gram-positive bacteria (HGC-GPB), low-G+C Gram-positive bacteria (LGC-GPB), Proteobacteria, and Bacteriodetes. The most dominant groups in the roots of the SS, HN, and TP cultivars were LGC-GPB (48.3%), Proteobacteria (50.2%), and HGC-GPB (38.2%), respectively. Importantly, most of the isolates that produced cell-walldegrading enzymes belonged to the genus Bacillus. Bacillus sp. (HNR03, TPR06), Bacillus pumilus (SSR07, HNR11, TPR07), and Bacillus subtilis (TPR03) showed high antagonism against the tested food-borne pathogenic bacteria. In addition, Bacillus sp. (HNR03, TPR06), Bacillus pumilus (SSR07, HNR11, HNR17, TPR11), Microbacterium oxidans (SSR09, TPR04), Bacillus cereus HNR10, Pseudomonas sp. HNR13, and Bacillus subtilis (TPR02, TPR03) showed strong antagonistic activity against the fungi Phythium ultimum, Phytophthora capsici, Fusarium oxysporum, and Rhizoctonia solani. The endophytes isolated from the TP cultivar showed the strongest antagonistic reactions against pathogens. This study is the first report on endophytic bacteria from Chinese cabbage roots.     

Diversity of Culturable Bacteria Isolated from Highland Barley Cultivation Soil in Qamdo,Tibet Autonomous Region

The soil bacterial communities have been widely investigated. However, there has been little study of the bacteria in Qinghai-Tibet Plateau, especially about the culturable bacteria in highland barley cultivation soil. Here, a total of 830 individual strains were obtained at 4°C and 25°C from a highland barley cultivation soil in Qamdo, Tibet Autonomous Region, using fifteen kinds of media. Seventy-seven species were obtained, which belonged to 42 genera and four phyla; the predominant phylum was Actinobacteria (68.82%), followed by Proteobacteria (15.59%), Firmicutes (14.29%), and Bacteroidetes (1.30%). The predominant genus was Streptomyces (22.08%, 17 species), followed by Bacillus (6.49%, five species), Micromonospora (5.19%, four species), Microbacterium (5.19%, four species), and Kribbella (3.90%, three species). The most diverse isolates belonged to a high G+C Gram-positive group; in particular, the Streptomyces genus is a dominant genus in the high G+C Gram-positive group. There were 62 species and 33 genera bacteria isolated at 25°C (80.52%), 23 species, and 18 genera bacteria isolated at 4°C (29.87%). Meanwhile, only eight species and six genera bacteria could be isolated at 25°C and 4°C. Of the 77 species, six isolates related to six genera might be novel taxa. The results showed abundant bacterial species diversity in the soil sample from the Qamdo, Tibet Autonomous Region.     

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。     

The Antimicrobial Efficiency of Root Oil Against Human Pathogenic Bacteria and Phytopathogenic Fungi

The in vitro antimierobial activity of root oil of C. orchioides were studied against human pathogenic bacteria and phytopathogenic fungi by comparing the results with standard microbial susceptibility testing biodises. The oil of C. orchioides in case of bacteria shows significant activity against Bacillus anthracis, B. subtilis, Salmonella pullorum, S. newport, and Staph. aureus. While in case of fungi root oil had excellent activity against Fusarium monili forme, F. solani, Asprgullus flavus and Cladosporium and shows significant activity against all other tested fungi.     

Identification of antifungal substances secreted by Bacillus subtilis Z-14 that suppress Gaeumannomyces graminis var. tritici

Bacillus subtilis strain Z-14 has biological control activity against the take-all fungus Gaeumannomyces graminis var. tritici (Ggt). In Petri dishes, the crude extract from B. subtilis Z-14 culture filtrate reduced take-all severity in roots of wheat seedlings by 91.3% and potted plants by 69.8% compared to the Ggt-inoculated control. Treatment with the crude extract also significantly (P?<?.05) increased growth of roots’ average length, and fresh weight in comparison with those of the Ggt-inoculated control. B. subtilis Z-14 culture filtrate was relatively thermally stable with 88.2% of the antifungal activity being retained after being heated at 100°C for 30?min. Meanwhile, the antifungal activity remained almost unchanged (>95%) when the culture filtrate was exposed to a pH ranging from 3 to 8, but significantly reduced in basic conditions. This activity was not transferred to the organic solvent phase after treatment with organic extraction agents. B. subtilis Z-14 culture filtrate exhibited a broad spectrum of antifungal activities against various phytopathogenic fungi. Three homologs of iturin A (C14–16) were characterised by liquid chromatography-mass spectroscopy (LC-MS) and electrospray ionisation mass spectrometry/mass spectrometry collision-induced dissociation (ESI-MS/MS CID).     

Microbial communities of Solanum tuberosum and magainin-producing transgenic lines

Antimicrobial peptide magainin II, isolated from the skin of the African clawed toad, has shown activity in vitro against a range of micro-organisms. Transgenic potato lines expressing a synthetic magainin gene show improved resistance to the bacterial plant pathogen, Erwinia carotovora. Culturable bacterial and fungal communities associated with magainin-producing potato plants were compared with those communities from the non-transgenic parental control and with another potato cultivar. Total numbers of aerobic bacteria recovered from the leaves of the magainin-producing line, its non-transgenic parent line and an unrelated cultivar did not differ significantly. There were no detectable differences in the numbers of Gram-positive and Gram-negative bacteria, pseudomonad populations or fungi recovered from foliage from the three plant lines. Bacterial populations recovered from the roots of a magainin-expressing plant line did not differ significantly from populations recovered from the unmodified parental line. Tubers from the magainin-expressing transgenic potatoes, however, had significantly lower total numbers of bacteria than tubers produced by unmodified plants. In vitro testing of rhizosphere isolates against magainin analogues found that bacterial isolates varied in their susceptibility to the peptides. There were no significant differences in the total numbers of fungi and yeasts recovered from the various plant lines, with one exception: higher numbers of fungi were recovered from roots of magainin-expressing plants than the unmodified control plants.     

Endophytic fungi from Chilean native gymnosperms: antimicrobial activity against human and phytopathogenic fungi

Thirty-eight endophytic fungi were isolated from eight Chilean gymnosperms. Isolates were characterized and grouped according to culture characteristics, colony growth, and conidia morphology. Thirteen isolates were identified: Acremonium bacillisporum, A. bactrocephalum, A. strictum, Alternaria alternata, Aureobasidium pullulans, Chaetomium funicola, Cladosporium tenuissimum, Curvularia protuberata, C. tritici, Microsphaeropsis olivacea, Penicillium chrysogenum, P. janczewskii, and Triblidiopycnis pinastri. Malbranchea and Stegonosporium were identified at the genus level. Fourteen isolates, considered to be sterile mycelia, did not fructify in the culture medium. Crude extracts of liquid cultures from endophytes were examined for antibacterial and antifungal activity against bacteria and phytopathogenic fungi using agar diffusion. Antifungal activity against pathogenic fungi was determined by microdilution assays. Extracts of Acremonium bactrocephalum, Microsphaeropsis olivacea, and isolate E-3 inhibited growth of selected pathogenic organisms, indicating they merit further study. This is the first comparative report on the antimicrobial activity of endophytic fungi from Chilean gymnosperms.     

Characteristics and antimicrobial activity of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strains isolated from soil

Antagonistic Bacillus strains were isolated from soil and analyzed for the purpose of determining whether they could be used as natural biological agents. Primary in vitro screening for antagonism of the isolates was performed against five phytopathogenic mould fungi. Strains TS 01 and ZR 02 exhibited the most pronounced inhibitory effects. They were identified as Bacillus subtilis on the basis of their morphological, cultural and physiology-biochemical properties as well as their hierarchical cluster analysis conducted by means of computer program SPSS. The antimicrobial activity of the strains from cultural medium and sterile filtrate were determined in vitro against a great number of predominantly phytopathogenic fungi and bacteria. TS 01 and ZR 02 strains exhibited very broad and at the same time degree varying antibiotic spectra of activities against both Gram-positive and Gram-negative microorganisms. Many of them were tested against sensitivity to the antimicrobial action of B. subtilis for the very first time. B. subtilis TS 01 and ZR 02 showed highest antifungal activity (sterile zone in diameter over 37 mm) against Alternaria solani, Botrytis cinerea, Monilia linhartiana 869, Phytophthora cryptogea 759/1 and Rhizoctonia sp. The most sensitive bacterial species were found to be Pseudomonas syringae pv. tomato Ro and Xanthomonas campestris with sterile zones 48.0 and 50.0 mm in diameter, respectively. The latter draws a conclusion that the isolated and identified Bacillus subtilis strains are promising natural biocontrol agents and should be further studied and tested for control of numerous plant diseases.     

Evaluation of the Effects of Biological Preparations on Phytopathogenic Fungi Didymella applanata and Botrytis cinerea

Fungicidal and fungistatic effects of biological preparations involving bacteria of the genera Pseudomonas and Bacillusand fungi of the genusChaetomium on phytopathogenic fungi Didymella applanata and Botrytis cinereawere evaluated. All the biological preparations under study inhibited the growth of colonies of the fungi; however, the degree of the inhibition depended on the nature of each particular microorganism and the concentration of each particular preparation. A preparation containing Bacillus subtilis at a concentration of 0.2% effected maximum suppression ofB. cinerea (the diameter of the colonies decreased sevenfold). Preparations containing bacteria of the genus Pseudomonas and fungi of the genus Chaetomium were most efficient in suppressing D. applanata.Preparations containingB. subtilis and Chaetomium spp. showed promise as agents against simultaneous development of spur blight and Botrytis blight.     

Characterization of bacterial communities from activated sludge: Culture-dependent numerical identification versus in situ identification using group- and genus-specific rRNA-targeted oligonucleotide probes

The structures of bacterial communities were studied in activated sludge samples obtained from the aerobic and anaerobic zones of a wastewater treatment plant showing enhanced phosphorous removal. Samples were analyzed by in situ hybridization with oligonucleotide probes complementary to selected regions of the 16S and 23S ribosomal RNA (rRNA) characteristic for defined phylogenetic entities (genera and larger groups). The microbial community structures revealed by molecular techniques were compared with the compositions of culturable bacterial communities, obtained from the characterization of 255 isolates from tryptone-soy (TS) agar and R2A agar. These isolates were characterized by 89 physiological tests and their cellular fatty acid patterns, and identified. Culture-dependent techniques indicated the following distribution: different Aeromonas spp. (2.7–8.3% on R2A agar; 45.0–63.7% on TS agar), Acinetobacter spp. (5.4–9.0% on R2A agar; 5.0–9.1% on TS agar), Pseudomonas spp. (up to 10% on R2A agar) and Shewanella putrefaciens (up to 3.0% on R2A agar), all members of the gamma subclass of Proteobacteria, were isolated most frequently. The relatively rare isolates of the beta subclass were identified as Acidovorax spp., Alcaligenes spp., and Comamonas spp., The Gram-positive bacteria (high DNA G+C) were assigned mainly to Arthrobacter spp., Microbacterium spp., and Mycobacterium phlei. In order to assess the in situ abundance of the most frequently isolated genus, Aeromonas, two rRNA-targeted oligonucleotide probes were developed. The two gamma proteobacterial genera Aeromonas and Acinetobacter constituted less than 5% of all bacteria. In situ, Proteobacteria belonging to the beta subclass and high G+C Gram-positive bacteria were dominant. From filamentous bacteria, Sphaerotilus spp. and Leptothrix spp. could be detected occasionally. In addition, one sample contained a high proportion of the morphologically distinct filaments of Microthrix parvicella.As for the genus Acinetobacter, the relative abundance of the most frequently gamma-proteobacterial genus Aeromonas was overestimated by the intrinsic selectivity of cultivation. Cultivation on nutrient-rich medium (TS-agar) especially supported an enhanced isolation of bacteria belonging to these two genera. Correspondence to: P. Kämpfer.     

Isolation of Paenibacillus sp. and Variovorax sp. strains from decaying woods and characterization of their potential for cellulose deconstruction

Prospection of cellulose-degrading bacteria in natural environments allows the identification of novel cellulases and hemicellulases that could be useful in second-generation bioethanol production. In this work, cellulolytic bacteria were isolated from decaying native forest soils by enrichment on cellulose as sole carbon source. There was a predominance of Gram positive isolates that belonged to the phyla Proteobacteria and Firmicutes. Many primary isolates with cellulolytic activity were not pure cultures. From these consortia, isolation of pure constituents was attempted in order to test the hypothesis whether microbial consortia are needed for full degradation of complex substrates. Two isolates, CB1-2-A-5 and VG-4-A-2, were obtained as the pure constituents of CB1-2 and VG-4 consortia, respectively. Based on 16S RNA sequence, they could be classified as Variovorax paradoxus and Paenibacillus alvei. Noteworthy, only VG-4 consortium showed measurable xylan degrading capacity and signs of filter paper degradation. However, no xylan or filter paper degrading capacities were observed for the pure cultures isolated from it, suggesting that other members of this consortium were necessary for these hydrolyzing activities. Our results indicated that Paenibacillus sp. and Variovorax sp. as well as VG-4 consortium, might be a useful source of hydrolytic enzymes. Moreover, although Variovorax sp. had been previously identified in metagenomic studies of cellulolytic communities, this is the first report on the isolation and characterization of this microorganism as a cellulolytic genus.     

Biodiversity of endophytic fungi in different leaf ages of Calotropis procera and their antimicrobial activity

Calotropis procera has many important medicinal properties with proven pharmacological potential. Some of these properties may be mediated by its fungal endophytes. This study analyzed, for the first time, the community of endophytic fungi of C. procera outside its region of origin. A total of 156 fungal isolates distributed across 19 taxa were obtained from 468 fragments of C. procera leaves at different stages of maturation. The rate of endophyte colonization increased with the leaf age/development. The dominant species of endophytic fungi of C. procera introduced in Northeast Brazil were different from those found in studies on the same species and other species of the same genus in native regions. The dominant endophyte was Phaeoramularia calotropidis (63.5 %), followed by Guignardia bidwellii (21.1 %). Six isolates of endophytic fungi showed antimicrobial activity against human pathogenic micro-organisms and one plant pathogenic fungus. The antibacterial activity was more intense than the antifungal activity. The endophytic Curvularia pallescens (URM 6048) stood out inhibited Gram-positive bacteria, Staphylococcus aureus, Streptococcus pyogenes, the plant pathogenic fungus Colletotrichum dematium. Ecological and biotechnological aspects of endophytic mycota are discussed.     

Site-Directed Mutagenesis,Heterologous Expression of Cyanamide Hydratase Gene and Antimicrobial Activity of Cyanamide

Site-directed mutagenesis on a recombinant plasmid, pUC8, that contained the cah gene, was conducted and confirmed by sequence analysis. Single base substitution, G to A at nucleotide position 81 or T to C at nucleotide position 84 of cah gene does not change the amino acid sequence of cah enzyme but eliminates the HindIII site. The wild-type cah and its mutants were cloned and overexpressed in pQE-60 Escherichia coli expression system. Western blot analysis confirmed the production of 27.7-kDa cah enzyme by all the recombinants. The mutated cah gene devoid of HindIII site was used to generate a recombinant plant transformation vector (pCAMBIA-cah). Agrobacterium-mediated transformation was performed in Nicotiana tabaccum cv. Samsun plants by employing the leaf-disc method. The integration and expression of cah gene in transgenic plants were confirmed by polymerase chain reaction, Southern and Western blot analyses. Antimicrobial activity of cyanamide against phytopathogenic fungi and bacteria was determined. Cyanamide can be used as fertilizer as well as an antimicrobial salt against phytopathogenic fungi and bacteria. The present investigation reports the heterologous expression of the cah marker gene. Due to its innate ability to convert cyanamide to urea and the broad-spectrum antimicrobial activity of cyanamide, the cah gene can be used to facilitate plant growth promotion and biocontrol of phytopathogens.     

Prokaryote diversity in the rumen of yak (Bos grunniens) and Jinnan cattle (Bos taurus) estimated by 16S rDNA homology analyses

Prokaryote diversity in the rumen of yak (Bos grunniens) and Jinnan cattle (Bos taurus) was estimated by 16S rDNA homology analysis. Two rumen 16S rDNA libraries were constructed. Of the 194 clones in the library of yak rumen, the sequences were mainly clustered to two phyla, low G+C Gram-positive bacteria (LGCGPB, 54.12% total clones) and Bacteroidetes (30.93%), respectively. While in the 197 clone-library of the cattle rumen, the sequences were mainly related to three phyla, Bacteroidetes (39.59%), gamma-Proteobacteria (26.9%) and LGCGPB (22.34%), respectively. The sequence analysis indicated that more than half of the species harbored in yak rumen belonged to the not-yet-cultured groups at <90% 16S rDNA similarity levels with cultured species, while 36% 16S rDNA sequences amplified from the rumen of Jinnan cattle fell in these catalogues. By comparing the uncultured sequences in yak rumen with those in Jinnan cattle and cow, the former formed distinct clusters loosely related to the later, implying that yak rumen could harbor some special prokaryote phyla. 10.8% sequences retrieved in yak rumen were related to the known rumen fibrolytic bacterial species; however none was related to the known amylolysis species. While 4% and 17.8% sequences retrieved from Jinnan cattle rumen were related to cultured fibrolytic and amylolysis species, respectively. The bacterial structures seemed to be in accordance with the feed of the two kinds of animals. In both rumens, retrieved methanogenic Archaea-related 16S rDNA sequences were at an unreasonable low level; in addition, none sequence was related to Ruminococcus albus, a classical rumen fibrolytic species. The reason can be due to the experimental biases.