Inhibitory activity against plant pathogenic fungi of extracts from Myoporum bontioides A. Gray and indentification of active ingredients

BACKGROUND: In order to understand the bioactivity of Myoporum bontioides A. Gray against plant pathogens and determine its active ingredients, the inhibitory activities of methanol extracts from M. bontioides against Fusarium oxysporum f. sp. niveum (E. F. Smith) Snyder & Hansen, Pestalotia mangiferae P. Henn., Thielaviopsis paradoxa (De Seynes) v. Hohnel, Colletotrichum musae (Berk. & M. A. Curtis) v. Arx, Alternaria alternata (Fr.) Keissler, Mycosphaerella sentina (Fr.) Schroter and Sphaceloma fawcettii Jenk. were evaluated using a growth rate method, and the active ingredient was isolated by activity-directed isolation and identified by determination and analysis of IR, (1)H NMR, (13)C NMR and mass spectra and correlative physical constants. RESULTS: The results showed that the extracts from stems and leaves of M. bontioides exhibited inhibitory activity against the seven fungi, with > 58% inhibition at 10 g L(-1) after 72 h. The active compound was isolated and identified as (-)-epingaione, and showed inhibitory activity against the above seven fungi. The inhibitory activity against P. mangiferae was the highest, with an EC(50) value of 77 mg L(-1). The EC(50) values against the other six fungi were 147-245 mg L(-1). (-)-Epingaione also inhibited spore germination of F. oxysporum f. sp. niveum, T. paradoxa and S. fawcettii.CONCLUSION: (-)-Epingaione demonstrated broad-spectrum inhibitory activity against plant pathogenic fungi and is promising for exploitation as a fungicide.     

德国小蠊肠道细菌抗真菌的初步研究

初步研究表明来自德国小蠊Blattella gemmnica Linnaeus肠道的细菌能产生广谱的抗真菌活性物质，对白色念珠菌Candlida albican、深红酵母Rhodotorula rubra（Demme）Lodder、丝核菌Rhixoctonia solani Huhn、黄萎轮枝孢菌Verticillium alboatrum Reinke、镰刀菌Fusarium solani（mart．）Sacc．、黄曲霉Aspergillus flavus Link、黑曲霉Aspergillus，niger Van Tieghem都有明显的抑制作用。推测蜚蠊肠道细菌有助其抵抗病原真菌，同时也为开发用于医药、农业的广谱抗真菌的活性物质提供新途径。     

海洋真菌的筛选及其对离体荔枝果霜霉病和炭疽病的防效

从广西北部湾红树林区的近海海底沉积物与海水样品中分离出64株海洋真菌,以荔枝霜疫霉菌Peronophythora litchii和荔枝炭疽病菌Colletotrichum gloeosporioides为拮抗对象,发现有8株菌株对病原菌菌丝生长有较强的抑制作用,其中菌株MF-3、MF-13、MF-15和MF-16的发酵滤液对荔枝霜疫霉菌的抑制效果较好,菌丝生长抑制率达到94%以上;菌株MF-3和MF-16的发酵滤液对荔枝炭疽病菌抑制效果较好,菌丝生长抑制率均在70%以上.用菌株MF-3、MF-6、MF-8、MF-13和MF-9的发酵滤液进行荔枝离体果防病保鲜试验,结果表明常温下保鲜处理2～4d后,鲜果的褐变指数小于对照组,其中菌株MF-9防褐变效果最好.在26～32℃下,用5株菌的发酵滤液分别浸泡鲜果1d后接种荔枝霜疫霉,3d后菌株MF-6、MF-8、MF-9和MF-13处理的发病率分别是68.3%、71.67%、66.18%和80%,显著低于对照组的87.2%和烯酰玛琳的84.2%(P＜0.05),能推迟鲜果晚发病1d.接种炭疽菌3d后,菌株MF-6处理的发病率仅为64.44%,与多菌灵(62.76%)和代森锌(62.54%)效果相当.形态学观察结合分子鉴定,确定菌株MF-3、MF-6、MF-8、MF-9和MF-13分别为布雷正青霉Eupenicillium brefeldianum、微紫青霉Penicillium janthinellum、阿达青霉Penicillitun adametzii、草酸青霉Penicillium oxalicum和短棒曲霉Aspergillus clavatonanicus.     

Compounds inhibitory to nematophagous fungi produced by Bacillus sp. strain H6 isolated from fungistatic soil

Soil fungistasis can cause inconsistent control by nematophagous fungi of plant-pathogenic nematodes in field situations. Recent studies have shown that production of fungistatic compounds by bacteria was the principal explanation for soil fungistasis. The culture filtrate of Bacillus sp. strain H6, a strain representative of the dominant colony types isolated from fungistatic soils, showed strong inhibitory activity against nematophagous fungi by inducing unusual swelling in the conidia and the germ tubes of nematophagous fungi, thereby preventing the fungi from proliferating. This inhibitory mechanism is novel in comparison with other known mechanisms. Antifungal activity of the culture filtrate of strain H6 was maximal after culture in Luria Bertani (LB) broth (pH 7.0) at 36°C for 36 h. The inhibitory effect of the compounds produced by Bacillus sp. strain H6 was not significantly influenced by pH, and the inhibitory compounds in the culture filtrate were thermostable. After being partially purified by thin layer chromatography (TLC) on silica gel plates, characterization by colour reactions and positive fast atom bombardment mass spectrometry indicated that the inhibitory compounds showed similarity to iturin A group compounds.     

Antiphytoviral activity of bruceine-D from Brucea javanica seeds

BACKGROUND: Brucea javanica (L.) Merr. is widely distributed throughout the southern parts of China and has been used in traditional medicine to treat a variety of diseases. The objective of the present study was to identify the active antiphytoviral compound in the seeds of B. javanica and evaluate the inhibitory activity of the compound against plant virus. RESULTS: Bioassay-guided fractionation of the most active extract from the seeds led to the isolation of an antiphytoviral compound which was identified as bruceine-D by conventional spectroscopy methods. The compound exhibited significant inhibitory activity against the infection and replication of tobacco mosaic virus (TMV), with IC(50) values of 13.98 and 7.13 mg L(-1) respectively. The compound also showed a strong inhibitory effect on the infectivity of potato virus Y (PVY) and cucumber mosaic virus (CMV). Furthermore, the compound could effectively inhibit systemic TMV infection in the host tobacco plant under glasshouse conditions.CONCLUSION: The results suggested that bruceine-D from Brucea javanica may have the potential to be used as a natural viricide, or a lead compound for new viricides.     

Note:In vitro antagonism of actinomycetes isolated from fungus-growing ants against plant pathogenic fungi

Fungus-growing ants have been found recently to be symbiotic with actinomycetes living on the ant’s cuticle; these bacteria are inhibitory to soil fungi that are detrimental to the ants’ fungus gardens. In order to investigate whether actinomycetes found on the cuticle of attine ants also had inhibitory properties against plant pathogenic fungi, we isolated 32 strains of actinomycetes from fungus-growing ants (Atta, Trachymyrmex, andCyphomyrmex), from the Mexican states of Coahuila, Nuevo León and Tamaulipas. Of the actinomycetes tested against selected plant pathogenic fungi (Alternaria solani, Aspergillus flavus, Colletotrichum lindemuthianum, Rhizoctonia solani, Sclerotium sp.) on Czapek-Dox agar medium, 13 isolates inhibited at least one of the fungi.C. lindemuthianum was inhibited by 11 actinomycetes, andRhizoctonia by three. An actinomycete strain isolated fromCyphomyrmex rimosus inhibited all five fungi tested. http://www.phytoparasitica.org posting July 30, 2008.     

臭椿抗烟草花叶病毒活性物质的提取及其初步分离

以烟草花叶病毒为供试病毒,采用生物活性跟踪的方法对臭椿抗病毒活性物质进行了提取与初步分离。结果表明,臭椿乙醇提取物、甲醇提取物和丙酮提取物均有较好的抗烟草花叶病毒作用;通过系统溶剂提取、硅胶柱层析分离和活性筛选,从臭椿乙醇提取物的氯仿极性部位获得两个抗烟草花叶病毒活性组分Fr3与Fr6;经硅胶柱层析进一步分离,氯仿/甲醇梯度洗脱,得到4种相对较纯的活性成分C1、C2、C3、C4。生测结果显示,4种活性成分对烟草花叶病毒具有一定程度的抑制作用,但效果均低于原乙醇提取物,在活体条件下对烟草花叶病毒系统侵染的防治作用不明显。     

Biological evaluation of neopeptins isolated from a Streptomyces strain

BACKGROUND: Microbial secondary metabolites are a rich source of antifungal agents and have merit as alternatives to synthetic fungicides. To develop disease control agents against powdery mildew, the lipopeptide antibiotic neopeptins were identified from the culture broth of a Streptomyces sp., and in vivo control efficacy of the compounds was evaluated on cucumber plants under glasshouse conditions. RESULTS: The Streptomyces sp. KNF2047 antagonistic against powdery mildew development in cucumber plants was isolated from a soil sample. Antifungal compounds were purified from the culture broth and identified as neopeptin A and B. In vitro microtitre assays revealed the inhibitory activities of the compounds in the range 128-512 microg mL(-1) against the mycelial growth of Alternaria mali, Botrytis cinerea, Cladosporium cucumerinum, Colletotrichum lagenarium, Didimella bryoniae and Magnaporthe grisea. Although neither compound showed remarkable in vitro antifungal activity against other plant pathogenic fungi, a mixture of neopeptins (484 mg of neopeptin A and 290 mg of neopeptin B per gram of partially purified powder) showed potent protective and curative activity against cucumber powdery mildew in vivo. The disease control activity of the neopeptins at a concentration of 2.4 mg L(-1) was 92.1%, which was similar to that of the commercial fungicide fenarimol (89.3% at 63 mg L(-1)) and that of the commercial biocontrol agent Actinovate (67.4% at 2 x 10(7) cfu L(-1)). CONCLUSION: Neopeptin mixtures isolated from Streptomyces sp. KNF2047 showed potent disease control activity against powdery mildew development on cucumber plants. .     

根结线虫拮抗放线菌的筛选及菌株HA10002的鉴定与活性物质分析

为了筛选获得对根结线虫具有拮抗能力的强活性放线菌菌株,并从菌株中发现新的杀线虫活性化合物,从海南东寨港红树林采集海泥样品9份,从五指山原始森林等地采集土壤样品51份,用平板稀释法对海泥和土壤样品进行分离;以南方根结线虫2龄幼虫为靶标线虫,采用24孔细胞培养板液体线虫法进行初筛和复筛;根据形态特征、培养特征、生理生化特征和16S rDNA序列对复筛出的活性菌株进行鉴定;菌株发酵液经溶媒萃取、硅胶柱层析、Sephadex LH 20凝胶过滤和制备薄层板层析,采用活性跟踪法从中分离纯化杀线虫活性化合物;化合物经光谱及波谱分析和文献对照,进行结构鉴定。从60份样品中分离得到356株菌落形态有差异的放线菌。初筛得到16株对根结线虫校正死亡率在80%以上的菌株,复筛获得3株线虫校正死亡率在95%以上的菌株,其中分离自海泥样品中的菌株HA10002抗线虫活性最强,发酵原液的校正死亡率达100%。HA10002经鉴定为白浅灰链霉菌,从其发酵液中分离获得2个活性化合物,其中A26 3为Nocardamine,A22 1的结构正在进一步鉴定中。上述结果表明,筛选出的放线菌HA10002能合成2种以上杀根结线虫的活性代谢产物,是一株遗传稳定且具有开发潜力的活性菌株。     

两种海洋真菌混合发酵的次级代谢产物及杀虫活性研究

为了研究海洋真菌哈茨木霉Trichoderma harzianum ABC19819和短柄青霉菌Penicillium Brefeldianum ABC190807混合发酵产生的次级代谢产物,利用硅胶柱色谱、Sephadex LH-20凝胶柱色谱和薄层色谱等多种分离纯化手段对混合发酵所产生的次级代谢产物进行分离纯化,结...     

3,4-二氯异噻唑-5-甲酰胺衍生物的合成及生物活性

异噻唑类杂环化合物具有广泛的生物活性。为了扩大先导结构的发现范围,以3,4-二氯异噻唑-5-甲酸为原料合成了10个未见文献报道的3,4-二氯异噻唑-5-甲酰胺衍生物;其结构经核磁共振氢谱、红外光谱和元素分析确证。初步生物测试结果表明,化合物Ⅲe对9种病原真菌表现出了广谱的抑菌活性,在50 μg/mL下其抑制率均超过50%;在100 μg/mL下,大部分化合物展现了较好的抗烟草花叶病毒(tobacco mosaic virus,TMV)或诱导烟草抗TMV活性,其中化合物Ⅲa和Ⅲi的诱导活性分别为41.88%和42.92%。     

内生解淀粉芽孢杆菌BEB17脂肽类和聚酮类化合物的抑菌活性分析

BEB17是本实验室从健康香蕉植株根部中分离得到的一株具有较强抑制真菌活性的内生解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。在本研究中,我们通过PCR检测多个脂肽类和聚酮类化合物的合成相关基因,并结合基质协助激光解吸附离子化-飞行时间质谱(MALDI-TOF-MS)和液相色谱-质谱联用(LC-MS)分析,发现该菌株能产生表面活性素Surfactin、泛革素Fengycin和伊枯草菌素Iturin三类脂肽类化合物以及Bacillibactin、Difficidin和Bacillaene三类聚酮类化合物。通过比较两种发酵培养基(M9和Landy培养基)和两种提取方法(酸沉淀法和大孔树脂抽提法),结果发现酸沉淀法提取较为高效,Landy培养基更易于BEB17脂肽类和聚酮类化合物的发酵。活性检测表明,两种提取方法提取的粗提物对香蕉枯萎病菌(Fusarium oxysporum f.sp.cubense race 4,Foc4)都具有较好的抑制作用,其中大孔树脂抽提法提取的物质抑制效果最好,当其浓度为300μg·m L-1时抑制率可达80.75%,说明该方法制备的粗提物的活性物质含量相对较高。进一步研究表明,该提取物能引起Foc4菌丝膨大和畸形,并抑制其孢子萌发。分析表明,该提取物对Foc4细胞膜具有破坏作用。     

拮抗内生芽孢杆菌BEB17分离鉴定及其挥发性物质抑菌活性分析

 项目组从健康香蕉植株根部中分离得到一株对多种植物病原真菌具有抑菌活性的内生细菌。结合形态特征观察、生理生化分析及分子鉴定,鉴定该菌株为解淀粉芽孢杆菌(Bacillus amyloliquefaciens),命名为BEB17。双皿对峙实验结果表明,该菌株产生的挥发性有机物(Volatile organic compounds, VOCs)可抑制多种病原真菌生长,其中对香蕉枯萎病菌(Fusarium oxysporum f. sp. cubense race 4,Foc4)抑制效果最强,抑制率达66.86%±0.90%,并能抑制其孢子萌发。用BEB17产生的挥发性物质做熏蒸处理后,Foc4菌丝发生缢缩膨大,细胞膜被破坏。采用顶空法固相微萃取结合气质联用(HS-SPME-GC-MS)对BEB17产生的挥发性有机物(VOCs)进行收集鉴定,得到10类33种单体有机物组分。     

Structure-activity relationships of carboxamide fungicides and the succinic dehydrogenase complex of Cryptococcus laurentii and Ustilago maydis

The systemic fungicide, carboxin (5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilide) and a variety of carboxamide compounds exhibit a marked specificity for Basidiomycete fungi. This unique specificity resides in the mitochondrial succinic dehydrogenase complex (SDC) of sensitive Basidiomycetes such as Ustilago maydis, the corn smut fungus. The present study examines in detail the structure-activity relationships of 93 carboxamide compounds and the SDC of two carboxin-sensitive organisms, U. maydis and a Basidiomycetous yeast, Cryptococcus laurentii. It has been possible to elucidate substantially the requirement in molecular structure needed for inhibition of the mitochondrial SDC. With few exceptions, a good correlation exists between the inhibitory activity of carboxamides towards the SDC of U. maydis and C. laurentii and the inhibition of growth of carboxamide-sensitive fungi, both in vitro and in vivo on the diseased plant. The structure-activity results were used as a basis for the synthesis of new, fungicidally-active carboxamides. The compounds found to be most active against the mycelial growth of Rhizoctonia solani were also tested on spore germination or mycelial growth of non-Basidiomycete fungi. Three carboxanilides (3-methyl-thiophene-2-carboxanilide, 3′-methyl-2-methylbenzanilide and 3′-methyl-2-ethylbenzanilide) had a fungitoxic spectrum which extended beyond Basidiomycetes. The spectrum of fungicidal activity of carboxanilides appears to be altered not only by substitution in the aniline ring, but by the nature of the ring attached to the carbonyl. No correlation was found between the inhibitory activity of oxathiins and benzanilides and their calculated partition coefficients.     

Inhibition of Septoria tritici and other phytopathogenic fungi and bacteria by Pseudomonas fluorescens and its antibiotics

Pseudomonas fluorescens strain PFM2, antagonistic to Septoria tritici , also inhibited growth of several other phytopathogenic bacteria and fungi in vitro. Three inhibitory compounds were extracted from liquid glycerol-peptone-phosphate medium in which strain PFM2 was grown for 4 weeks. The compound recovered in greatest quantity (70 mg/1) was identified as 2,4-diacetylphloroglucinol. The other two antibiotics, which were produced in small amounts (< 5 mg/l), were not identified. A similar pattern of inhibition against the different bacteria and fungi was exhibited by both 2,4-diacetylphloroglucinol and strain PFM2 when grown in a medium of the same composition as that in which the antibiotic was produced.     

Evaluation of aqueous Moringa seed extract as a seed treatment biofungicide for groundnuts

In a search for alternatives to currently used fungicides, the potential of aqueous Moringa seed extract (AMSE) as a seed treatment was evaluated. Seeds of groundnut, Arachis hypogea L cv Dakar, were soaked in AMSE at concentrations of 1, 5, 10, 15 and 20 g litre(-1) for 24 h. Comparison was made with Apron Plus (metalaxyl+carboxin+furathiocarb), until recently a recommended seed-treatment chemical, and distilled water, which was the medium for extraction of Moringa seeds. The results showed that AMSE has potential for use as a biofungicide on groundnut seeds, since all the concentrations used except 1 g litre(-1) brought about significant reduction in the incidence of fungi on the seeds, such reduction increasing as the dosage of AMSE increased. There were no significant differences in control between the highest concentration of AMSE (20 g litre(-1)) and Apron Plus at the manufacturer's recommended level. Water also produced slight reductions in the incidence of fungi, although this was not significant at P = 0.05. The sensitivity to AMSE of the fungi tested varied, Mucor sp being the most sensitive and Aspergillus niger the least, with Rhizopus stolonifer and Aspergillus flavus intermediate.     

United States Department of Agriculture-Agricultural Research Service research programs on microbes for management of plant-parasitic nematodes

Restrictions on the use of conventional nematicides have increased the need for new methods of managing plant-parasitic nematodes. Consequently, nematode-antagonistic microbes, and active compounds produced by such organisms, are being explored as potential additions to management practices. Programs in this area at the USDA Agricultural Research Service investigate applied biocontrol agents, naturally occurring beneficial soil microbes and natural compounds. Specific research topics include use of plant growth-promoting rhizobacteria and cultural practices for management of root-knot and ring nematodes, determination of management strategies that enhance activity of naturally occurring Pasteuria species (bacterial obligate parasites of nematodes), studies on interactions between biocontrol bacteria and bacterial-feeding nematodes, and screening of microbes for compounds active against plant-parasitic nematodes. Some studies involve biocontrol agents that are active against nematodes and soil-borne plant-pathogenic fungi, or combinations of beneficial bacteria and fungi, to manage a spectrum of plant diseases or to increase efficacy over a broader range of environmental conditions. Effective methods or agents identified in the research programs are investigated as additions to existing management systems for plant-parasitic nematodes.     

Potential inhibitors against <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>, produced by the fungus <Emphasis Type="Italic">Myrothecium</Emphasis> sp. associated with the marine sponge <Emphasis Type="Italic">Axinella</Emphasis> sp.

Sclerotinia sclerotiorum is a worldwide ascomycete fungal plant pathogen, which causes enormous yield losses on major economic crops such as crucifers, grain legumes and several other plant families. The objective of this research was to isolate and characterise some bioactive products from cultures of fungi associated with the marine sponge Axinella sp. In total, nine fungal isolates were obtained from the marine sponge Axinella sp. collected from the South China Sea. A group of test strains, including two G⁺ strains (Bacillus subtilis and Staphylococcus aureus), two G⁻ strains (Escherichia coli and Pseudomonas aeruginosa) and three fungi including two plant pathogenic fungi Sclerotinia sclerotiorum and Magnaporthe grisea and Saccharomyces cerevisiae, were employed as the indicator organisms for bioactivity screening. Using antagonistic tests and bioactive screening of the ethyl acetate (EtOAc) extracts of the corresponding cultures, fungal isolate JS9 showed the stronger efficacy against the test indicator strains, especially the indicator fungal pathogens. Isolate JS9 was further identified as Myrothecium sp. by a combination of morphological features and 18S rDNA BLAST on GenBank. Two macrocyclic trichothecenes, roridin A (compound 1) and roridin D (compound 2) were purified by tracking the activity of the EtOAc extract fractions and characterised with spectral analyses including MS, ¹H-NMR, ¹³C-NMR and disortionless enhancement by polarization transfer (DEPT). In vitro antifungal tests showed that the two macrocyclic trichothecenes were bioactive against S. cerevisiae, M. grisea and S. sclerotiorum with minimal inhibitory concentrations of 31.25, 125 and 31.25 μg ml⁻¹ for roridin A, and 62.5, 250 and 31.25 μg ml⁻¹ for roridin D, respectively. The present investigation demonstrated that two antifungal trichothecenes including roridin A and roridin D produced by the fungus Myrothecium sp. isolated from the marine sponge Axinella sp. could be potential inhibitors against the plant pathogen S. sclerotiorum. Lian Wu Xie and Shu Mei Jiang contributed equally to this work.     

柠檬桉内生真菌的分离及其提取物的抗细菌活性

采用组织块分离法分离柠檬桉中的内生真菌,通过菌落观察和分子生物学相结合的方法对分离到的内生真菌进行鉴定;进一步采用薄层层析-生物自显影法测定了柠檬桉内生真菌次生代谢产物的抗细菌活性。从柠檬桉中共分离鉴定得到10株内生真菌,主要分布于附球菌属Epicoccum、镰刀菌属Fusarium、炭疽菌属Colletotrichum、Rhytidhysteron、半壳霉属Leptostroma、Campylocarpon、Neofusicoccum、白腐菌属Phlebia和Dinemasporium等9个属中。抗细菌活性的测定结果表明,内生真菌Ecf-4(Rhytidhysteron sp.,MK211261)和Ecf-1(Epicoccum sp.,MK211258)表现出最强的抑菌活性,两菌对桉树青枯病菌的抑菌斑最大直径均大于10 mm;依据其R_f值的范围和抑菌斑的最大直径,内生真菌Ecf-4的抗菌活性高于Ecf-1,表现出更强的抗细菌活性,可作为候选菌株,进一步分离和鉴定其中的活性成分。     

The properties of RH 3866, a new triazole fungicide

2-Butyl-2-(4 chlorophenyl)-3-(1H-1,2,4-triazol-1-yl)propanenitrile, RH 3866, a new broad-spectrum fungicide was particularly effective against fungi in the subclass loculoascomycetidae and against powdery mildews and rusts. Some activity against fungi in the genera Fusarium, Monilinia and Rhizoctonia was also observed. The compound was translocated upwards when applied as a soil drench to control wheat-leaf rust caused by Puccinia recondita f.sp. tritici. Fenarimol-resistant Aspergillus nidulans was cross-resistant to RH 3866. Using a cell-free yeast enzyme extract RH 3866 strongly inhibited desmethyl sterol production.