Broad bean wilt virus 2 in yams

We identified Broad bean wilt virus 2 (BBWV-2) in yams based on particle morphology, test plant symptoms, protein features, aphid transmission, and molecular classification using nucleotide sequences of coat protein genes.     

嗜酸性PGPR菌株CLB-17的筛选、鉴定及其对烟草青枯病菌的生防活性

为提高生防菌在酸性土壤中对烟草青枯病的生防效果,采用室内酸性平板快速筛选法从烟草根际土壤中筛选嗜酸性拮抗细菌,应用Biolog GENⅢ自动微生物鉴定系统结合16S rDNA序列分析进行种类鉴定,并采用室内琼脂扩散法在pH 5.5和pH 7.0培养条件下比较其对烟草青枯病菌Ralstonia solanacearum的抑制活性,通过盆栽试验测定其对烟草青枯病的防效及对烟草的促生作用,通过测定胞外酶活性和铁载体含量分析其生防特性。结果表明:从烟草根际土壤中筛选获得1株具有高拮抗活性的嗜酸性细菌,命名为CLB-17,并将其鉴定为枯草芽胞杆菌Bacillus subtilis。在pH 5.5条件下,菌株CLB-17的拮抗活性最强,其发酵液对烟草青枯病菌的抑菌带宽度为7.11 mm,而在pH 7.0条件下抑菌带宽度为6.37 mm;在pH 5.5条件下,CLB-17菌株的铁载体相对含量(2.12)及蛋白酶活性(40.12 U/mL)、葡聚糖酶活性(142.52 U/mL)和酸性磷酸酯酶活性(6.42 U/mL)均高于pH 7.0条件下各指标(1.51、36.14 U/mL、110.24 U/mL和6.11 U/mL);该菌株在pH 5.5条件下对烟草青枯病的防效为76.99%,高于在pH 7.0条件下的防效66.18%;且烟草株高、整株干重、根干重和叶绿素含量均显著高于pH 7.0处理和空白对照。表明枯草芽胞杆菌CLB-17在酸性条件下的生防活性高,在酸性土壤中对烟草青枯病的生防效果更好,且对烟草生长具有促进作用。     

Nitric oxide-overproducing transformants of Pseudomonas fluorescens with enhanced biocontrol of tomato bacterial wilt

The relation between nitric oxide (NO) production and the protective ability of Pseudomonas fluorescens T5 against bacterial wilt disease in tomato was examined. The endogenous nitric oxide reductase gene of T5 was disrupted by homologous recombination using a suicide plasmid. Three disruptants were obtained, and all had higher levels of NO production. Infection with Ralstonia solanacearum was reduced in tomato plants treated with the NO-overproducing transformants compared with the wild type. These results suggest that the modification of pseudomonads to increase their level of NO production is a new approach to enhancing their biocontrol efficacy.     

Analysis of genetic relations between <Emphasis Type="Italic">Broad bean wilt virus 1</Emphasis> and <Emphasis Type="Italic">Broad bean wilt virus 2</Emphasis>

We determined the complete nucleotide sequence of RNA-1 and the 5-terminal region of RNA-2 from Broad bean wilt virus 1 (BBWV-1) isolate PV132. This report is the first analysis of the genome organization of BBWV-1. We also determined the complete nucleotide sequence of RNA-1 from Broad bean wilt virus 2 (BBWV-2) isolate IP and analyzed the genetic relations between BBWV-1 and BBWV-2. Similar to the BBWV-2 isolates, both RNAs of PV132 encoded a single large polyprotein, which was predicted to contain some functional proteins in a manner similar to those of comovirus. With respect to the deduced amino acid sequences of the mature proteins, PV132 and IP had only 20%–40% homology to comovirus. On the other hand, IP was 73%–98% homologous to BBWV-2 isolates, but PV132 was 39%–67% homologous to the isolates. Although the extent of the homologies differed, the homologies were limited between BBWV-1 and BBWV-2 not only for the coat protein but also for the other proteins. These results clearly support the placement of BBWV-1 and BBWV-2 in the genus Fabavirus as distinct species, proposed on the basis of double immunodiffusion tests.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB084450 (RNA-1 of isolate PV132), AB084451 (RNA-2 of isolate PV132), and AB023484 (RNA-1 of isolate IP)     

Phenotypic characterization of Pseudomonas fluorescens PfG32R and its spontaneous gacS mutants and biocontrol activity against bacterial wilt disease of tomato

Pseudomonas fluorescens strain PfG32R actively suppresses the occurrence of bacterial wilt disease of tomato caused by Ralstonia solanacearum. To determine the characteristics affected by spontaneous mutations in the gacS gene, three spontaneous mutants NR1, NR9, and ASW6 were tested for production of several enzymes, antimicrobial activity, and biocontrol activity against bacterial wilt disease of tomato in a greenhouse. Production of biosurfactant and of enzymes such as Tween-80 lipase, gelatin protease, and lecithinase; swarming activity; and antifungal activities against Fusarium oxysporum f. sp. radicis-lycopersici and F. graminearum were inactivated by mutations in the gacS gene, indicating that they are under the regulation of the GacS/GacA two-component regulatory system. Levan production, swimming, antifungal activity against Magnaporthe grisea, and antibacterial activity against R. solanacearum and Erwinia carotovora ssp. carotovora were not controlled by the GacS/GacA system. Biocontrol activities of the three spontaneous mutants varied from strain to strain, indicating that the biocontrol activities of PfG32R are influenced not only by the GacS/GacA system but also by other complex factors, which may not be regulated by the system.     

番茄疫霉根腐病生防菌的分离鉴定及其防治效果

 利用平板对峙法从山东寿光采集的土样中筛选出1株对番茄疫霉根腐病菌Phytophthora capsici Leonian具有较好拮抗作用的生防菌株ZF50,抑菌率为76.26%。经形态学、生理生化特性以及系统发育树分析,确定菌株ZF50为贝莱斯芽胞杆菌Bacillus velezensis。该菌株具有较广谱的抑菌能力,且在活体盆栽和田间小区条件下,将浓度为1×10⁸ cfu·mL^-1 ZF50菌悬液10 mL灌根后,对番茄疫霉根腐病的防治效果最好,盆栽防效为55.55%,田间小区防效为43.52%;且该菌株还具有明显的促生效果和产吲哚乙酸的能力。     

番茄疫霉根腐病生防菌的分离鉴定及其防治效果

 利用平板对峙法从山东寿光采集的土样中筛选出1株对番茄疫霉根腐病菌Phytophthora capsici Leonian具有较好拮抗作用的生防菌株ZF50,抑菌率为76.26%。经形态学、生理生化特性以及系统发育树分析,确定菌株ZF50为贝莱斯芽胞杆菌Bacillus velezensis。该菌株具有较广谱的抑菌能力,且在活体盆栽和田间小区条件下,将浓度为1×10⁸ cfu·mL^-1 ZF50菌悬液10 mL灌根后,对番茄疫霉根腐病的防治效果最好,盆栽防效为55.55%,田间小区防效为43.52%;且该菌株还具有明显的促生效果和产吲哚乙酸的能力。     

枯草芽胞杆菌B1409对番茄和辣椒的防病促生作用

为探讨枯草芽胞杆菌Bacillus subtilis菌株B1409对番茄早疫病和辣椒疫霉病的防效和生防机制,采用平板对峙法和盆栽法测定了该菌株对番茄早疫病菌和辣椒疫霉病菌菌丝生长的抑制作用、对2种病害的盆栽防效以及对番茄和辣椒植株促生长效果和防御酶活性的影响。结果表明:菌株B1409能明显抑制番茄早疫病菌和辣椒疫霉病菌菌丝生长,且导致菌丝发生畸变。10~8CFU/mL菌株B1409菌液对番茄早疫病和辣椒疫霉病的预防效果分别为67.82%和61.22%,治疗效果分别为41.22%和56.43%。不同浓度B1409菌液均能促进番茄和辣椒植株生长,并能增强其体内超氧化物歧化酶、过氧化物酶和过氧化氢酶活性,且浓度越高促进效果越明显。番茄和辣椒植株的平均干重分别在10~2CFU/mL和10~4CFU/mL B1409菌液处理后显著高于对照,增长率分别为42.35%和4.87%。番茄和辣椒植株经10~2CFU/mL B1409菌液处理后,体内超氧化物歧化酶活性比对照显著增加,增长率分别为91.23%和19.58%。研究表明枯草芽胞杆菌B1409菌株可通过直接抑制菌丝生长及诱导植物体自身抗病性等方式来有效防治番茄早疫病和辣椒疫霉病。     

枯草芽孢杆菌微囊剂的制备及其对番茄立枯病的防治效果

为开发环境相容性好、贮藏稳定性高的生物农药新剂型,采用喷雾干燥法制备了枯草芽孢杆菌微囊剂。通过单因素试验确定该微囊剂壁材麦芽糊精与芯材枯草芽孢杆菌发酵液(以下简称发酵液)的最佳配比;通过正交试验优化了喷雾干燥条件;通过田间试验验证了该微囊剂对番茄立枯病的防治效果。结果表明:制备枯草芽孢杆菌微囊剂时,壁材与芯材的最佳配比为m(麦芽糊精)∶V(发酵液)=1∶1;喷雾干燥的最优条件为进风温度125℃,进样流量750 m L/h,喷雾压力0.20 M Pa;所制备微囊剂常温贮存360 d后,菌体存活率仍高达91.36%,显著高于对照枯草芽孢杆菌可湿性粉剂。田间药效试验表明,该枯草芽孢杆菌微囊剂用量为300 g/hm2(芽孢浓度为1.5×107cfu/m2)时,对番茄立枯病的防效最高,为72.76%。     

植物青枯病高效生防菌株PBW1培养工艺研究

植物青枯病生防菌株PBW1对植物青枯病的防效可高达70%以上。本文首先获得了PBW1 500 mL摇瓶培养的最佳条件:30℃、初始pH7、接种量为1%、装液量为120 mL、摇床转速为220 r/min;在最佳条件下培养48 h的菌体浓度达4.2×10¹²cfu/mL;同时还对500 mL摇瓶培养过程中菌体生长、还原糖、总糖及pH变化特征进行了研究。此外,本文还在6L全自动发酵罐上对PBW1培养过程中的菌体生长、还原糖、氨基氮、pH和DO的变化特征进行了研究,培养48h的菌体浓度达4.76×10¹²cfu/mL。     

Granular formulation of Fusarium oxysporum for biological control of faba bean and tomato Orobanche

BACKGROUND: Orobanche spp. represent a serious threat to a wide range of crops. They are difficult targets for herbicides, and biological control could provide a possible solution. This work therefore aimed to formulate mycoherbicides of Fusarium with adequate shelf life and virulence against Orobanche but safe to faba bean and tomato. RESULTS: Only two isolates of Fusarium oxysporum Schlecht. (Foxy I and Foxy II) obtained from diseased Orobanche shoots were found to be pathogenic to Orobanche crenata Forsk. and Orobanche ramosa L. Conidial suspension of both isolates significantly decreased germination, attachments and tubercles of Orobanche. Microconidia and chlamydospores of both isolates were formulated as mycoherbicides encapsulated in a wheat flour–kaolin matrix (four different formulations). All formulations greatly diminished Orobanche emerged shoots, total shoot number, shoot height, attachment of emerged shoots, the germinated seeds that succeeded in emerging above the soil surface and dry weight. Meanwhile, disease incidence and disease severity of emerged shoots were enhanced. The shelf life was adequate, particularly for coarse, freshly prepared, low‐temperature‐stored, microconidia‐rich formulations. The induced growth reduction of Orobanche‐infected host plants seemed to be nullified by formulations, particularly at the highest dose. CONCLUSION: These formulations seemed to destroy Orobanche but appeared harmless to host plants. Hence, they could be efficiently used as mycoherbicides for biological control of Orobanche in faba bean and tomato. Copyright © 2008 Society of Chemical Industry     

青枯雷尔氏菌无致病力突变菌株的构建及其防效评价模型分析

 利用青枯雷尔氏菌(Ralstonia solanacearum)无致病力菌株防治番茄青枯病具有很好的应用潜力。作者通过分离筛选自然弱毒株、⁶⁰Co辐射诱变和EZ-Tn5插入诱变,分别获得3、12和40株青枯雷尔氏菌无致病力突变菌株。经盆栽番茄苗致病性检测,15 d后均未发病,证实均为无致病力青枯雷尔氏菌。进一步对番茄青枯病的防治试验表明,从番茄青枯病发病田块分离的无致病力突变菌株FJAT1458的防治效果最好,防效达100%。该菌株能定殖番茄植株根系土壤、根部和茎部,定殖数量均表现为“先增后减”的趋势,并且接种浓度越大、苗龄越小,定殖数量越大。从构建的防效模型可以看出,不同接种浓度条件下,植株发病率随时间变化符合的回归方程不同,相关系数R值也不同,接种浓度越大,R值越小。本研究获得的青枯雷尔氏菌无致病力突变菌株FJAT1458对番茄青枯病具有很好的防病效果。     

产香真菌GS-1菌株鉴定及其挥发性物质对番茄灰霉病的生防效果

为鉴定产香真菌GS-1菌株物种分类和评价其挥发性物质对番茄灰霉病的生防效果,通过形态学和ITS(internal transcribed spacer)序列分析对菌株GS-1进行物种鉴定,以对扣法测定其挥发物对番茄灰霉病菌Botrytis cinerea、水稻胡麻斑病菌Bipolaris oryzae、小麦赤霉病菌Fusarium graminearum、白菜黑斑病菌Alternaria brassicae和莴苣菌核病菌Sclerotinia sclerotiorum共5种植物病原真菌的熏蒸抑制活性,以果实针刺法测定挥发物对番茄灰霉病的抑制作用,并观察对其菌丝生长的影响。结果显示,菌株GS-1为普通瘤座孢Tubercularia vulgaris;其挥发物对5种植物病原真菌均有明显的熏蒸抑制作用,72 h的抑制率为58.05%~75.81%;其挥发物可造成番茄灰霉病菌菌丝畸形、分枝增多、原生质外渗等,对番茄果实灰霉病的防效72 h后达53.62%。表明产香真菌普通瘤座孢GS-1菌株挥发物对番茄灰霉病有良好的生防效果。     

苯甲酸和肉桂酸促进蚕豆枯萎病发生的生理生化机制

为探讨苯甲酸和肉桂酸胁迫促进蚕豆枯萎病发生的生理生化机制,在接种蚕豆枯萎病菌Fusarium oxysporum f. sp. fabae的条件下,研究了苯甲酸和肉桂酸4个浓度处理对蚕豆枯萎病发生、植株抗氧化酶活性、膜质过氧化程度及病程相关蛋白的影响。结果表明,与对照相比,不同浓度苯甲酸处理下蚕豆枯萎病的病情指数提高了25.0%～362.5%;蚕豆根系和叶片中过氧化物酶(POD)活性分别降低了15.7%～31.4%和21.3%～38.7%,过氧化氢酶(CAT)活性分别降低了37.7%～42.8%和28.4%～44.8%,丙二醛(MDA)含量分别提高了28.9%～42.6%和16.4%～45.0%,根系几丁质酶和β-1,3-葡聚糖酶活性降低了23.6%～39.4%和17.4%～38.7%。与对照相比,不同浓度肉桂酸处理下蚕豆枯萎病的病情指数提高了37.5%～350.6%,蚕豆根系和叶片中POD活性分别降低了17.1%～48.6%和13.4%～36.0%,CAT活性分别降低了15.6%～61.0%和18.5%～57.9%,MDA含量分别提高了24.5%～51.8%和42.0%～94.1%,根系几丁质酶和β-1,3-葡聚糖酶活性分别降低了29.1%～48.9%和21.3%～40.2%。苯甲酸和肉桂酸浓度高于50 mg/L时显著降低蚕豆抗氧化酶性能,加剧细胞破损程度,抑制病程相关蛋白表达,降低自身生理生化抗性,有利于病原菌入侵,促进枯萎病发生,其中肉桂酸的促进效应大于苯甲酸。     

Potential of a novel antibiotic, 2-methylheptyl isonicotinate, as a biocontrol agent against fusarial wilt of crucifers

Screening for newer bioactive compounds from microbial metabolites resulted in the isolation of a novel antibiotic from the culture filtrate, Streptomyces sp 201, of a soil. The bioactive compound, with antifungal and antibacterial activity, was identified as 2-methylheptyl isonicotinate. The antifungal activity of live culture, culture broth and the isolated bioactive compound showed marked inhibition against dominant soil-borne phytopathogens such as Fusarium oxysporum Schlect, F moniliforme Sheldon, F semitectum Berkeley & Ravenel, F solani (Martius) Sacc and Rhizoctonia solani Kuehn. The compound had no effect on seed germination and seedling development as displayed by root and stem growth of the test plant species. In pot experiments with seedlings of cruciferous plants such as Raphanus sativus L (radish), Brassica campestris L (yellow mustard), Brassica oleracea var botrytis L (cauliflower), the antibiotic compound showed promising protective activity of 92% when seeds of the test plants were treated at a dose of 50 micrograms ml-1 prior to sowing. Seed treatment with a spore suspension (3 x 10(8) spores ml-1) of the Streptomyces sp 201 displayed protective activity in the range of 56-60%. Seeds coated with 2.5% methyl cellulose-amended spores of the antagonist showed protective activity in the range of 64-72%. Further, seed treatment with the culture filtrate of the antagonist also showed promising protective activity in the range of 64-84%.     

Involvement of jasmonic acid signalling in bacterial wilt disease resistance induced by biocontrol agent Pythium oligandrum in tomato

When the biocontrol agent Pythium oligandrum (PO) colonizes the rhizosphere, it suppresses bacterial wilt disease in tomato (Solanum lycopersicum cv. Micro‐Tom) caused by Ralstonia solanacearum, and a homogenate of its mycelia exhibits elicitor activity, inducing an ethylene (ET)‐dependent defence response in Micro‐Tom. Since salicylic acid (SA) and jasmonic acid (JA) play an important role in plant defence responses to pathogens, the involvement of SA‐ and JA‐dependent signal transduction pathways in resistance to R. solanacearum was investigated in tomato roots treated with a mycelial homogenate of PO. Bacterial wilt disease was also suppressed in tomato cv. Moneymaker treated with the PO homogenate. However, the SA‐inducible PR‐1(P6) gene was not up‐regulated in either Micro‐Tom or Moneymaker. SA did not accumulate in homogenate‐treated roots in comparison with distilled water‐treated controls, even 24 h after inoculation. Induced resistance against R. solanacearum was not compromised in SA‐non‐accumulating NahG transgenic plants treated with the PO homogenate. On the other hand, the expression of the JA‐responsive gene for the basic PR‐6 protein was induced in both tomato cultivars treated with the PO homogenate. Furthermore, quantitative disease assays showed that the induced resistance against R. solanacearum was compromized in PO homogenate‐treated jai1‐1 mutant plants defective in JA signalling. These results indicated that the JA‐dependent signalling pathway is required for PO‐induced resistance against R. solanacearum in tomato.     

番茄青枯病植物疫苗胶悬菌剂的制备及其对病害的防治效果

研究了植物疫苗胶悬剂的制备工艺,确定胶悬剂制备的适宜参数为:琼脂终浓度为2.0‰、氯化钠终浓度为2.0%和pH为7.0。在此条件下,胶悬效果最好,质地均匀,无上下分层。试验了植物疫苗胶悬剂对番茄青枯病的防治效果,结果表明,该胶悬剂能较好地抑制番茄青枯病的发生,其防效(77.45%)与化学农药72%农用硫酸链霉素SP(77.23%)相当,配合生防菌剂20亿活芽孢/g蜡质芽孢杆菌WP施用,效果更佳,防效达81.81%。     

Soil solarization as a substitute for methyl bromide fumigation in greenhouse tomato production in Cyprus

Preplant soil fumigation with methyl bromide (MB) is presently standard practice in greenhouse tomato production. Since this compound is scheduled to be phased out by 2005, the possibility of using solarization as an alternative soil disinfestation method was examined in four greenhouse tomato trials. Solarization was applied for 8 weeks in July-August, using transparent polyethylene sheets for soil mulching, and compared with MB fumigation applied in September, before planting, at 80 g/m². Solarization raised the maximum soil temperature by 9°C and reduced the population density ofFusarium spp. in soil by 91–98%. Similar reductions of soil inoculum (95–99%) were obtained with MB fumigation. Both methods provided effective control of Fusarium wilt, Verticillium wilt and corky root rot on tomato plants. MB fumigation was in addition highly effective against root-knot nematodes, whereas nematode control with solarization did not exceed 50%. Both treatments resulted in similar fruit yield increases, ranging within 90–140% compared with plants grown in untreated soil. During the second cropping season following soil treatment, solarization exhibited two times higher residual effectiveness against vascular wilt diseases compared with MB fumigation. The latter treatment, however, was superior to solarization in its residual effectiveness against root-knot nematodes and to a lesser extent against corky root rot. Fruit yields from solarized and MB-fumigated soil during the second cropping season were higher than those obtained from untreated soil by approximately 35% and 60%, respectively. In Cyprus, solarization appears to be an effective alternative to MB fumigation in greenhouse tomato production, especially if integrated with other approaches enabling more effective nematode control.     

小麦全蚀病菌拮抗细菌的筛选及生防效果

为筛选小麦全蚀病菌Gaeumannomyces tritici的拮抗细菌,从健康小麦根际土壤中分离和筛选拮抗细菌,采用对峙培养法和室内盆栽试验测定拮抗细菌对小麦全蚀病菌的抑制活性及生防效果,通过形态学特征观察、生理生化特性测定和保守序列分析确定其分类地位。结果表明,从小麦根际土壤中分离并筛选获得2株具有较好拮抗效果的菌株XJ-3和XJ-4,对小麦全蚀病菌菌丝生长的抑制率分别为64.31%和65.25%。基于菌株XJ-3和XJ-4的形态学特征、生理生化特性和gyrB基因系统进化分析结果,将其均鉴定为贝莱斯芽胞杆菌Bacillus velezensis。菌株XJ-3和XJ-4的发酵液中均含有一类具抗真菌活性、耐热性的非蛋白类次生代谢产物;经菌株XJ-3和XJ-4发酵液处理小麦幼苗后,株高较对照分别显著增加了6.48%和13.12%,干重较对照分别增加了1.30%和14.29%,其中菌株XJ-4的促生效果相对较好;对小麦全蚀病的盆栽防效分别达到51.29%和52.46%。表明筛选得到的贝莱斯芽胞杆菌XJ-3和XJ-4可用于小麦全蚀病的生物防治,具有开发为生防制剂的潜力。