一株北里孢菌株的分离鉴定及其对松材线虫的致病性

【目的】筛选、鉴定出对松材线虫杀灭活性较高的放线菌菌株,并确定生防菌株的毒力因子。【方法】采用平板活性测试及代谢杀虫活性检测方法进行筛选,采用形态学及16S rDNA序列分析等进行鉴定。对发酵液中的活性物质稳定性分析后,利用醇沉、萃取、层析、气相色谱/质谱分析等方法分离纯化出杀虫毒力因子。【结果】从河南南阳宝天曼的腐木及枯枝落叶样品中共分离获得了79株放线菌,从中筛选出对松材线虫有灭活作用的放线菌6株,其中分离株C620菌株对松材线虫的灭活性最高:该菌株的发酵液处理松材线虫48、60 h后线虫的死亡率分别达到60.0%、81.5%。结合该菌株的形态学、生理学特征及16S rDNA序列分析等结果将其归为北里孢菌属中的一个种,菌株编号Kitasatospora sp.strain C620。该菌株的发酵液中杀线虫活性物质的热稳定性、光稳定性及耐储藏性均较强,在中性偏碱性环境较稳定;经pH纸电泳层析初步确定该物质属于碱性水溶性物质。对菌株C620发酵液分离纯化,得到活性化合物为1-苯基-3-(2-吡啶)-5-吡唑啉酮。【结论】获得一株松材线虫高效生防菌Kitasatospora sp.strain C620,其活性物质为1-苯基-3-(2-吡啶)-5-吡唑啉酮。     

具杀线虫活性植物内生细菌的筛选和活性产物

【目的】植物寄生线虫是危害植物的重要病原物,为了筛选到能在植物体内稳定定殖并且对植物寄生线虫具有较高杀线虫活性的植物内生细菌生防菌。【方法】以松材线虫为靶标,用直接触杀法进行筛选。对高活性菌株采用正交实验优化发酵条件,测定发酵液杀线虫活性的稳定性,并对菌株进行鉴定。【结果】从6种植物中分离筛选出13株对松材线虫具有较高杀线虫活性的植物内生细菌菌株,这些菌株的发酵上清液对松材线虫处理24h杀线虫率均达到了100%;其中BCM2、SZ5、CCM7和DP1这4个菌株的杀线虫活性较高,发酵上清液稀释3倍处理24h杀线虫率均达到95%以上,DP1和SZ5菌株达到了100%;并发现部分菌株发酵液能使线虫虫体发生渗漏或消解。发酵条件优化后能使发酵液杀线虫效果提高4倍。4株高活性菌株产生的杀线虫物质均对蛋白酶稳定、耐热不耐酸碱且长时间保藏活性不下降。经过鉴定DP1和CCM7是枯草芽孢杆菌(Bacillus subtilis),BCM2和SZ5是蜡样芽孢杆菌(Bacillus cereus)。【结论】经济作物体内存在一定数量的能产生杀线虫活性物质的内生细菌,其中一些细菌产生的杀线虫物质具有较强的稳定性。认为杀线虫活性的植物内生细菌具有很大的生防潜力。     

线虫生防细菌W3对线虫侵染活性的测定

目的:筛选以体壁穿透方式进入线虫体内而引起宿主致病的杀线虫细菌,为开发新的植物寄生线虫生防战略奠定基础.方法:通过毒性测试和平板法筛选杀线虫细菌,利用显微镜和扫描电镜研究观察细菌对线虫的作用方式和侵染过程.结果:筛选获得的杀线虫细菌菌株W3具有明显的侵染活性.生测结果表明,细菌培养上清液和上清蛋白粗提物对腐生线虫致死率分别达到95%和100%.在平板测试中,测试线虫体壁被严重破坏,在48h内超过80%的线虫被杀死和降解.扫描电镜观察可以明显的看到细菌穿透线虫体壁后在线虫体壁上留下的孔.结论:细菌W3具有显著的杀线虫活性和线虫体壁降解活性,其杀线虫活性物质主要存在于细菌培养上清液中.     

松材线虫拮抗细菌的杀线活性及其发酵培养特性

旨为筛选具有高效杀松材线虫活性的拮抗细菌。采用浸测法对3株芽孢杆菌进行杀线活性测定,并对高效杀线菌株发酵滤液处理线虫后的虫体进行形态观察及该菌株培养条件和杀线物质稳定性测定。结果表明3株细菌发酵滤液处理松材线虫(Bursaphelenchus xylophilus)48 h后,线虫校正死亡率均达100%,且松材线虫在蜡样芽孢杆菌(Bacillus cereus)JK-XZ3发酵滤液4倍和8倍稀释液中处理48 h后,其校正死亡率分别达99.55%和88.32%,明显高于短小芽孢杆菌(B. pumilus)HR10和瓦雷兹芽孢杆菌(B. velezensis)YH-20;该JK-XZ3菌株发酵滤液处理过的线虫虫体断裂,内含物外溢,体壁消解;该菌株在30℃培养4 d的发酵滤液杀线活性最高,且松材线虫在发酵滤液4倍和8倍稀释液中处理48 h后,其校正死亡率均达100%;松材线虫在40℃、60℃、80℃和100℃处理后的发酵滤液8倍稀释液中处理48 h后,其校正死亡率均在84%以上;在pH为8-10的发酵滤液8倍稀释液处理48 h后,其校正死亡率均在88%以上;说明该菌株发酵滤液具有耐高温和耐碱特性。蜡样芽孢杆菌JK-XZ3是一株具有开发和应用潜力的高毒力杀线菌株。     

杀松材线虫细菌的分离、鉴定及其培养条件研究

为了对松萎蔫病进行安全、无污染的生物防治,从青岛大学校园内采集的样品中分离出120株菌。采用浸渍法从中筛选出1株具有较高杀线虫活性的细菌G8-1。杀线虫活性测定结果表明,该菌株发酵上清液48 h后的杀线虫率为90.81%。通过16S rRNA的序列分析,将其鉴定为Flectobacillus rhizosphaerae。通过单因素分析法和正交实验对该菌株的培养条件进行研究,确定最佳培养条件:最适初始pH为8.0,最适培养温度为35℃,最佳接种菌龄为9 h,最佳培养时间为3 d,菌株G8-1发酵上清液的杀线虫率可达到96.31%。对菌株产生的活性物质的稳定性进行初步分析,结果表明该菌株的活性物质都具有较好的热稳定性,并且在弱碱性条件下具有较高的杀线虫活性稳定性。本研究为松材线虫的生物防治提供参考。     

杀松材线虫海洋放线菌HT-8的鉴定及培养条件的研究（英文）

松树萎蔫病(Pine wilt disease,PWD)是由松材线虫(Bursaphelenchus xylophilus)引起的一种毁灭性疾病,该病对世界上许多国家和地区的针叶林资源造成严重破坏。为了寻找具有高杀松材线虫活性的海洋放线菌,对分离自海洋环境中的放线菌菌株进行杀线虫活性测定。选用稀释涂布平板法对采自青岛近海海域的样品进行放线菌分离,采用浸渍法测定菌株的杀松材线虫活性,并对所筛选出的高杀线虫活性菌株的形态学、培养特征和16S rD NA序列进行测定,运用单因素试验测定菌株产生杀线虫活性物质的最适培养条件。结果显示,从采集的样品中共分离到28株放线菌,经杀线虫活性筛选得到1株具有高杀线虫活性的放线菌HT-8。该菌株分离自海沙,其培养上清液处理松材线虫30 h时,线虫的校正死亡率高达88.30%。根据形态学、培养特征和16S rD NA序列测定及其系统发育分析结果,将菌株HT-8鉴定为Streptomyces termitum。单因素试验结果表明,该菌株产生杀线虫活性物质的最适培养条件为接种体菌龄48 h,接种量6%,培养基海水浓度100%,初始pH 7.5,培养温度25℃,培养时间7 d。该研究为海洋微生物资源的开发及其杀松材线虫天然活性物质的利用提供理论依据。     

松材线虫伴生菌中产纤维素酶细菌的筛选、鉴定及其相关基因的克隆

[目的]从松材线虫伴生菌中筛选出高效降解纤维素的细菌菌株,初步鉴定后,对其相应的纤维素酶基因尝试克隆.[方法]首先从河南南阳松材线虫病疫区采集到的木材样本中,分离获得松材线虫.采用刚果红平板初筛法,从松材线虫伴生菌中获得具有分泌较高活性纤维素酶的细菌菌株.基于该菌株的形态学、生理学及16 s rDNA序列特征等对高活性菌株进行分类鉴定.设计兼并引物,从高活性菌株中克隆该菌株的纤维素酶基因,并进行序列分析.[结果]获得7株具有分泌较高活性纤维素酶的细菌菌株,其中编号为C8的菌株酶活最高.经鉴定该菌株归为肠杆菌属,命名为Enterobacter sp.C8.进一步从C8菌株中成功克隆出该菌株的一个全长1104 bp的纤维素酶基因(GenBank JQ845065),在NCBI比对后发现该基因分别与产气肠杆菌( Enterobacter aerogenes) KCTC 2190的纤维素合成酶亚单位BcsC的核苷酸序列有97％的同源性,氨基酸序列有92％的同源性 ;与克雷白氏肺炎菌(Klebsiella pneumoniae)的endo-1,4-D-glucanase基因有82％的同源性,与不可培养的细菌内切纤维素酶基因有82％的同源性.[结论]本文首次从松材线虫伴生菌中筛选到了一株简单的产纤维素酶的细菌菌株并从中克隆出了一个新型纤维素酶基因,为下一步进行新能源的利用奠定了理论基础.     

一株植物病原拮抗细菌的分离筛选及拮抗物质

从黄豆酱中分离筛选对植物病害有防治效果的拮抗菌,提取发酵液中的拮抗物质,研究其抑菌机理及基本性质。采用稀释平板法分离细菌、平板对峙法筛选拮抗菌,通过形态特征、生理生化特征及16S rRNA鉴定菌种,硫酸铵沉淀法提取发酵液中的拮抗物质,考马斯亮蓝法测得蛋白含量,扫描电镜观察拮抗物质抑菌作用,琼脂孔扩散法测定拮抗物质抑菌谱及其活性的稳定性。从黄豆酱中分离出18株细菌,通过筛选,得到一株拮抗效果最好的菌株,菌体抑菌率62.78%,发酵液抑菌率53%,命名为4-z-3,经鉴定为Bacillus siamensis。4-z-3发酵液在硫酸铵饱和度为20%~30%时提取的粗提物抗菌活性最大,为60.8%。最适饱和度下提取的粗提物的蛋白质含量为3.45%。拮抗物质对多种病原菌有良好的拮抗效果。经过4-z-3粗提物处理的病原真菌,菌丝受到破坏,细胞形状改变,细胞完整性缺失。粗提物的稳定性方面,对热不敏感,仅在90和100℃时抑菌活性下降明显;对酸碱不敏感,pH值为4时仍有81.6%的抑菌活性;对蛋白酶不敏感,抑菌活性最低时降低了19.6%;对紫外线不敏感,照射12 h之后抑菌活性仍有92.5%。本研究表明,菌株4-z-3是一株具有发展潜力的农业生防菌,分泌的胞外拮抗物质能够有效防治多种植物病害,且抑菌活性稳定。     

动物乳杆菌的分离鉴定及其抑菌蛋白的特性分析

通过滤纸片法从健康肉猪猪大肠、小肠中分离得到212株抗生物质产生菌, 以杯碟法复筛, 得到1株对溶壁微球菌(Micrococcus lysodeikticus)、金黄色葡萄球菌(Staphylococcus aureus)、枯草芽孢杆菌(Bacillus subtilis)、蜡状芽孢杆菌(Bacillus cereus)等革兰氏阳性菌和大肠杆菌(Escherichia coli)、鼠伤寒沙门氏菌(Salmonella typhimurium)等革兰氏阴性菌以及部分真菌如禾谷镰刀霉(Fusarium graminearum)均有强烈抑制作用的乳酸菌。经形态学、生理生化特征及16S rDNA序列同源性分析等手段鉴定该菌株为动物乳杆菌(Lactobacillus animalis)。排除酸和过氧化氢的干扰后, 该菌株的发酵上清液对指示菌仍有明显抑菌活性; 用蛋白酶处理该菌株的发酵上清液后, 抑菌活性丧失; 发酵液粗提物具有较好的热稳定性(经121°C处理20 min仍有较强抑菌活性)以及较宽的抑菌活性pH值范围(3.5~5.5), 因此初步认为该菌株产生一类具有广谱抑菌活性的类细菌素物质。     

植物根际益生细菌代表性菌株贝莱斯芽孢杆菌FZB42对松材线虫的抑杀性

【目的】由松材线虫导致的松树萎蔫病是松树的毁灭性病害,也是我国最主要的林业病害之一。本研究测评了在农业上广泛使用的、我国微生物肥料行业主要菌种资源之一——贝莱斯芽孢杆菌,对松材线虫的潜在抑杀性能。【方法】选用贝莱斯芽孢杆菌的代表性菌株FZB42为材料,测定对不同条件下的菌液上清、不同菌株的菌液上清、细菌素plantazolicin的提取物以及菌体直接接触等方式,对松材线虫死亡率/存活率的影响,并构建FZB42生物膜合成缺损菌株,测定比较其对松材线虫存活率的影响。【结果】相比于Landy培养基,使用LB培养基得到的菌液上清,对松材线虫具有明显抑杀性,培养48h的菌液上清比培养24h的菌液上清抑杀性更强,同时,菌液上清的抑杀效能随浓度升高及处理时间增长而有所增加。其中,使用培养48h后收集的LB菌液上清处理48h,松材线虫死亡率可高达约50%。之前报道对秀丽隐杆线虫有杀线虫活性的细菌素plantazolicin,经不同突变株上清以及plantazolicin提取物测试,被证实对松材线虫无抑制作用。直接接触实验表明,尽管FZB42的生物膜形成作用会刺激松材线虫提高抗胁迫能力,但整体而言,菌体接触对松材线虫亦有明显抑杀效果。【结论】本研究通过严格细致的测试证明,贝莱斯芽孢杆菌FZB42对松材线虫具有抑杀性,该抑杀作用的分子基础和作用机理有待深入挖掘,但其与plantazolicin无关。作为安全性好、研究开发程度高的一类生防菌株,贝莱斯芽孢杆菌在防治松材线虫病方面的潜在价值值得关注。     

Nematicidal metabolites produced by the endophytic fungus Geotrichum sp. AL4

From the endophytic fungal strain Geotrichum sp. AL4, cultivated from the leaves of the neem tree (Azadirachta indica), four compounds, 1-4, were isolated from the AcOEt extract, including two new, chlorinated, epimeric 1,3-oxazinane derivatives. All compounds were assessed for their nematicidal activities against the nematodes Bursaphelenchus xylophilus and Panagrellus redivivus, and three out of the four isolates showed noticeable bioactivities.     

Proteases from Bacillus: a new insight into the mechanism of action for rhizobacterial suppression of nematode populations

AIMS: The aim of this study was to investigate the role of proteases in Bacillus spp. of rhizobacteria in suppressing nematode populations and to understand their mechanism of action. METHODS AND RESULTS: Rhizobacteria with nematicidal activity were isolated from soil samples of five root knot nematode-infested farms. Among these strains, nematotoxicities of Bacillus strains were intensively analysed. Further assays of nematicidal toxins from Bacillus sp. strain RH219 indicated an extracellular cuticle-degrading protease Apr219 was an important pathogenic factor. The Apr219 shared high similarity with previously reported cuticle-degrading proteases from Brevibacillus laterosporus strain G4 and Bacillus sp. B16 (Bacillus nematocida). The cuticle-degrading protease genes were also amplified from four other nematicidal Bacillus strains isolated from the rhizosphere. In addition to Apr219, a neutral protease Npr219 from Bacillus sp. RH219 was also investigated for activity against nematodes. CONCLUSIONS: The wide distribution of cuticle-degrading proteases in Bacillus strains with nematicidal activity suggested that these enzymes likely play an important role in bacteria-nematode-plant-environment interactions and that they may serve as important nematicidal factors in balancing nematode populations in the soil. SIGNIFICANCE AND IMPACT OF THE STUDY: Increased understanding of the mechanism of action of Bacillus spp. against nematodes could potentially enhance the value of these species as effective nematicidal agents and develop new biological control strategies.     

Fumiquinones A and B, nematicidal quinones produced by Aspergillus fumigatus

New nematicides named fumiquinones A (1) and B (2), together with spinulosin (3), LL-S490beta (4), and pseurotin A (5), were isolated from Aspergillus fumigatus and their structures were established by spectroscopic methods including 2D-NMR. Compound 1 showed effective nematicidal activities against Bursaphelenchus xylophilus and Pratylenchus penetrans without inhibiting plant growth except for lettuce seedlings. Compound 2 showed effective nematicidal activity against B. xylophilus, but had no inhibitory activity against P. penetrans. Compounds 3-5 showed effective nematicidal activities against B. xylophilus without any plant growth inhibition. Compounds 1-5 had no nematicidal activity against Caenorhabditis elegans. This is the first report of the nematicidal activities of compounds 3-5.     

Selective inhibition of toxic cyanobacteria by β-carboline-containing bacterium Bacillus flexus isolated from Saudi freshwaters

A bacterial strain SSZ01 isolated from a eutrophic lake in Saudi Arabia dominated by cyanobacterial blooms, showed an antialgal activity against cyanobacteria species. Based on the analysis of the 16S rDNA gene sequence, the isolated strain (SSZ01) most likely belonged to the genus Bacillus with a 99% similarity to Bacillus flexus strain EMGA5. The thin layer chromatography (TLC) analysis of the ethyl acetate extract of this bacterium revealed that this strain can produce harmine and norharmane compared to different β-carboline analog standards. Harmine and norharmane were also detected in considerable amounts in bacterial growth medium, indicating a potential excretion of these compounds into the aquatic environment. The crude extract of Bacillus flexus as well as pure materials of harmine and norharmane inhibited the growth of tested species of cyanobacteria. However, the bacterial crude extract has a higher toxicity against tested species of cyanobacteria than harmine and norharmane. In addition, harmine was more toxic to cyanobacteria than norharmane. On the other hand, neither pure compounds of harmine and norharmane nor crude bacterial extract showed any antialgal activity against tested species of green algae. The results of the present study suggest that B. flexus SSZ01 or its crude extract containing harmine and norharmane could be a candidate for the selective control of cyanobacterial blooms without affecting other algal species.     

Nematicidal activity of Trichoderma spp. and isolation of an active compound

Trichoderma spp. play an important role in biotic control, and several are efficacious against nematodes. To study the potential of Trichoderma species in controlling nematodes, fungal filtrates of 329 Trichoderma strains were evaluated for their nematicidal activity against Panagrellus redivivus and Caenorhabditis elegans. Fifteen strains exhibited nematicidal activity against P. redivivus, and 14 strains showed activity against C. elegans. The strain YMF1.02647 showed strong nematicidal activity against both nematodes, and the culture broth could cause more than 90% mortality to the tested nematodes within 48 h. A nematicidal compound was isolated from ethyl acetate extract of Trichoderma YMF1.02647 based on bioassay-guided fractionation. The compound was identified as trichodermin according to the spectroscopic data, which could kill more than 95% both P. redivivus and C. elegans in 72 h at 0.4 g l⁻¹.     

Nematicidal coumarin from Ficus carica L.

The methanol extracts from 40 plant species were screened for their nematicidal activity against the nematodes Bursaphelenchus xylophilus, Panagrellus redivivus and Caenorhabditis elegans. The leaf extract of Ficus carica L. exhibited the strongest nematicidal activity, causing 74.3%, 96.2% and 98.4% mortality, respectively, within 72 h. By bioassay-guided fractionation, a coumarin was obtained. The compound was determined to be psoralen based on spectroscopic data. It showed nematicidal activity against the tested nematodes. This is the first report of the nematicidal activity of F. carica and psoralen.     

Genetic and Proteomic Characterization of rpoB Mutations and Their Effect on Nematicidal Activity in Photorhabdus luminescens LN2

Rifampin resistant (Rif(R)) mutants of the insect pathogenic bacterium Photorhabdus luminescens LN2 from entomopathogenic nematode Heterorhabditis indica LN2 were genetically and proteomically characterized. The Rif(R) mutants showed typical phase one characters of Photorhabdus bacteria, and insecticidal activity against Galleria mellonella larvae, but surprisingly influenced their nematicidal activity against axenic infective juveniles (IJs) of H. bacteriophora H06, an incompatible nematode host. 13 out of 34 Rif(R) mutants lost their nematicidal activity against H06 IJs but supported the reproduction of H06 nematodes. 7 nematicidal-producing and 7 non-nematicidal-producing Rif(R) mutants were respectively selected for rpoB sequence analysis. rpoB mutations were found in all 14 Rif(R) mutants. The rpoB (P564L) mutation was found in all 7 mutants which produced nematicidal activity against H06 nematodes, but not in the mutants which supported H06 nematode production. Allelic exchange assays confirmed that the Rif-resistance and the impact on nematicidal activity of LN2 bacteria were conferred by rpoB mutation(s). The non-nematicidal-producing Rif(R) mutant was unable to colonize in the intestines of H06 IJs, but able to colonize in the intestines of its indigenous LN2 IJs. Proteomic analysis revealed different protein expression between wild-type strain and Rif(R) mutants, or between nematicidal-producing and non nematicidal-producing mutants. At least 7 putative proteins including DsbA, HlpA, RhlE, RplC, NamB (a protein from T3SS), and 2 hypothetical proteins (similar to unknown protein YgdH and YggE of Escherichia coli respectively) were probably involved in the nematicidal activity of LN2 bacteria against H06 nematodes. This hypothesis was further confirmed by creating insertion-deletion mutants of three selected corresponding genes (the downregulated rhlE and namB, and upregualted dsbA). These results indicate that the rpoB mutations greatly influence the symbiotic association between the symbionts and their entomopathogenic nematode hosts.     

Nematicidal Activity of Plant Essential Oils and Components From Ajowan (Trachyspermum ammi), Allspice (Pimenta dioica) and Litsea (Litsea cubeba) Essential Oils Against Pine Wood Nematode (Bursaphelenchus Xylophilus)

Commercial plant essential oils from 26 plant species were tested for their nematicidal activities against the pinewood nematode, Bursaphelenchus xylophilus. Good nematicidal activity against B. xylophilus was achieved with essential oils of ajowan (Trachyspermum ammi), allspice (Pimenta dioica) and litsea (Litsea cubeba). Analysis by gas chromatography-mass spectrometry led to identification of 12, 6 and 16 major compounds from ajowan, allspice and litsea oils, respectively. These compounds from three plant essential oils were tested individually for their nematicidal activities against the pinewood nematode. LC₅₀ values of geranial, isoeugenol, methyl isoeugenol, eugenol, methyl eugenol and neral against pine wood nematodes were 0.120, 0.200, 0.210, 0.480, 0.517 and 0.525 mg/ml, respectively. The essential oils described herein merit further study as potential nematicides against the pinewood nematode.