土壤杆菌K1026对果树根癌病的生物防治

由致病性根癌土壤杆菌Agrobacterium spp.侵染引起的根癌病对山东及我国其他省份的樱桃、桃及其他果树生产造成严重的影响.土壤杆菌Agrobacterium rhizogenes K1026在澳大利亚已制成生防菌剂并在全球多个国家实现商品化,但在我国果树育苗和生产行业中还未得到广泛应用.本文从山东樱桃和桃树根癌组织及感病土壤中分别分离到100余株致病性土壤杆菌,经鉴定均属于生物Ⅱ型土壤杆菌;利用双层平板抑菌试验,检测了37株病原菌对菌株K1026的敏感性,结果显示,所有菌株均对K1026产生的抗生物质敏感,可以在Stonier,s培养基上产生5种以上不同类型的抑菌圈;胡萝卜切片试验显示,菌株K1026可完全抑制37株病原菌对胡萝卜的致瘤活性.上述结果为土壤杆菌K1026用于山东省樱桃及桃树根癌病的生物防治奠定了基础.     

K84的生物防治效果与土壤杆菌Ti质粒类型的关系

用K84对我国不同寄主来源的68个Agrobacterium tumefaciens菌株进行生物防治试验,结果表明:K84对我国桃树菌株的防治效果显著,对新疆的啤酒花菌株也有效;但对浙江萧山的部分啤酒花菌株无效;对甜菜菌株的防治效果不佳;对葡萄菌株基本上无效。对致病菌株的质粒类型的研究结果表明:桃树、啤酒花和甜菜菌株基本上都是胭脂碱(Nopaline)型质粒、葡萄菌株基本上是章鱼碱(Octopine)型质拉。试验中还发现不少胭脂碱型菌株不能被K84所防治。此外,在甜菜和毛白杨菌株中也发现了对细菌素A84(agrocin84)敏感,但不能被K84防治的菌株。     

芽胞杆菌种衣剂对玉米茎基腐病的防治效果及对玉米的促生作用

为明确玉米内生细菌YY4和YG2菌株的生防应用潜力,采用滤纸片法测定两者对禾谷镰孢菌Fusarium graminearum和拟轮枝镰孢菌F. verticillioides的抑制作用,结合形态学特征、16S r DNA及gyrA基因序列分析对其进行鉴定,将菌株YY4与YG2的发酵液以体积比4∶1混合,再添加助剂制成生物种衣剂,设置3种药种质量比(1∶40、1∶50和1∶60)进行田间促生作用及防治效果的测定,并分析其对玉米产量的影响。结果表明,YY4菌株对禾谷镰孢菌和拟轮枝镰孢菌的抑制率分别为62.32%和59.83%,YG2菌株的抑制率则分别为63.40%和60.72%;YY4菌株被鉴定为贝莱斯芽胞杆菌Bacillus velezensis,YG2菌株被鉴定为耐盐芽胞杆菌B. halotolerans;这2株芽胞杆菌混合制成的生物种衣剂按药种质量比1∶50包衣处理后15 d,玉米的出苗率和株高分别为74.43%和5.16 cm,均显著高于空白对照和化学种衣剂50%克菌丹可湿性粉剂及15.5%福美双·克百威悬浮种衣剂包衣处理;且该处理对玉米茎基腐病的防治效果达68.89%,玉米产量分...     

芽孢杆菌对黄瓜霜霉病的防治效果研究

经黄瓜霜霉病孢子囊萌发抑制试验明确,芽孢杆菌菌株Z-X-3、Z-X-10对黄瓜霜霉病菌有较强拮抗作用。离体叶片法和温室防病试验表明,两菌株对黄瓜霜霉病有较好的防治效果,相对保护效果分别达46.42％、41.55％;治疗效果分别达46.30％、44.44％,高于农药克霜氰的31.81％、37.03％。     

枯草芽孢杆菌B-903菌株的研究Ⅰ.对植物病原菌的抑制作用和防治试验

本文报道枯草芽孢杆菌Ｂ－９０３菌株对棉枯萎等多种土传镰刀菌和苹果轮纹病等的抑菌活性。电镜和光学镜观察证实,该菌抗菌物质可造成病菌细胞畸形,胞内物质外泄和细胞壁崩溃。盆栽和田间试验,证明以Ｂ－９０３ 菌液浸种可显著降低棉炭疽病等棉苗病害和菠菜枯萎病的苗期发病率,防治效果分别为５１．６７％ ～６９．４４％ 和６８．１％ ～７６．２％ 。果园喷雾可使苹果轮纹病和叶斑病的防效相当于化学药剂处理,以Ｂ－９０３菌液３０～６０ 倍稀释液浸果可使贮藏期１５天和３０天的烂果率比清水对照降低５０％ 以上。     

枯草芽孢杆菌B—903菌株的研究Ⅰ.对植物病原菌的抑制作用和防治 …

本文报道枯草芽孢杆菌Ｂ－９０３菌株对棉枯萎等多种土传镰刀菌和苹果轮纹病等的抑菌活性。电镜和光学镜观察证实,该菌抗菌物质可赞成病菌细胞畸形,胞内物质外泄和细腻壁崩溃。盆栽和田间试验,证明以Ｂ－９０３菌液浸种可显著降低棉炭疽病等棉苗丰和菠菜枯萎病的苗期发病率,防治效果分别为５１．６７％～６９。４４％和６８．１％～７６．２％。果园喷雾可使苹果轮纹病和叶斑病的防效相当于化学药剂得到处理,以Ｂ－９０３菌液３     

两株荧光假单胞杆菌菌株对烟草黑胫病病原菌的抑制作用

烟草黑胫病是一种典型的土传病害，在我国，除个别较寒冷的地区，各主要产烟省均有不同程度的发生。其中安徽、河南、山东为历史上的重病区；云南、贵州、福建、广东、湖南、四川等烟区发生也相当普遍，且多与烟草青枯病混合发生，危害更为严重。根际细菌防治土传病害是     

枯草芽胞杆菌发酵液对苹果树腐烂病的防治效果

为明确来自苹果树上的1株生防菌LF17对腐烂病菌的抑制作用及其发酵液对腐烂病的防治效果,采用形态学结合16S rDNA和gyrA基因序列分析对菌株LF17进行了鉴定,并通过滤纸法测定了发酵液对腐烂病菌的抑制率,利用涂抹发酵液的方法测定了发酵液对腐烂病的防效。结果表明,菌株LF17发酵液对腐烂病菌的抑菌率为93.80%,与甲基硫菌灵(94.20%)抑菌率相当,显著高于嘧菌酯(42.60%)、辛菌胺(52.80%)和苯醚甲环唑(81.30%);离体枝条防治结果表明,发酵液水剂和膏剂对腐烂病的防效为78.59%～80.40%,与甲基硫菌灵(76.93%)相比差异不显著,与嘧菌酯(44.15%)、辛菌胺(68.25%)、苯醚甲环唑(74.45%)相比差异显著;田间试验表明,涂有发酵液的伤口愈合面积为9.37～9.79 cm~2,病疤复发率为3.82%～4.56%,与其他4种药剂相比差异显著。研究表明菌株LF17发酵液对腐烂病菌有明显抑制作用,且可促进伤口的愈合并显著降低腐烂病的复发。     

稻瘟病生防芽胞杆菌的筛选及防治效果

筛选稻瘟病生防芽胞杆菌,为水稻可持续发展中稻瘟病的防治提供生防种质资源。通过平板对峙培养和离体叶片法筛选稻瘟病菌的拮抗细菌,通过形态特征观察、biolog和16S rDNA序列分析法进行鉴定,田间试验明确供试菌株对稻瘟病的防治效果和促生作用。从280株分离菌中筛选获得3株拮抗细菌对稻瘟病菌具有较好的抑制作用,在番茄燕麦培养基平板上与稻瘟病菌Magnaporthe oryzae P131对峙培养2 d后,菌株SYX04和SYX20的菌丝生长抑制率分别是95.6%和97.5%,菌株S?22菌丝生长抑制率是75%;离体叶片法明确SYX04和SYX20防治效果分别是89.6%和91.9%,与20%三环唑溶液没有显著差异,菌株S?22的防治效果是80.1%。根据16S rDNA序列分析比对结果,菌株SYX04和SYX20与枯草芽胞杆菌属基因序列的同源性最高,结合形态观察和biolog鉴定结果,确定这2株菌为枯草芽胞杆菌Bacillus subtilis,菌株S?22鉴定为多食鞘氨醇杆菌Sphingobacterium multivorum。田间试验结果表明,菌株SYX04和SYX20对水稻有较好的促生作用,分别促进水稻根长6.7%和13.2%、增加株高7.8%和8.2%、增加穗重均为23.1%、增加千粒重分别达到10.7%和11.1%。菌株SYX20对叶瘟的田间防效是73.5%～83.5%,对穗瘟的防治效果是64.0%～85.6%,与75%三环唑可湿性粉剂之间没有显著差异。菌株SYX04对叶瘟的防治效果是71.5%～82.6%,对穗瘟的防治效果是63.5%～83.8%,与绿地康处理之间没有显著差异,但与75%三环唑可湿性粉剂有差异。菌株S?22对叶瘟的防治效果是69.2%～73.3%,对穗瘟的防治效果是61.6%～77.9%。     

短小芽胞杆菌AR03对烟草炭疽病的抑制作用

为测定短小芽胞杆菌AR03菌株对烟草炭疽病菌的室内拮抗活性和盆栽防治效果,采用平板拮抗法初步筛选菌株拮抗活性,采用抑制菌丝生长法及抑制孢子萌发法筛选该菌株菌悬液的抑菌浓度,采用喷雾法进行防治效果测定。AR03菌株对烟草炭疽病菌的拮抗活性很强,能显著抑制菌丝生长、孢子萌发和病害发生,浓度为1×107 cfu/mL的AR03发酵液能有效抑制菌丝的生长和孢子萌发,AR03菌悬液(1×108 cfu/mL)对炭疽病的盆栽防治效果为83.03%,与对照药剂25%三唑酮可湿性粉剂的防治效果相当。AR03菌株是一株优良的广谱高效生防菌株。     

Evidence of pAgK84 transfer from Agrobacterium rhizogenes K84 to natural pathogenic Agrobacterium spp. in an Italian peach nursery

Nine Italian peach nurseries, which use Agrobacterium rhizogenes strain K84 to protect plants from crown gall, were monitored for three years with the aim of determining whether transconjugant populations may arise following plasmid exchanges between K84 and autochtonous soil agrobacteria. Six hundred and seventy-eight Agrobacterium isolates were obtained from 120 tumours developed on apricot and peach rootstocks that had been treated in pre-planting with the antagonist. Agrobacteria were characterized for pathogenicity, biovar, opine catabolism and agrocin 84 sensitivity. Colony hybridization was used for screening the isolates harbouring plasmids pTi and/or pAgK84. Analysis of plasmid content and Southern blotting were performed on putative transconjugant agrobacteria found in tumours collected from one nursery where a biological control breakdown was observed. The RFLP analysis of 16S + IGS regions showed that pAgK84 was transferred from the antagonist to virulent and avirulent soil agrobacteria belonging to different ribotypes. Pathogenic transconjugants, inoculated on GF677 rootstocks, were not controlled in vivo by K84 and stably maintained pTi and pAgK84 in the bacterial cells for at least one year. At the end of a biocontrol trial, new transconjugant tumorigenic agrobacteria originated by the transfer of pAgK84 to the pathogen. Virulent and avirulent transconjugants may represent a real threat for biological control by K84 strain since all of them produced agrocin and were insensitive to it. Survival in soil of these populations could make the future application of K84 ineffective.     

Mutations affecting chemotaxis of Agrobacterium vitis strain E26 also alter attachment to grapevine roots and biocontrol of crown gall disease

Crown gall in grapevine, caused by tumorigenic Agrobacterium vitis strains, can cause severe losses in most viticulture regions worldwide. The only effective means of control is through cultivation practices. One non-tumorigenic A. vitis strain, E26, can prevent crown gall infection when applied to wounds on grapevine prior to or simultaneous with inoculation of tumorigenic strains. ME19, a Tn5 mutant of strain E26, was impaired in terms of its ability to be chemo-attracted by grapevine root tissue extracts and its attachment to grapevine roots, and had reduced biological control activity; it did not significantly differ from the wild-type strain of E26 in phenotypes of agrocin production, growth in minimum medium or swarming activity. Complementation of ME19 with the cosmid clone of CP1543 from an E26 DNA library restored the chemotaxis, attachment, and biocontrol phenotypes. A 7·3-kb Kpn I fragment from CP1543 was cloned and sequenced, and sequence analysis revealed that the Tn 5 insertion occurred in a region that shares a significant homology with genes coding for methyl-accepting chemotaxis proteins (MCPs) in many bacteria. Complementation of the mcp gene mutants restored the affected phenotypes to the level of wild-type E26. An in-frame deletion mutant of the mcp gene was generated and was determined to have the same phenotypes as the original Tn5 mutant.     

The Pathogen of Crown Gall Disease on Flowering Cherry and its Sensitivity to Strain K1026

Six strains of crown gall bacteria were isolated from flowering cherry. It was revealed by Otten paper electrophoresis that of the six strains, only BYH18-4 possessed the octopine type Ti plasmid, the remainder having nopaline type Ti plasmid. BYH5-1 was identified by physiological and biochemical tests to be Agrobacterium tumefaciens (originally biovar 1). The other five were A. rhizogenes (originally biovar 2). It was demonstrated with Stonier's method of double layer medium that flowering cherry crown gall bacteria exhibited different sensitivities to agrocin produced by biocontrol strain K1026. Strain K1026 on greenhouse-grown sunflower seedlings exerted a relatively potent inhibitory action on flowering cherry crown gall bacteria. Artificial inoculation showed that K1026 produced 67–99% inhibition of flowering cherry crown gall disease, compared with the treatment of inoculation with crown gall bacteria only.     

Inhibition of crown gall formation by Agrobacterium radiobacter biovar 3 strains isolated from grapevine

Graft unions of nursery stock of grapevine (Vitis vinifera L.) collected in Japan yielded pathogenic and nonpathogenic strains of Agrobacterium. On the basis of classical diagnostic tests, a sequence analysis, and a multiplex polymerase chain reaction method previously reported, the pathogenic strain was identified as Agrobacterium tumefaciens biovar 3, whereas the nonpathogenic strains were assigned to Agrobacterium radiobacter biovar 3. Stems of tomato (Lycopersicon esculentum Mill.) seedlings were inoculated with both A. tumefaciens biovar 3 strain G-Ag-27 as a pathogen and one of the control strains isolated from grapevine or A. radiobacter biovar 2 strain K84 as competitors to assay the suppression of gall formation caused by the pathogen. In a test with a 1 : 1 pathogen/nonpathogen cell ratio, all A. radiobacter biovar 3 strains reduced gall incidence and size compared to that of the positive control inoculated only with the pathogen. Strain VAR03-1 was especially effective in reducing the incidence of gall formation on grapevine and reduced gall size by 84%–100% of those on the positive control. Many tested nonpathogenic biovar 3 strains were bacteriocinogenic, causing an inhibition zone against A. tumefaciens biovar 3 strains on YMA medium. Strain VAR03-1 was the most effective against indicator strains and appears to be a promising agent for controlling crown gall of grapevine.     

A short‐length single chimeric transgene induces simultaneous silencing of Agrobacterium tumefaciens oncogenes and resistance to crown gall

Transformation with self‐complementary oncogene sequences was used to silence the Agrobacterium tumefaciens oncogenes ipt and iaaM. The silencing response was triggered by using a very short chimeric sequence where conserved fragments from both oncogenes were fused in one unique transgene. Most T₀ transgenic tobacco lines and T₁ seedlings evaluated in vitro had intermediate or very low susceptibility to A. tumefaciens as compared with the wildtype plants. A greenhouse evaluation of whole plants confirmed the lines that were resistant. Low levels of transgene hairpin RNA (hpRNA) coupled with small interfering RNA (siRNA) accumulation correlated with oncogene silencing and, therefore, resistance to crown gall. After infection with the oncogenic strain, much lower levels of the oncogenes’ mRNA were found in resistant lines than in wildtype plants. The frequency of resistant lines, with few or no symptoms, produced with the chimeric construct was similar to the highest reported efficiencies obtained by using sense and antisense whole oncogene sequences.     

泰安樱桃根癌病致病菌分离及生物型鉴定

由致病性土壤杆菌侵染引起的根癌病严重影响了樱桃树的生长?利用选择性培养基, 以阿糖醇和赤藓糖醇作为不同碳源, 从山东泰安食用樱桃根部瘤状组织和感病根际土壤中分离到土壤杆菌100余株, 通过胡萝卜切片法和番茄茎部针刺接种法从中筛选到31株有致瘤活性的菌株?经16S rDNA和recA基因序列分析, 结合生物型检测鉴定出96%以上的致病菌属原生物Ⅱ型(Agrobacterium rhizogenes), 只有1株致病菌为原生物Ⅰ型(A. tumefaciens)?根据Ti质粒上致病相关基因保守序列设计3对引物, 12株有致瘤活性菌株中11株PCR扩增到目的条带?经分子生物学测定和高压纸电泳检测, 所有致病菌株的质粒均为胭脂碱型?建立了致病型土壤杆菌的分子生物学快速鉴定方法, 为定向选择生防菌株提供基础?     

Efficacy of olive mill waste water and its derivatives in the suppression of crown gall disease of bitter almond

Olive mill waste water (OMW) and some of its indigenous bacterial strains were tested in vitro and in planta for their efficacy against crown gall disease caused by Agrobacterium tumefaciens. OMW and polyphenols displayed a high level of antibacterial activity, however the volatile fraction was less efficient as only a bacteriostatic effect was observed. In pot experiments, the percentage of bitter almond rootstock showing symptoms of crown gall was significantly reduced with the dosage rate of OMW 1% as compared to the control (highly natural infected soil treated with water). Five indigenous bacterial strains isolated from OMW exhibited an antagonistic effect against the bacterium. Based on the gene 16S rRNA sequence analysis, one isolate showed 99.2% similarity to known sequences of Bacillus subtilis, one isolate demonstrated high percentage similarities (99.3%) to the genera Bacillus pumilis, and two isolates were associated with Stenotrophomonas maltophilia and Pseudomonas putida 100% and 99.6% similarities respectively. Among these bacteria, the strain B1 proved efficient against the soil borne pathogen in vitro and pot experiments. Our study in controlled conditions suggested that the addition of OMW to soil exerts significant disease suppressiveness against A. tumefaciens. Thabet Yangui and Ali Rhouma contributed equally to this work and are regarded as joint first authors.     

月季根癌病病原菌分离及抗病资源初步筛选

从月季品种金玛丽、曼海姆、杏花村、梅郎口红的根部肿瘤组织中纯化了9株分离物,根据其在MW选择性培养基上的单菌落形态初步判断其为根癌土壤杆菌。以根癌土壤杆菌高度保守的virD2和ipt基因的部分序列设计引物对分离的菌株进行PCR检测,其中有6株菌株能扩增出virD2和ipt基因片段,为根癌土壤杆菌毒性菌株。采用针刺涂抹法接种向日葵、荷花蔷薇幼茎,6株菌株均能在供试植株上形成肿瘤,一个月后肿瘤大小有显著差异,说明分离获得的根癌土壤杆菌株毒性有差异。以强毒性菌株J-5-1在田间接种了部分蔷薇属野生资源,以鉴定其对根癌病的抗性。根据发病率、肿瘤大小、肿瘤干质量以及感病后植株生长状况将植物的根癌病抗性分为高度抗病、中度抗病、中度感病、高度感病4个类型。