10株绿僵菌菌株分类地位的多基因系统进化分析

为确定10株高效杀虫绿僵菌菌株的分类地位, 通过克隆、测序10株绿僵菌菌株的ef1-α、rpb1、rpb2、 β-tubulin部分序列进行多基因系统进化分析, 并结合形态特征确定10株绿僵菌菌株的分类地位。结果表明多基因分子鉴定在绿僵菌菌种的划分上较单基因更为准确, 结合多基因系统进化分析结果和形态学特征, 将CQMa114、CQMa117、CQMa125、CQMa126、CQMa128归为大孢绿僵菌, CQMa102归为蝗绿僵菌, CQMa132、CQMa135归为贵州绿僵菌, 菌株CQMa138、CQMa140与其他绿僵菌亲缘关系较远, 有可能为绿僵菌属的新种或新的分类单位。     

四株球孢白僵菌菌系的初步研究

从西北地区杨树的蛀干性害虫上分离得到球孢白僵病的黄斑星天牛菌系、云斑天牛菌系、青杨天牛菌系及蒙古木囊蛾菌系。通过交互感染试验:不同菌系对不同昆虫的致病力有显著差异,各菌系对原寄生的致病力明显高于其它菌系,在形态上及生物学特征上也表现出明显的差别。为此,定向选择对目标害虫致病力较强的自然菌系,在害虫防治上有实际意义。     

GA1-2菌株的分离鉴定及其对香蕉尖孢镰刀菌的抑菌效果

为了从土壤中分离筛选对香蕉尖孢镰刀菌具有良好拮抗作用的放线菌,采用平板稀释涂布法从四川省会理县干热河谷小麦根际土壤中进行放线菌分离,并采用平板对峙法和孢子萌发法进行筛选,通过形态特征、培养特征、生理生化特征以及16S r DNA序列分析对筛选菌株进行鉴定。结果表明,从四川省会理县干热河谷小麦根际土壤筛选获得一株对香蕉尖孢镰刀菌4号生理小种Fusarium oxysporum f.sp.cubense race 4(FOC4)菌丝和孢子萌发都有很强抑制作用的菌株GA1-2,对FOC4菌丝和孢子萌发抑制率分别为36.34%和94.81%。菌株GA1-2与薰衣草灰链轮丝菌Streptoverticillium lavenduligriseum的亲缘关系最近,相似率达99.85%,且其形态特征、培养特征、生理生化特征也与薰衣草灰链轮丝菌基本相符,因此将菌株GA1-2初步鉴定为薰衣草灰链轮丝菌。     

内生细菌B_(47)菌株的鉴定及其对番茄青枯病的防效测定

通过形态特征、生理生化特征和16SrDNA序列分析,对分离于番茄茎部能较强抑制番茄青枯病菌生长的内生细菌B47菌株进行了鉴定。结果表明,该菌为枯草芽孢杆菌,其最适生长pH5~6,最适生长温度为35℃。室内防治试验结果表明,用淋根法先接种B47菌后接种病原菌和用注射法先接种B47菌后接种病原菌的处理可取得81.25%和92.10%的防效,而用淋根法、注射法同时接种B47菌与病原菌的处理防效较低。     

褐腐病菌三种分子鉴定方法的比较

为了筛选出适用于口岸检疫的、可快速鉴定三种褐腐菌,即美澳型核果褐腐菌(Monilinia fructicola)、核果褐腐菌(M. laxa)和仁果褐腐菌(M. fructigena)的方法,采用Lane等基于形态学特征的方法对采自北京、山东、河北等省市的58株褐腐菌进行了种类鉴定,并以这些菌及三个种的标准菌株为材料比较了已报道的三种分子检测方法的可靠性。研究发现,58株菌中有53株是美澳型核果褐腐菌,2株为核果褐腐菌,3株为仁果褐腐菌。采用Ioos等的PCR方法,从3个标准菌株、53株美澳型核果褐腐菌和2株核果褐腐菌中都只扩增出相应种的特征条带,而从3株仁果褐腐菌中的2株中扩增出了两个种的特征条带。采用Ma等的方法,从美澳型核果褐腐菌标准菌株、采自国内的53株美澳型核果褐腐菌和核果标准菌株中,只扩增到相应种的特征条带,而从采自国内的2株核果褐腐菌株中不仅扩增出了核果褐腐菌的条带还扩增出了美澳型核果褐腐菌的特征条带,3株仁果褐腐菌株产生了核果褐腐菌的特征条带,仁果褐腐菌标准菌株中没有得到产物。采用Cote等的方法,只从16株美澳型核果褐腐菌中扩增出该种的特征条带,从其余菌株(包括标准菌株)中没有获得产物。这些研究结果表明:Ioos等的检测方法可用于检测美澳型核果褐腐菌和核果褐腐菌;Ma等的检测方法可用于检测美澳型核果褐腐菌,而不适用于检测其它两个种;而仁果褐腐菌的分子检测方法需要进一步研究和完善。     

拮抗放线菌JD211的抑菌活性及其鉴定

本实验室从江西庐山珙桐树中分离筛选出的一株拮抗放线菌,编号为JD211。为明确放线菌JD211菌株在植物病害生物防治中的应用潜力,采用菌丝生长速率法测定其抑菌作用,通过多相分类法研究其分类地位。结果表明：JD211的粗提物可显著抑制水稻纹枯病菌、稻瘟病菌、烟草黑胫病菌、根霉、胶孢炭疽病菌、犁头霉、西瓜枯萎病菌等真菌的生长,其中对稻瘟病菌、烟草黑胫病菌等植物病原真菌的相对抑制率高达90％以上。根据形态特征、培养特征、生理生化特征及16SrDNA序列分析结果,将菌株JD211初步鉴定为奈良链霉菌（Streptomyees naraensis）。     

三株拮抗细菌的鉴定及抑菌作用研究

为了充分发掘和利用有益微生物资源,本文以草莓根腐尖孢镰刀菌Fusariumoxysporum为靶标菌,从草莓根际土壤中分离筛选得到3株拮抗细菌,通过培养性状与形态特征、生理生化特征以及16SrDNA同源序列比对分析进行了鉴定,菌株1-9和9—4为多粘类芽孢杆菌Paenibacilluspolymyxa、菌株12—4为枯草芽孢杆菌Bacillussubtilis。这3株细菌对草莓根腐尖孢镰刀菌的抑菌作用进行了初步的研究,结果表明,3株拮抗细菌对草莓根腐尖孢镰刀菌的生长有很好的抑制作用,镜下观察对菌丝有致畸作用,其培养滤液还能够抑制草莓根腐病尖孢镰刀菌分生孢子的产生与萌发。     

水稻纹枯病生防菌株的筛选、鉴定及其防治效果

为获得对水稻纹枯病有生防潜力的菌株,从湖北省恩施土家族苗族自治州植物根际土壤中分离获得菌株,筛选对水稻纹枯病菌有拮抗作用的菌株,并测定其发酵液的抑菌活性、抑菌谱及生物学特性;基于形态学特征、生理生化特征及分子生物学特征对其进行分类鉴定,并通过离体叶片、室内盆栽和田间试验进一步评价其生防潜力。结果显示,从根际土壤中共分离获得162株菌株,其中生防菌株E12对水稻纹枯病菌的抑制率最高,达94.93%,且该菌株对常见的12种植物病原菌均有抑制作用,抑制率在59.84%~93.14%之间;生防菌株E12能分泌蛋白酶、嗜铁素和淀粉酶,具有固氮及形成生物被膜的能力;结合形态学特征、生理生化特征及分子生物学特征,将菌株E12鉴定为越南伯克霍尔德氏菌Burkholderia vietnamiensis;生防菌株E12处理离体水稻叶片后,其相对病斑长度较对照显著减小,对水稻纹枯病的室内盆栽和田间防治效果分别为62.51%和67.78%。表明越南伯克霍尔德氏菌E12在防控水稻纹枯病方面有较好的潜力。     

一株内生拮抗细菌的分离鉴定及其抗菌机理研究

本研究通过平板稀释法、平板对峙法从核桃根部皮层筛选出一株对核桃根腐病有拮抗作用的内生菌株1A,通过形态特征测定、理化特性分析和16SrDNA序列分析鉴定该菌为芽胞杆菌(Bacillus sp.),GenBank序列登录号为KJ865856;其抑菌特性研究结果表明:该拮抗菌对核桃根腐病菌有显著的抑制效果,抑菌率达63.33%;此外,该菌对板栗疫病菌等几种不同的林木病原菌均有较强的抑制作用。抗生机理研究结果表明,该菌株通过分泌蛋白酶和纤维素酶降解真菌细胞壁中蛋白质和纤维素,破坏病菌菌丝,病菌生长受到抑制。该拮抗菌能够显著抑制核桃根腐病菌菌丝生长和孢子萌发,孢子萌发抑制率达74.89%,病菌菌丝表现为菌丝扭曲、断裂,分支增多并缠绕,菌丝颜色加深等。盆栽生防效果研究显示,该拮抗菌对各组核桃根腐病具有显著的防治效果,采用该拮抗菌预处理具有相对较好的生防效果。     

边缘假单胞菌的分离鉴定及其特性

边缘假单胞菌Pseudomonas marginalis是假单胞菌科假单胞菌属植物病原细菌,能引发百合软腐病、草莓花疫病、番茄细菌性髓部坏死病、马蹄莲块茎软腐病等,还可引发洋葱、莴苣、中国白菜、大蒜、姜、花椰菜、水稻等软腐病。边缘假单胞菌具有如此广泛的宿主致病性取决于其生理特点,尤其是在0 ℃生长良好、5 ℃能大量繁殖等特点。2007年在我国甘肃发现由边缘假单胞菌边缘致病变种引发的马铃薯腐烂病,而有关边缘假单胞菌分离鉴定和特性的研究则鲜有报道。本试验从苗木中分离得到边缘假单胞菌,对其形态特征、生理生化特性和致病性进行研究,并通过16S rRNA序列分析进一步验证其分类地位。     

内生真菌HND5菌株抗香蕉枯萎病菌相关非核糖体多肽合成酶基因簇的鉴定

为明确帚枝霉属内生真菌Sarocladium brachiariae HND5菌株具体的抑菌活性物质,在全基因组测序数据的基础上,利用生物信息学对抑菌活性物质合成基因簇进行定位,通过聚类分析和系统发育树分析对基因簇合成产物进行鉴定,并通过基因缺失突变构建及代谢组分析确定具体的抑菌活性物质。结果显示,HND5菌株共含有7个非核糖体多肽合成酶(non-ribosomal peptide synthetase,NRPS)基因簇,聚类分析结果表明基因簇29合成产物为噬铁素,基因簇30合成产物为类环孢霉素类抗菌多肽;系统发育树结合生物信息学结果显示基因簇30合成产物与已知环孢霉素结构差异大,为一个新型非核糖体多肽;将基因簇30 NRPS基因缺失突变后,HND5菌株丧失抑菌活性;同野生型菌株相比,基因簇30 NRPS基因缺失突变体缺失一个分子量为887.54 Da的多肽类物质。表明HND5菌株的抑菌活性物质为一个分子量为887.54 Da的新型类环孢霉素非核糖体多肽,基因簇30负责该多肽的生物合成。     

Antigenic characterization of rat louse Polyplax spinulosa

Number and relative molecular weights of proteins in whole body homogenates of the louse, Polyplax spinulosa were determined using SDS-PAGE. 13 protein components from the total of 28 bands were defined as Con A binding glycoproteins. In immunoblotting sera of lice infested laboratory rats of BN strain specifically recognized 3 major protein components with r.m.w. of 77,000, 105,000 and 230,000 Da and from 6 to 8 minor protein components with r.m.w. of 31,000-180,000 Da.     

Isolation and characterisation of fusaricidin-type compound-producing strain of <Emphasis Type="Italic">Paenibacillus polymyxa</Emphasis> SQR-21 active against <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f.sp. <Emphasis Type="Italic">nevium</Emphasis>

A bacterial strain was isolated from the rhizosphere of healthy watermelon plants in a heavily wilt-diseased field. This isolate was tentatively identified as Paenibacillus polymyxa (SQR-21) based on biochemical tests and partial 16S rRNA sequence similarity. The purified antifungal compounds were members of the fusaricidin group of cyclic depsipeptides having molecular masses of 883, 897, 947, and 961 Da with an unusual 15-guanidino-3-hydroxypentadecanoic acid moiety, bound to a free amino group. The strain SQR-21 was not able to produce antifungal volatile compounds but was able to produce cellulase, mannase, pectinase, protease, β-1,3-glucanase and lipase enzymes. However, the strain did not show any chitinase activity. Biocontrol potential of this strain was evaluated against Fusarium oxysporum cause of Fusarium wilt disease of watermelon in a greenhouse experiment. This strain combined with organic fertiliser decreased the disease incidence by 70% and increased the dry plant weight by 113% over the control.     

莲腐败病生防细菌3-b培养条件的研究

从土壤中筛选的经抑菌实验证明对镰刀菌(Fusarium oxysporum f.sp.nelumbicola)有拮抗作用的细菌3-b,测定其在不同培养基、培养温度和pH条件得到发酵液对镰刀菌的抑菌效果。结果表明:在32℃pH 7的条件下用PDB培养液培养得到的发酵液抑菌效果最好,抑菌直径为18 mm。该培养条件的获得为进一步开发防治白莲腐败病的生防菌剂提供理论依据。     

Similarity between Copper Resistance Genes from Pseudomonas syringae pv. actinidiae and P. syringae pv. tomato

Twenty-eight strains of Pseudomonas syringae pv. actinidiae isolated in 1984, 1987 and 1988 from kiwifruit orchards in Japan were tested for their resistance to copper sulfate. All strains isolated in 1984 were copper sensitive with a minimum inhibitory concentration (MIC) of cupric sulfate of 0.75 mM. However, some strains isolated in 1987 and 1988 were resistant, with the MIC ranging from 2.25 to 3.0 mM. All copper-resistant strains contained at least one of two plasmids, pPaCul (about 70.5 kb) or pPaCu2 (about 280 kb), or both. In a copper-resistant strain Pa429, the location of the copper-resistance gene(s) was examined by insertional inactivation with Tn5. The MIC of copper sulfate in the copper-sensitive mutant obtained by Tn5 tagging decreased from 2.75 to 0.75 mM. The 14.5 kb BamHI fragment, designated pPaCuB14, containing the same locus mutagenized with Tn5 was cloned from pPaCu1. However, pPaCuB14 did not confer copper resistance in the transformant of copper-sensitive strain Pa21R, suggesting that this clone did not contain a full set of copper-resistance gene(s). Then a cosmid library of pPaCu1 was constructed and six cosmid clones hybridized with pPaCuB14 were selected. One of the six cosmids, designated pPaCuC1, conferred a near wild-type level of copper resistance in the transformant of the copper-sensitive strain. pPaCuC1 had a homologous region that hybridized with all of the PCR-amplifled fragments of copA, copB, copR, and copS genes of P. syringae pv. tomato. DNA sequence analysis of the homologous region revealed the existence of four open reading frames (ORF A, B, R and S) oriented in the same direction. The predicted amino acid sequences of ORF A, B, R and S had 80, 70, 97 and 95% identity with CopA, B, R and S of P. syringae pv. tomato, respectively. Received 5 July 2001/ Accepted in revised form 27 September 2001     

抗稻瘟病菌株—诺卡氏菌A11的诱变育种

从四川农业大学农场土壤中分离得到的诺卡氏菌(Norcardia sp.),其代谢产物对稻瘟病菌有强烈的抑制作用。以此菌株为出发菌株,进行紫外线(UV)、硫酸二乙酯(DES)、微波(MV)诱变,诱变处理后获得的突变株中以UV处理120 s时正变率最高,其摇瓶复筛试验中,该变异株代谢产物拮抗活性比出发株提高43.4%。MV处理在20 s剂量下提高了32.8%;DES处理在浓度为0.2%时提高了24.8%。经连续传代8次,拮抗活性稳定。     

小麦根部内生细菌ZY-1对小麦白粉菌的抑菌活性及拮抗机理

从小麦根部分离到6株代谢表面活性剂的菌株,比较各菌株排油性和抑菌性,结合PCR方法优选出一株具广谱抗菌性且含有srfA、fenA、ituB和bacD等4个关键基因的细菌ZY-1。经BIOLOG生理生化试验测定和16S rDNA序列系统发育学分析,将其鉴定为蜡样芽孢杆菌。平板和盆栽试验结果显示,菌株ZY-1的发酵液对活体专性寄生菌小麦白粉菌的生防效果分别达到（97.94±1.25）%和（82.56±2.11）%,优于10 mg/L的三唑酮化学药剂（P<0.05）。组织学显微观察表明,菌株ZY-1发酵液对小麦白粉病菌的分生孢子萌发和吸器形成具有极显著的抑制作用。产脂肽细菌ZY-1是一株应用前景广阔的生防菌。     

Characterization of a novel potyvirus isolated from maize in Israel

A potyvirus (proposed name of Zea mosaic virus [ZeMV]) isolated from maize in Israel was analyzed by serology, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of capsid proteins, symptomatology, and sequencing. Parts of the nuclear inclusion b, coat protein, and 3' regions were sequenced; the amino acid sequence of ZeMV capsid was determined by time-of-flight mass spectrometry (TOFMS). The results of these analyses were compared with those of similar analyses of the following potyviruses: Maize dwarf mosaic virus (MDMV), Sugarcane mosaic virus strain MDB (SCMV-MDB), Johnsongrass mosaic virus(JGMV), Sorghum mosaic virus (SrMV), and an isolate of MDMV from Israel. Indirect enzyme-linked immunosorbent assay using ZeMV antiserum detected only ZeMV, and reciprocal tests using MDMV, JGMV, or SrMV antisera failed to detect ZeMV. ZeMV cross-reacted weakly when SCMV-MDB antiserum was used. The mass of ZeMV capsid was determined to be 36,810 Da by SDS-PAGE and 34,216 Da by TOFMS. The ZeMV systemically infected johnsongrass (Sorghum halepense), but did not infect oat (Avena sativa), pearl millet (Pennisetum glaucum), barley (Hordeum vulgare), or rye (Secale cereale). Necrosis was caused in 19 sorghum lines by SrMV, in 15 by ZeMV, in 14 by MDMV, and in 5 by JGMV and SCMV-MDB. The nucleic acid and amino acid sequences of ZeMV clearly showed that it is not a strain of JGMV, MDMV, SCMV, or SrMV.     

黄瓜枯萎病菌拮抗菌的筛选、鉴定和防效测定

为获得对黄瓜枯萎病菌Fusarium oxysporum f.sp.cucumerinum（FOC）具有拮抗作用的生防菌株,采用稀释涂布平板和平板生长对峙法从健康黄瓜根际土壤中分离、筛选对FOC具有强拮抗能力的拮抗细菌,并从形态学观察、生理生化特性、全细胞脂肪酸分析及16S rRNA和gyrB基因序列相似性对拮抗能力最强的菌株进行鉴定,同时测定拮抗菌无菌过滤发酵液对FOC菌丝生长、孢子萌发的抑制作用,并评价其发酵液对盆栽和大田黄瓜枯萎病的防治效果。研究结果显示,从健康黄瓜根际土壤中分离获得23株对FOC具有拮抗作用的细菌,其中菌株FJ17-4对FOC抑菌作用最强,鉴定为贝莱斯芽胞杆菌Bacillus velezensis。FJ17-4抑制FOC引起菌丝畸形、扭曲、膨胀、皱缩、缠绕等异常现象。10%无菌过滤液对FOC菌丝生长和孢子萌发的抑制率分别为70.96%和85.40%。50倍发酵液、菌体悬浮液和无菌过滤液盆栽防治效果分别为70.20%、58.87%和47.80%,大田防治效果分别为69.53%、58.46%和36.12%。综上,FJ17-4能有效抑制FOC,对黄瓜枯萎病具有显著的生防效果,具有广泛的应用前景。     

西农No.24菌株发酵产物对反枝苋的除草活性初步研究

西农No.24菌株是从陕西秦岭山区采集的200份土壤中分离得到的一株放线菌,经初步鉴定为链霉菌属(Streptomyces)。采用微量小杯法,通过发酵、洗脱和高效液相色谱(HPLC)分离,测定西农No.24菌株发酵液对反枝苋Amaranthus retroflexus L.的除草活性。结果表明,该菌株的发酵产物对反枝苋有较强的抑制生长作用。其发酵液对反枝苋种子萌发抑制率为26.7%,主根生长抑制率为80.6%,主茎生长抑制率为66.5%;离子交换液对反枝苋的种子萌发抑制率为42.9%,主根生长抑制率为93.1%,主茎生长抑制率为86.2%。经HPLC分离后得到8个馏份,其中馏份4的除草活性最强,对反枝苋的种子萌发抑制率为86.36%,主根生长抑制率为95.84%,主茎生长抑制率为93.76%。