山药炭疽病菌拮抗放线菌30702菌株的初步鉴定及发酵培养基优化

为了寻求一种高效、环保的防治山药炭疽病菌的新方法,采用对峙平板法从热带药用植物根际土壤中筛选到一株对山药炭疽病菌有较好拮抗作用的放线菌30702。经形态特征培养观察和16Sr RNA基因序列分析鉴定为紫黑链霉菌[Streptomyces violaceoniger]16Sr RNA基因进化分枝的放线菌。通过单因子和多因子正交实验对其发酵培养基进行了优化,通过测定菌体湿重和抑菌圈直径,应用SAS9.3软件进行分析,获得最佳发酵培养基为:可溶性淀粉25 g/L、黄豆粉20 g/L、MgSO_4·7H_2O 0.6 g/L、CaCO_30.2 g/L、初始pH为7.0,优化后菌株发酵产物对山药炭疽病菌抑菌活性提高了31.5%。     

海洋放线菌Streptomyces sp. HNWSW-49的次生代谢产物研究

对1株具有抗芒果炭疽病菌活性的海口湾海泥来源放线菌Streptomyces sp.HNWSW-49发酵液的化学成分进行研究。采用多种色谱技术对化合物进行分离纯化,以波谱数据确定化合物的结构。从来源于海口湾海泥的放线菌Streptomyces sp.HNWSW-49发酵液的乙酸乙酯提取物中分离鉴定了7个含氮化合物,分别为:环-(苯丙-丙)二肽(1),环-(亮-甘)二肽(2),环-(苯丙-缬)二肽(3),环-(羟脯-苯丙)二肽(4),N-(2-苯基)乙酰胺(5),β-腺苷(6),和2-哌啶酮(7)。所有化合物均为首次从该放线菌中分离得到,其中化合物6为首次从微生物中分离得到。      

露兜果实的抗氧化及抗菌活性研究

采用分光光度法,考察露兜果实浸膏的多酚含量及其对1,1-二苯基-2-三硝基苯肼(DPPH)自由基、2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵(ABTS)自由基和羟自由基的清除活性,并采用生物自显影技术,考察露兜果实浸膏对马铃薯炭疽病菌、草莓炭疽病菌及黄柏炭疽病菌的抑制作用。结果表明：露兜果实浸膏中多酚含量为152.6 μg/mg,对DPPH自由基、ABTS自由基和羟自由基均具有一定的清除活性,且呈现量效关系;此外,还发现其对马铃薯炭疽病菌和草莓炭疽病菌的生长均有一定的抑制作用。     

琯溪蜜柚炭疽病拮抗放线菌的筛选和鉴定

为筛选出对琯溪蜜柚炭疽病有防治效果的生防菌,以琯溪蜜柚炭疽病菌（Colletotrichum gloeosporioides）为靶标菌,采用平板对峙法进行初筛,用生长速率法和人工接种法对抑菌效果较好的菌株FX28进行抑菌谱及防治效果测定;通过形态学、生理生化特征,结合16S rDNA序列分析等方法研究其分类地位。结果表明：从25份土样中共分离到放线菌105株,对琯溪蜜柚炭疽病菌有较好拮抗作用的放线菌有16株,其中菌株FX28抑菌活性最高,其抑菌带宽度为15.3 mm,抑制率达86.4%,抑菌谱广,对琯溪蜜柚炭疽病菌、琯溪蜜柚黑点病菌、琯溪蜜柚黑斑病菌等14种供试植物病原菌均有抑制作用;其作用机制表现为菌丝分枝增多、变粗、顶端膨大等,对琯溪蜜柚炭疽病的预防效果和治疗效果分别为83.8%和71.1%。根据形态、生理生化特性及16S rDNA序列分析,初步确定菌株FX28为深红紫链霉菌（Streptomyces violaceorubidus）。     

对玉米螟具有生物活性的海洋放线菌筛选与鉴定

通过平板稀释法对来自青岛不同地区的海洋样品进行分离,共得到18株海洋放线菌。采用浸虫法测定海洋放线菌发酵液对玉米螟2龄幼虫的室内毒力,筛选得到2株活性较高的菌株YC3和SLR3,其48 h发酵液对玉米螟2龄幼虫的校正死亡率分别为86.21%和81.04%。根据形态特征、培养特征及生理生化性状将活性菌株鉴定为链霉菌属(Streptomyces)。     

西湖龙井产区茶树炭疽菌分离鉴定及其植物源抑菌剂筛选

为筛选出对西湖龙井产区茶树炭疽病菌具有较好抑菌活性的植物提取物，对该地区的茶树炭疽病菌进行了分离鉴定，采用菌丝生长速率法测定了12种植物提取物的抑菌活性，进一步采用浓度梯度试验测定了3种抑菌活性较好的植物提取物的抑菌毒力。研究结果表明，引起西湖龙井产区茶树炭疽病的主要病原为山茶炭疽菌(Colletotrichum camelliae)；在200 mg·L^-1时香荆芥酚、牛至精油和紫苏醛对该菌的抑菌活性较好，菌丝生长抑制率均在68%以上，有效中浓度(EC₅₀)分别为71.459、77.155、110.753 mg·L^-1。显微观察发现，在添加有效中浓度的香荆芥酚、牛至精油和紫苏醛的培养基上进行病原菌培养时，菌丝形态发生明显改变，呈现变形扭曲、皱缩干瘪等现象。综上所述，香荆芥酚、牛至精油和紫苏醛对山茶炭疽菌具有较强的抑菌活性，具有开发为植物源杀菌剂的应用潜力。     

茶炭疽病拮抗放线菌的分离筛选与鉴定

为获得对茶炭疽病有生防效果的拮抗放线菌,采用平板对峙法,从湖南茶园土壤分离筛选到1株对茶炭疽病菌具有显著拮抗效果的放线菌株F2,其中菌株对指示菌拮抗效果显著。经形态观察,培养性状,16S r DNA PCR扩增,初步鉴定F2菌株为链霉菌属。该研究结果为茶炭疽病的生物防治提供科学依据。     

铜绿金龟子肠道链霉菌Streptomyces sp. BCa1 菌体的抗菌成分研究

为从昆虫来源的放线菌次级代谢产物中寻找药物先导分子，对铜绿金龟子幼虫肠道链霉菌Streptomyces sp. BCa1菌丝体化学成分进行了研究。通过活性跟踪，从其菌丝体的氯仿甲醇萃取物中分离得到主要成分1。利用高分辨质谱、一维和二维核磁波谱技术，将该主要成分1鉴定为具有双重旋转对称结构的不饱和大环内酯类抗生素阿扎霉素（elaiophylin），具有显著的抗金黄色葡萄球菌活性。该类化合物为首次从昆虫来源的放线菌中发现。     

水茄提取物对香蕉和芒果炭疽病菌的活性研究

前期研究发现水茄甲醇提取物有优良的抑菌活性,为明确水茄甲醇提取物的活性分布和进一步直接开发利用作技术储备,采用生长速率法研究水茄甲醇提取物不同溶剂萃取相及水茄植株不同部位提取物对香蕉炭疽病菌和芒果炭疽病菌的抑制活性。结果表明：氯仿萃取相对2种病原菌的抑制活性显著高于其它溶剂萃取相,在5 mg/mL氯仿萃取相的抑菌率分别为64.50％、73.28％,EC50分别为2.104 9、1.642 6 mg /mL;对不同部位甲醇提取物的活性研究表明,在1 mg/mL叶部提取物对香蕉炭疽病菌和芒果炭疽病菌抑制率分别82.70％和84.41％,EC50分别为0.379 8、0.195 2 mg/mL,茎部提取物对二者的抑制率分别为68.27％和71.54％,EC50分别为0.476 4、0.431 4 mg/mL,叶部提取物对二者抑制活性最好,其次为茎部,根部提取物活性最低。     

茶树油控制香蕉采后炭疽病害的研究

为了探讨植物提取物对香蕉采后病害的防治效果,以香蕉果实为材料,采用菌丝生长速率方法测定不同剂量的茶树油(TTO)对香蕉炭疽病菌的离体抗菌活性,并用TTO熏蒸处理接种炭疽病菌的香蕉果实,测定果实的发病情况。结果显示,20～40μL TTO能够显著抑制离体炭疽病菌的菌丝生长,浓度越高,抑制效果越明显;接种炭疽病菌的果实经TTO处理后,病斑直径显著小于未处理果实。另外,TTO处理的果实在常温贮藏10 d后果实果皮颜色和硬度也显著优于未处理果实。初步研究表明TTO在控制香蕉果实采后腐烂和保鲜上具有应用可行性。     

解淀粉芽孢杆菌HAB-7对18株植物病原真菌的抑制作用

HAB-7是一株分离自豇豆根际土壤、具有较强抑菌作用的解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。研究HAB-7对18株植物病原真菌及其发酵液粗提蛋白对植物病原的抑制作用，并测定其对番茄种子的促生作用。采用平板对峙法测定其对18株植物病原真菌的抑制活性，以橡胶树炭疽菌为指示菌，对其发酵上清液经60%、80%硫酸铵盐析获得的抗菌粗蛋白进行测试；并将HAB-7菌液稀释成6个浓度梯度，分别用来对番茄种子进行促生测试。结果显示：HAB-7对18株植物病原真菌的平均抑菌率为61.03%；     

拮抗香蕉枯萎病菌的解淀粉芽孢杆菌LX1菌株的鉴定及其抗菌蛋白基因的克隆

收集海南不同盐碱地的土样进行拮抗香蕉枯萎病菌的微生物分离,对具有较强抑菌作用的菌株进行形态特征、生理生化和分子鉴定,并克隆其抗菌蛋白基因.结果分离到1株具有较强抑制香蕉枯萎病菌能力的细菌LXl,经鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens).抑菌实验发现LX1菌株的发酵上清液中粗蛋白有一定的抑菌作用,经过Sephacryl S-200HR柱层析、DEAE Sepharose Fast Flow离子交换层析分离纯化了其中抑菌作用最强的抗菌蛋白.质谱鉴定结果表明,抗菌蛋白与B.amyloliquefaciens FZB42内切葡聚糖酶同源性最高.根据质谱结果克隆了该抗菌蛋白的编码基因,该基因的核苷酸和氨基酸序列与B.amyloliquefaciens FZB42的内切葡聚糖酶的核苷酸和氨基酸序列同源性分别达99％和100％.拮抗香蕉枯萎病菌的芽孢杆菌LX1可作为潜在的防治香蕉枯萎病的的生防制剂,其抗菌蛋白基因也可通过遗传工程应用于香蕉枯萎病的防治.     

Antifungal activity of polyacetylenes isolated from Cirsium japonicum roots against various phytopathogenic fungi

Antifungal substances from a methanol extract of Cirsium japonicum roots were purified and characterized, and their antifungal activities against various plant pathogens were evaluated. Three polyacetylene substances were isolated from roots of C. japonicum using repeated column chromatography; these were identified as ciryneol A, ciryneol C and 1-heptadecene-11,13-diyne-8,9,10-triol by mass and nuclear magnetic resonance spectral analyses. In vitro antifungal activity of the three substances varied according to compound and target species. Magnaporthe oryzae, Colletotrichum coccodes, Colletotrichum acutatum, Pythium ultimum and Botrytis cinerea were relatively sensitive to the three polyacetylenes, with IC₅₀ values below 50 μg mL⁻¹. In vivo, they all showed similar and broad antifungal spectra against the seven plant diseases tested. At 500 μg mL⁻¹, all three compounds effectively suppressed the development of rice blast, rice sheath blight, tomato late blight, wheat leaf rust and red pepper anthracnose, with control values over 90%. They were highly active especially against wheat leaf rust; they controlled the development of this disease more than 88% even at a concentration of 125 μg mL⁻¹. In addition, ciryneol C effectively suppressed barley powdery mildew. This is the first report on the antifungal activities of the three polyacetylenes from roots of C. japonicum against plant pathogenic fungi. Polyacetylenes from roots of C. japonicum may contribute to the development of environmentally safer alternatives to protect crops from various phytopathogenic fungi.     

Metabolomic Profiling and Genomic Study of a Marine Sponge-Associated Streptomyces sp

Metabolomics and genomics are two complementary platforms for analyzing an organism as they provide information on the phenotype and genotype, respectively. These two techniques were applied in the dereplication and identification of bioactive compounds from a Streptomyces sp. (SM8) isolated from the sponge Haliclona simulans from Irish waters. Streptomyces strain SM8 extracts showed antibacterial and antifungal activity. NMR analysis of the active fractions proved that hydroxylated saturated fatty acids were the major components present in the antibacterial fractions. Antimycin compounds were initially putatively identified in the antifungal fractions using LC-Orbitrap. Their presence was later confirmed by comparison to a standard. Genomic analysis of Streptomyces sp. SM8 revealed the presence of multiple secondary metabolism gene clusters, including a gene cluster for the biosynthesis of the antifungal antimycin family of compounds. The antimycin gene cluster of Streptomyces sp. SM8 was inactivated by disruption of the antimycin biosynthesis gene antC. Extracts from this mutant strain showed loss of antimycin production and significantly less antifungal activity than the wild-type strain. Three butenolides, 4,10-dihydroxy-10-methyl-dodec-2-en-1,4-olide (1), 4,11-dihydroxy-10-methyl-dodec-2-en-1,4-olide (2), and 4-hydroxy-10-methyl-11-oxo-dodec-2-en-1,4-olide (3) that had previously been reported from marine Streptomyces species were also isolated from SM8. Comparison of the extracts of Streptomyces strain SM8 and its host sponge, H. simulans, using LC-Orbitrap revealed the presence of metabolites common to both extracts, providing direct evidence linking sponge metabolites to a specific microbial symbiont.     

Antifungal textile dyeing with<Emphasis Type="Italic">Mahonia napaulensis D.C.</Emphasis> leaves extract based on its antifungal activity

The antifungal activity of the methanolic extract ofMahonia napaulensis D.C. or Taming (local name) leaves was evaluated with four species of common pathogenic fungi e.g.Colletotrichum capsici (MTCC No. 2071),Leptosphaerulin trifoli (MTCC No. 2328),Alternaria brassicicola (MTCC No. 2102) andHelminthosporium solani (MTCC No. 2075). The present investigation also aims at developing an eco friendly natural antifungal finish from plant extract ofMahonia for textile application. The antifungal textile dyeing was also carried out with aqueous extract of stem and leaves ofMahonia and the dyed fabric was tested against fungal speciesTrichoderma for its antifungal activity in vitro.Mahonia extract showed substantial antifungal activity of 83.33% forLeptosphaerulin trifoli andAlternaria brassicicola by 80 ppm dose in 24 hours and 46% antifungal activity inMahonia dyed pieces in broth againstTrichoderma.     

一株木薯拮抗内生细菌CEB33的鉴定及田间防效初步测定

以木薯细菌性萎蔫病病菌为靶标,从健康木薯组织中分离获得一株有较好抑菌作用的内生细菌。生理生化测定、形态观察及16S rDNA序列分析表明其为类芽孢杆菌属。拮抗试验表明该菌株对木薯细菌性萎蔫病菌、疫霉根腐病菌、棒孢霉叶斑病菌和胶孢炭疽病菌均有较强的抑制作用。田间防控试验表明该菌株对木薯细菌性萎蔫病有较好的防治效果,具有良好的开发前景。     

128种南药植物提取物对6种病原菌的生长抑制作用

以水稻稻瘟病菌(Pyricularia oryzae Cav.)、香蕉枯萎病菌[(Fusarium oxysporum sp.cubense(E.F.Smith)Syn.et Han)]、香蕉炭疽病菌[Colletotrichum musae(Berk&Curt)Arx]、杧果炭疽病菌(Colletotrichum gloeosporioides PenZ)、辣椒炭疽病菌[Colletotrichum capsici(syd.)Butl.]和西瓜枯萎病菌(Fusarium oxysporum f.sp.niveum)6种病原菌为供试菌种,对47科128种南药植物的丙酮提取物进行室内抑菌活性试验。结果表明:有36种植物提取物对6种病原菌菌丝生长抑制率在60%以上;22种植物提取物对至少4种病原菌菌丝生长抑制率在80%以上;经方差分析,荜茇、艾纳香、白背叶、香茅、阴香、九里香、花椒、高良姜、益智、山柰、姜等11种植物提取物的抑菌效果与化学农药0.5mg/mL施保功的抑菌效果差异不显著。      

A Thermotolerant Marine Bacillus amyloliquefaciens S185 Producing Iturin A5 for Antifungal Activity against Fusarium oxysporum f. sp. cubense

Fusarium wilt of banana (also known as Panama disease), is a severe fungal disease caused by soil-borne Fusarium oxysporum f. sp. cubense (Foc). In recent years, biocontrol strategies using antifungal microorganisms from various niches and their related bioactive compounds have been used to prevent and control Panama disease. Here, a thermotolerant marine strain S185 was identified as Bacillus amyloliquefaciens, displaying strong antifungal activity against Foc. The strain S185 possesses multiple plant growth-promoting (PGP) and biocontrol utility properties, such as producing indole acetic acid (IAA) and ammonia, assimilating various carbon sources, tolerating pH of 4 to 9, temperature of 20 to 50 °C, and salt stress of 1 to 5%. Inoculation of S185 colonized the banana plants effectively and was mainly located in leaf and root tissues. To further investigate the antifungal components, compounds were extracted, fractionated, and purified. One compound, inhibiting Foc with minimum inhibitory concentrations (MICs) of 25 μg/disk, was identified as iturin A5 by high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) and nuclear magnetic resonance (NMR). The isolated iturin, A5, resulted in severe morphological changes during spore germination and hyphae growth of Foc. These results specify that B. amyloliquefaciens S185 plays a key role in preventing the Foc pathogen by producing the antifungal compound iturin A5, and possesses potential as a cost-effective and sustainable biocontrol strain for Panama disease in the future. This is the first report of isolation of the antifungal compound iturin A5 from thermotolerant marine B. amyloliquefaciens S185.     

In vitro activity of eighteen essential oils and some major components against common postharvest fungal pathogens of fruit

The development of natural crop protective products as alternatives to synthetic fungicides is currently in the spotlight. In vitro experiments are valuable precursors to more costly in vivo trials, allowing the identification of effective essential oils and establishing the concentrations required for inhibition of a specific, or spectrum of decay pathogens. In this study, the antifungal properties of eighteen essential oils were evaluated in vitro by addition to the fungal growth medium of five pathogens (Lasiodiplodia theobromae, Colletotrichum gloeosporioides, Alternaria citrii, Botrytis cinerea and Penicillium digitatum) isolated from mango, avocado, citrus, grapes and cactus pear. The inhibitory properties of some of the major compounds of the oils, identified and quantified by gas chromatography-mass spectroscopy and gas chromatography-flame ionization detection were also determined. Most of the oils were selected on the basis of commercial availability and for containing a predominant compound. Visual inspection of fungal growth was done and the lowest concentration where fungal growth was completely inhibited on all replicates was recorded. Thyme oil proved to be the most effective inhibitor, totally inhibiting all of the pathogens tested at concentrations of 1000 μl/l and lower, with the exception of a resistant Penicillium strain. Cinnamon oil, rich in eugenol (81.2%), demonstrated good fungicide potential, while the carvone-rich oils displayed promising activity against the citrus pathogens. Oils characterized by high concentrations of S-carvone were less effective than those containing the R-enantiomer. Essential oil of Lippia citriodora was active against all of the pathogens, excluding L. theobromae from avocado. These essential oils, applied alone or in combination, are good candidates for further in vivo testing and for investigations concerning their modes of action.     

Dereplication Strategies for Targeted Isolation of New Antitrypanosomal Actinosporins A and B from a Marine Sponge Associated-Actinokineospora sp. EG49

High resolution Fourier transform mass spectrometry (HRFTMS) and nuclear magnetic resonance (NMR) spectroscopy were employed as complementary metabolomic tools to dereplicate the chemical profile of the new and antitrypanosomally active sponge-associated bacterium Actinokineospora sp. EG49 extract. Principal Component (PCA), hierarchical clustering (HCA), and orthogonal partial least square-discriminant analysis (OPLS-DA) were used to evaluate the HRFTMS and NMR data of crude extracts from four different fermentation approaches. Statistical analysis identified the best culture one-strain-many-compounds (OSMAC) condition and extraction procedure, which was used for the isolation of novel bioactive metabolites. As a result, two new O-glycosylated angucyclines, named actinosporins A (1) and B (2), were isolated from the broth culture of Actinokineospora sp. strain EG49, which was cultivated from the Red Sea sponge Spheciospongia vagabunda. The structures of actinosporins A and B were determined by 1D- and 2D-NMR techniques, as well as high resolution tandem mass spectrometry. Testing for antiparasitic properties showed that actinosporin A exhibited activity against Trypanosoma brucei brucei with an IC₅₀ value of 15 µM; however no activity was detected against Leishmania major and Plasmodium falciparum, therefore suggesting its selectivity against the parasite Trypanosoma brucei brucei; the causative agent of sleeping sickness.