炭团菌提取物对樟子松枯梢病菌的抑菌活性及稳定性研究

采用水提和酯提2种方式提取炭团菌液体培养菌液中高效抑制樟子松枯梢病菌活性物质。采用生长速率法测定提取物对病原菌菌丝生长的抑制率,采用悬滴法测定其对病原菌孢子萌发的抑制率。炭团菌液体培养菌液乙酸乙酯提取物稳定性试验选用紫外线、温度、pH、氧化剂与还原剂以及贮存时间5个因子。结果表明:炭团菌液体培养菌液的5种提取物对樟子松枯梢病菌均有一定的抑菌活性,乙酸乙酯提取物对病原菌菌丝生长的抑制率最高,达到73.20%;乙酸乙酯提取物、超声波提取物、正丁醇提取物对病原菌孢子萌发抑制率均超过90%。乙酸乙酯提取物对自然环境下的紫外线、温度具有很高的稳定性,并且有一定的抗氧化还原能力和良好的耐热性,长期贮存不影响其抑菌活性,具有很高的开发价值和很好的应用前景。     

鹅膏菌抑真菌活性及其活性物质的提取工艺优化(英文)

通过鹅膏菌抑菌活性物质对杨树烂皮病病原菌金黄壳囊孢菌的菌丝生长及其孢子抑制效果的研究,分析鹅膏菌的抗真菌活性。将培养15 d的鹅膏菌液体发酵物分成培养液和菌丝体两部分,然后分别将培养液、菌丝体提取物和发酵液本身对金黄壳囊孢菌菌丝生长和孢子萌发抑制效果进行检测。结果表明:培养液对金黄壳囊孢菌菌丝生长的抑制率最高,为68.90%;三者对孢子萌发都有明显的抑制效果,即在处理后10 d孢子都没有萌发。菌丝体在超声波、加热和浸泡的条件下,利用乙醇,丙酮、乙酸乙酯和正丁醇进行提取,其提取物用于病原菌的抑制效果检测,结果表明:用乙醇和丙酮提取时,超声波、浸泡及加热处理,对金黄壳囊孢菌的抑制效果都与对照有显著差异(a=0.01),而正丁醇的提取物的抑制效果较差,与对照没有显著差异。通过不同提取物对金黄壳囊孢菌菌丝生长和孢子萌发的抑制效果进行分析,并对抑菌活性物质提取工艺进行筛选,得到抑菌活性物质提取的最佳方法为摇床培养,乙醇是最适提取溶剂,处理方式(超声波、浸泡或者加热)相互间差异不显著。     

黄花草木樨提取物抑菌活性初探

采用离体和活体试验方法分别测定了黄花草木樨不同溶剂提取物对12种植物病原真菌的抑菌活性.结果表明:各溶剂提取物对12种植物病原真菌均具有不同程度的抑菌活性,其中以乙酸乙酯提取物的抑菌活性最高,对油菜菌核病菌、玉米大斑病菌和白菜黑斑病菌抑制菌丝生长的EC50分别为0.62、0.83、0.64g/L,对稻瘟病菌和玉米大斑病菌抑制孢子萌发的EC50分别为0.67、0.97g/L.离体组织法测定表明其乙酸乙酯提取物对番茄灰霉病菌具有较高的保护和治疗作用,在浓度为5.0g/L时,防治效果分别为75.41%和59.18%(6d).活体试验表明乙酸乙酯提取物对小麦白粉病和小麦条锈病也有一定的保护作用,在浓度为10.0g/L时,防治效果分别为73.39%和63.27%.     

菊苣根提取物的抑菌活性研究

采用离体的试验方法测定了菊苣根的石油醚、乙酸乙酯和乙醇提取物对7种植物病原真菌和3种细菌的抑制活性。采用盆栽试验方法测定了菊苣根提取物对小麦白粉病的防治效果。结果表明,乙醇和乙酸乙酯提取物均有一定的抑制植物病原真菌和细菌活性。且乙酸乙酯提取物效果更佳。在10 g.L-1浓度下,乙酸乙酯提取物能显著抑制小麦赤霉病菌、玉米大斑病菌和烟草赤星病菌3种病原真菌菌丝的生长,抑制率均在85%以上;对小麦根腐病菌、玉米大斑病菌和烟草赤星病菌的孢子萌发抑制率也均在80%以上;对枯草芽孢杆菌和金黄色葡萄球菌的抑菌圈直径达21.01 mm和17.23 mm;对盆栽小麦白粉病的预防和治疗作用分别为50.93%和65.82%。     

天然植物提取物对茄子黄萎病菌的抑制活性

以茄子黄萎菌为供试菌种,研究了54种天然植物的乙醇提取液对茄黄萎菌的抑菌活性. 结果表明, 有15种植物提取物对茄子黄萎菌菌丝生长抑制率大于50%,其中细辛、黄连、厚朴、儿茶、苦参、甘草、蛇床子、桔梗、洋葱的提取物对供试菌菌丝生长抑制作用较强,抑制效果均在65%以上,蛇床子的抑菌率达86.84%.有16种植物提取物对病菌孢子萌发的抑制作用大于70%,其中7种植物提取物对孢子萌发的抑制率大于95%以上,细辛、黄连、厚朴基本上抑制了孢子的萌发.儿茶提取物对菌丝生长的抑制效果较差,但却显著抑制了孢子萌发,抑制率达100%.蛇床子提取物虽然抑菌效果较高,但其处理的孢子萌发率为11.3%.研究发现天然植物提取物中存在着抑制黄萎菌的活性物质,这为生物防治黄萎病和进一步研究蔬菜抗病增产理论提供了新的思路与途径.     

黄瓜枯萎病生防菌HD-087产抗菌物质条件的 优化及抑菌作用初探

【目的】优化比基尼链霉菌HD-087摇瓶发酵条件,提高菌株发酵液对黄瓜枯萎病菌HU-M的抑制率,并通过拮抗试验初步评价发酵液的抑菌作用效果。【方法】采用单因素筛选及正交试验对HD-087的发酵培养基和发酵条件进行优化。经发酵液处理后,光学显微镜观察HU-M菌丝形态和孢子萌发抑制率,测定HU-M菌丝电导率。【结果】改进的发酵培养基配方为:淀粉1.00%、黄豆粉0.80%、酵母粉0.12%、CaCO30.40%。对发酵条件的研究表明:pH为6.8,180 r/min、28°C条件下,250 mL三角瓶装液量为40 mL,接种1 mL种龄为2 d的种子,发酵5 d为最佳培养条件。抑菌结果表明,HD-087产抗菌物质能造成病原菌HU-M菌丝细胞质渗漏,菌丝畸形,分生孢子萌发受抑制,5倍发酵稀释液孢子抑制率达72.1%;除此之外还能引起菌丝电解质渗漏,造成菌丝细胞膜受损。【结论】优化后的摇瓶发酵条件能提高生防菌HD-087发酵液抑菌效果,并且发酵液可破坏细胞膜明显抑制病原菌HU-M生长,具有较大开发应用潜力。     

Endomelanconiopsis microspora发酵产物乙酸乙酯提取物对人参核盘菌的抑制机理

【背景】人参菌核病是人参的主要病害之一,严重影响人参的产量。【目的】探索白花蒲公英内生菌(Endomelanconiopsis microspora)发酵产物乙酸乙酯提取物对人参核盘菌的抑制机理。【方法】采用人参核盘菌菌丝生长和孢子萌发试验测定抑制效果;采用显微镜观察菌丝形态变化,通过电导率和核酸含量的变化测定细胞膜通透性,通过丙二醛(malondialdehyde,MDA)含量和超氧化物歧化酶(superoxide dismutase,SOD)、过氧化物酶(peroxidase,POD)和过氧化氢酶(catalase,CAT)活力的变化测定膜脂过氧化程度。【结果】内生菌E. microspora发酵产物乙酸乙酯提取物能显著抑制人参核盘菌菌丝生长,最小抑菌浓度为3.75 mg/mL,培养6 d后抑制率为76.22%。该提取物能显著抑制人参核盘菌孢子萌发,15.00 mg/mL时抑制效果最好,抑制率达90.69%。提取物影响菌丝形态,增加人参核盘菌细胞膜通透性,造成菌丝内含物外渗,7.50 mg/mL处理10 h后电导率和核酸含量分别比对照组增加30.11%和62.85%。同时提取物显著增加人参核盘菌MDA含量和SOD、POD、CAT活力,7.50 mg/mL处理组呈现先上升后下降的变化趋势,并在12 h时达到最高值。【结论】内生菌E. microspora发酵产物乙酸乙酯提取物通过改变人参核盘菌细胞膜通透性,加剧膜脂过氧化,破坏细胞膜完整性,导致细胞内含物流失,显著抑制孢子萌发和菌丝生长。     

粘帚霉几丁质酶GcCHI1的结构鉴定及其抑菌作用

【目的】对分离纯化自链孢粘帚霉(Gliocladium catenulatum)HL-1-1菌株的几丁质酶Gc CHI1进行化学结构鉴定,并测定该酶对几种病原真菌的抑菌机制。【方法】几丁质酶Gc CHI1化学结构鉴定采用Nano-ESI-MS/MS技术。该酶对病原真菌菌丝生长、病原菌孢子萌发和病原菌菌核萌发的抑制作用采用牛津杯法等方法。【结果】获得几丁质Gc CHI1胰蛋白酶水解肽段的肽质量指纹谱图,较好的MS/MS图谱,以及3个肽段的氨基酸序列(均15个氨基酸),分别为LYNSNDAIEAFISR、VIGYFTQWGIYGR、LNLGIGYYGR。经Mascot数据库检索认为与来自Stenotrophomonas maltophilials 34S1的几丁质酶A具有高度的相似性。几丁质酶Gc CHI1能明显抑制立枯丝核菌、油菜菌核病菌、番茄灰霉病菌等多种植物病原真菌的菌丝生长、孢子萌发和菌核萌发。【结论】几丁质酶Gc CHI1对多种植物病原菌有抑制作用,因此几丁质酶Gc CHI1是HL-1-1菌株抑菌作用的机制之一。     

丁布对小麦赤霉病菌和玉米小斑病菌的抑制作用

采用菌丝生长速率法和孢子萌发试验法检测了抗菌化合物丁布对小麦赤霉病菌和玉米小斑病菌的抑菌作用。结果表明,丁布在PDA培养基中浓度为0.2-1.0 mg/ml时对两种供试病菌的菌丝生长无抑制作用;丁布浓度为0.4-1.0 mg/ml时对两种供试病菌孢子悬浮液中孢子的萌发具有显著抑制作用;1.0 mg/ml丁布药液中培育15h的小麦赤霉病菌和培育5h的玉米小斑病菌的孢子萌发抑制率分别达到100%和83.6%。     

20种植物提取物抑制植物病原菌活性研究

以小麦纹枯病菌(Rhizoctonia cerealis)、番茄灰霉病菌(Alternaria solani)这两种植物病原真菌为供试菌,对采自江西省吉安市的20种植物提取物的抑制菌丝生长活性及孢子萌发进行测定.结果表明,在供试质量浓度为1 mg/mL时,黄花草木樨乙醇提取物的乙酸乙酯萃取部分抑菌效果最好,对小麦纹枯病菌和番茄灰霉病菌两种菌的抑制率均达100%;窃衣、小白酒草、羊蹄和车前草的抑制作用次之,对两种菌的抑制率均大于80%;单从对一种病原菌的抑制作用看,还有黄荆对小麦纹枯病菌率为100%,空心莲子草、窃衣、小白酒草和鬼针草对番茄灰霉病菌的抑制率为100%,但是,这20种供试植物的石油醚相和水相萃取物对两种病原真菌的抑制效果均不强.抑制病原菌孢子萌发亦得到类似结果.以上结果提示植物抑菌活性成分主要存在于乙酸乙酯萃取部分,黄花草木樨、窃衣、小白酒草等的提取物作为植物源杀菌剂值得进一步研究.     

甘肃黑蛋巢抗松梢枯病菌物质的分离纯化及抑菌活性*

采用MS培养基培养甘肃黑蛋巢Cyathus gansuensis,以生测为导向,乙酸乙酯和氯仿从甘肃黑蛋巢MS培养液中萃取出抗菌活性组分。Rp-18反相色谱柱和高效液相色谱仪相结合,从抗菌代谢产物分离纯化出一种抗松梢枯病菌(Sphaeropsis sapinea)的活性物质CXL-I,高效液相色谱检测表明CXL-I为一纯物质。CXL-I对松梢枯病菌的菌丝生长和孢子萌发有较强的抑制活性,当CXL-I浓度为50礸/ml时对菌丝生长抑制率可达80%,对孢子萌发抑制率达95%以上。     

In vitro inhibition of Sphaeropsis sapinea by natural stilbenes

The effects of pinosylvin, pinosylvin monomethyl ether, pinosylvin dimethyl ether, and resveratrol on the fungal shoot blight and canker pathogen of conifers Sphaeropsis sapinea were examined in vitro. Effects of compounds, isolates, and concentrations on both conidial germination and mycelial growth were significant (values of P < 0.001), indicating inhibitory activity of these compounds.     

多噬伯克霍尔德氏菌WS-FJ9对林木病原菌物的拮抗作用及代谢产物的稳定性

【背景】伯克霍尔德氏菌(Burkholderia)是一类重要的植物根际促生细菌,许多菌株具有抑制植物病原菌生长和促进植物生长等功能。【目的】探究高效解磷促生细菌多噬伯克霍尔德氏菌(B. multivorans) WS-FJ9对不同林木病原菌物的抑菌作用。【方法】采用平板对峙法检测菌株WS-FJ9对5株林木病原真菌和卵菌的抑制效果;基于比色法检测经菌株WS-FJ9处理后病原菌菌丝细胞内含物的变化;使用antiSMASH 5.0在线预测网站对其次生代谢物质进行预测;通过菌丝生长抑制速率法对其无菌发酵滤液的抑菌活性和稳定性进行研究。【结果】菌株WS-FJ9对5种林木病原菌均具有不同程度的抑制作用,其中菌悬液对樟疫霉(Phytophthora cinnamomi)的抑制作用最好,抑菌带宽度为14.82±0.20mm,无菌发酵滤液对真菌拟茎点霉(Phomopsismacrospore)和松杉球壳孢(Sphaeropsis sapinea)的抑制效果显著,抑菌率分别为62.22%和62.78%;经无菌发酵滤液处理后的病原菌菌丝内的丙二醛含量增高,还原糖和可溶性蛋白含量显著降低。WS-FJ9菌株的基因组中含27个不同的次级代谢产物编码基因簇,其中包含编码嗜铁素、细菌素和抗生素等抑菌基因簇;该菌株发酵液在高温、紫外照射和强酸强碱环境条件下及经蛋白酶处理后,其抑菌活性均未受到影响。【结论】多噬伯克霍尔德氏菌WS-FJ9对林木病原菌物具有很好的生防潜力。     

Chemical and biological characterisation of sapinopyridione, a phytotoxic 3,3,6-trisubstituted-2,4-pyridione produced by Sphaeropsis sapinea, a toxigenic pathogen of native and exotic conifers, and its derivatives

A phytotoxic trisubstituted 2,4-pyridione, named sapinopyridione, was isolated from the culture filtrates of Sphaeropsis sapinea, a fungal pathogen of conifers occurring world-wide. Three strains were isolated from two cypress species. Strain D-55 isolated from Cupressus sempervirens resulted high producer of sapinopyridione (12.3 mg l(-1)), whereas strain D-54 isolated from the same cypress species was low producer (1.1 mg l(-1)); strain D-50 isolated from C. macrocarpa was intermediate producer (5.4 mg l(-1)). Sapinopyridione was characterised by spectroscopic and chemical methods, as the 6-methyl-2-(2-methyl-1-oxobutyl)-1-oxa-5-azaspiro[2.5]oct-6-ene-4,8-dione. The structure was supported by the preparation of three key derivatives, whose phytotoxic and antimycotic activities were also tested on host plants and on three Seiridium species, virulent fungal agents of cypress canker disease. Some structure-activity relationships were identified for both phytoxicity and antifungal activities. These activities appear related to the presence of both pyridione and oxiran rings. Also the carbonyl group of the side chain seems to play a role into impart activity.     

水霉拮抗菌的筛选及其拮抗活性物质稳定性初步研究

【目的】从海底沉积物中分离、筛选水霉拮抗放线菌菌株,鉴定目标菌株及其无菌发酵液对水霉生长的抑制效果,并初步分析拮抗活性物质的稳定性。【方法】用稀释涂布法从采集的海底沉积物中分离得到海洋放线菌,以水霉为靶菌,通过平板对峙法在PDA平板上筛选出对水霉有拮抗作用的菌株;利用其发酵液对水霉菌丝和孢子进行初步拮抗效果研究;通过16S rRNA基因序列分析对目标菌株的种属进行初步鉴定。【结果】从分离到的数十株海洋放线菌中筛选到5株水霉拮抗菌,其中拮抗效果最强的为S26菌株,16S rRNA基因序列分析结果显示其为链霉菌,并与紫色链霉菌具有较近的亲缘关系;S26马铃薯葡萄糖液体培养基发酵液在平板抑菌圈实验中,对水霉孢子萌发的抑菌圈直径达32.00 mm±0.81 mm,其5倍浓缩无菌发酵液对水霉菌丝的抑菌圈直径达39.75 mm±0.50 mm;5倍浓缩无菌发酵液抑菌活性的3.125%即能完全抑制水霉孢子的萌发;5倍浓缩液对温度具有较强耐受性,经100 °C高温30 min处理后平板抑菌圈直径为25.50 mm±0.58 mm;经不同pH值处理12 h后,pH 5.0–9.0之间仍保持较好的拮抗活性;在37 °C下蛋白酶处理2 h后实验组与对照组存在显著性差异,但平板抑菌圈直径仍可达33.25 mm以上,推测拮抗物质活性成分由多肽和非多肽类代谢物共同组成。【结论】海洋链霉菌株S26产生的活性物质对病原水霉真菌有较强的抑制作用,并对外界环境变化有较强的适应能力,因而在水霉病的生物防治中具有潜在的应用价值。研究结果同时也显示海洋链霉菌在水产病害生物防治应用领域有较好的发展前景和更广阔的挖掘空间。     

Studies on structure-activity relationship of sphaeropsidins A-F, phytotoxins produced by Sphaeropsis sapinea f. sp. cupressi

Six forms of sphaeropsidins (SA-SF), three- and tetra-cyclic unrearranged pimarane diterpenes produced by Sphaeropsis sapinea f. sp. cupressi, as well as eight derivatives obtained by chemical modification of SA-SC, were assayed for their bioactivity. The effect of each compound on plants which are host or non-host of the pathogen was investigated. Activity on some plant pathogenic fungi was also tested. Some structure-activity relationships have been identified for both phytotoxic and antifungal activity. It appears that the integrity of the tricyclic pimarane system, the preservation of the double bond C(8)-C(14), the tertiary hydroxyl group at C-9, the vinyl group at C-13, and the carboxylic group at C-10 as well as the integrity of the A-ring provide these molecules with non selective phytotoxic and antimycotic activity.     

一株真菌拮抗细菌Z21的筛选与鉴定及其发酵条件优化

【背景】芽孢杆菌属的许多细菌具有抗逆性强、安全等特点,一直以来都是开发新型活性物质的研究热点。【目的】筛选对食品腐败真菌有抑制作用的细菌,将其开发为天然食品防腐剂。【方法】采用平板分离法、平板对峙法、抑制菌丝生长速率法从空气、竹子内生细菌中筛选真菌拮抗菌,通过形态、生理生化特征及16S rRNA基因序列分析等方法对其进行鉴定,利用正交试验确定其最优生长条件。【结果】筛选到一株对6种常见霉菌均有较强抑制作用的细菌Z21。Z21与甲基营养型芽孢杆菌(Bacillusmethylotrophicus strain CBMB205~T)的相似性最高,且形态特征和生理生化特征与CBMB205~T菌株基本相符。Z21最佳发酵培养基配方和培养条件分别为:葡萄糖20.0 g/L、NaNO_3 20.0 g/L、MgSO_4 3.0 g/L,培养温度为32°C,培养时间为48 h。【结论】Z21为甲基营养型芽孢杆菌(Bacillus methylotrophicus),对黑曲霉、康氏木霉、绿色木霉、少根根霉、易脆毛霉、赭绿青霉的生长具有较强的抑制作用且抑菌效果稳定,为广谱真菌拮抗菌。     

Simple sequence repeat markers distinguish among morphotypes of Sphaeropsis sapinea

Sphaeropsis sapinea is a fungal endophyte of Pinus spp. that can cause disease following predisposition of trees by biotic or abiotic stresses. Four morphotypes of S. sapinea have been described from within the natural range of the fungus, while only one morphotype has been identified on exotic pines in the Southern Hemisphere. The aim of this study was to develop robust polymorphic markers that could be used in both taxonomic and population studies. Inter-short-sequence-repeat primers containing microsatellite sequences and degenerate anchors at the 5' end were used to target microsatellite-rich areas in an S. sapinea isolate. PCR amplification using an annealing temperature of 49 degrees C resulted in profiles containing 5 to 10 bands. These bands were cloned and sequenced, and new short-sequence-repeat (SSR) primer pairs were designed that flanked microsatellite-rich regions. Eleven polymorphic SSR markers were tested on 40 isolates of S. sapinea representing different morphotypes as well as on 2 isolates of the closely related species Botryosphaeria obtusa. The putative I morphotype was found to be identical to B. obtusa. Otherwise, the markers clearly distinguished the remaining three morphotypes and, furthermore, showed that the C morphotype was more closely related to the A than the B morphotype. The B morphotype was the most genetically diverse, and the isolates could be further divided based on their geographic origins. Sequencing of different alleles from each locus showed that the most polymorphic markers had mutations within a microsatellite sequence.     

Isolation, purification and characterization of an antifungal molecule produced by Bacillus licheniformis BC98, and its effect on phytopathogen Magnaporthe grisea

AIMS: Isolation of bacterial antagonist for use in the biological control of phytopathogenic fungi like rice blast fungus, Magnaporthe grisea, and to further purify and characterize the antifungal molecule produced by the antagonist. METHODS AND RESULTS: Bacterial antagonist exhibiting highest antifungal activity against the rice blast fungus M. grisea was isolated from soil and identified as Bacillus licheniformis BC98. Besides M. grisea, the isolate also inhibited the growth of other phytopathogens such as Curvularia lunata and Rhizoctonia bataticola. Biologically active fractions were isolated from the culture filtrate and further fractionated by reverse-phase high-performance liquid chromatography (HPLC) enabling detailed structural characterization of a component of molecular mass 1035 Da. The active peptide was identified as surfactin after 500 MHz (1)H NMR analysis. Microscopic analysis of the effect of the antagonist on M. grisea revealed bulbous hyphae showing patchy and vacuolated cytoplasm when observed under the electron microscope. CONCLUSIONS: The antagonistic lipopeptide secreted by B. licheniformis BC98 and identified as surfactin, induced morphological changes in M. grisea, inhibiting its further growth, and thus exhibiting fungicidal activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The antagonist inhibits germination of M. grisea, a potent rice phytopathogen, and therefore appears to be a potential candidate for control of rice blast disease.