烟草黑胫病菌培养特性的研究

研究了云南烟草黑胫病菌在不同培养基、不同温度的生长情况及不同菌龄与不同诱导时间孢子囊的产生数量。结果表明,菌株在8种供试培养基中,以燕麦培养基和选择性培养基上菌落生长最好,其次为胡萝卜培养基,在番茄汁和马铃薯培养基上生长最差。同一菌株在不同培养基上产孢量不同、产生的孢子囊大小亦有所不同。05P226菌株在玉米培养基上产孢量最高;05P55菌株在燕麦培养基上产孢量最高,在番茄汁培养基上产生的孢子囊最大,而05P62菌株在燕麦培养基上产孢量最高,在烟叶汁培养基上产生的孢子囊最大。在30℃菌丝生长速率最高,其次为28℃,以35℃生长最差。供试3个菌株间生长差异显著,以05P226生长最好,05P55菌株次之,05P62生长最差。05P226、05P55菌株皆以培养14d、诱导72h产孢量最高,最适宜试验中产孢培养。     

贵州省烟草黑胫病菌遗传多样性分析

运用RAPD分子标记技术分析了采自贵州各个地区的38个烟草黑胫病菌株的遗传多样性,探索了贵州省主要烟草种植区间的烟草黑胫病病菌的遗传分化关系.结果表明,受试38个菌株共产生65条谱带.其中多态性为59条,占90.7％.说明贵州省主要烟草种植区的烟草黑胫病病菌具有丰富的遗传多样性,不同地点烟草黑胫病病菌的遗传结构之间都有一定的差异.根据引物扩增的指纹图谱,运用UPGMA分析法,以遗传相似系数0.74,将供试38个菌株划分为9个基因型,发现除少数地区的菌株被划分为同一基因型外,其他不同地理区域的菌株基因型的划分与地理来源没有直接的相关性.     

贵州省烟草黑胫病菌对甲霜灵的抗药性

为了明确贵州地区烟草黑胫病菌对甲霜灵的抗药性水平, 从贵州省12个市、县采集黑胫病病株, 经分离纯化鉴定, 获得98株黑胫病菌株, 采用生长速率法测定其对甲霜灵的敏感性。结果表明, 采自不同地区的菌株对甲霜灵的敏感性差异较大, 各供试菌株的EC50 值分布范围为0.419 2～20.486 1 μg/mL; 各地区平均EC50 在0.551 9～10.986 0 μg/mL之间; 最低抗药性水平为1.00, 最高抗药性水平为48.87, 相差48.87倍, 各地区平均抗药性水平在1.32～26.21之间。遵义、兴仁、贵定、赫章和兴义有抗甲霜灵菌株出现, 除遵义部分菌株的抗性水平表现为高抗外, 其余菌株对甲霜灵产生的抗性均处于中抗水平。     

Differential Sensitivity of Phytophthora parasitica var. nicotianae and Thielaviopsis basicola to Monomeric Aluminum Species

ABSTRACT Aluminum (Al) is toxic to many plant pathogens, including Thielaviopsis basicola and Phytophthora parasitica var. nicotianae. Because fungi-toxicity of Al has been described in soils over a wide pH range, multiple species of Al may be responsible for pathogen suppression. The goals of this work were to determine the sensitivity of T. basicola and P. para-sitica var. nicotianae to Al over a range of pH values, quantify the toxicity of monomeric Al species to production of sporangia of P. parasitica var. nicotianae and chlamydospores of T. basicola, and detect the accumulation of Al in pathogen structures. A complete factorial treatment design was used with Al levels ranging from 0 to 100 muM and pH levels ranging from 4 to 6 in a minimal salts medium. The chemistry of test solutions was modeled using GEOCHEM-PC. Colonies were grown in 5% carrot broth, and after 1 or 2 days, the nutrient solution was removed, colonies were rinsed with water, and Al test solutions were added to each of four replicate plates. After 2 days, propagules were counted and colonies were stained with the Al-specific, fluorescent stain lumogallion. The oomycete P. parasitica var. nicotianae was sensitive to multiple monomeric Al species, whereas sensitivity of T. basicola to Al was pH-dependent, suggesting that only Al(3+) is responsible for suppression of this fungal pathogen. Chlamydospore production by T. basicola was inhibited at pH values <5.0 and Al levels >20 muM, whereas sporangia production by P. parasitica was inhibited at Al levels as low as 2 muM across all pH values tested. The lumogallion stain was an effective technique for detection of Al in fungal tissues. Aluminum accumulated in sporangia and zoospores of P. parasitica var. nicotianae and in nonmelanized chlamy-dospores of T. basicola, but not in cell walls of either organism. The differential sensitivity of the two organisms may indicate that true fungi respond differently to Al than members of the oomycota, which are more closely related to plants.     

拮抗细菌B8对烟草黑胫病菌的抑制作用及其菌株鉴定

从烟草根际土壤中分离到对烟草黑胫病菌及其它植物病原菌具有抑制作用的菌株B8,平板对峙培养抑菌带内菌丝畸形、原生质凝集或外渗。其发酵原液和发酵液的无菌滤液均能抑制烟草黑胫病菌和蜡状芽孢杆菌;离体叶片接种法测定其发酵液对烟草黑胫病的防效,结果表明预防作用达100%。室内盆栽试验表明B8菌株发酵液对烟草黑胫病的预防作用可达78.1%,优于治疗作用。该菌菌体杆状,芽孢侧生、椭圆形,经形态学、生理生化性状和16S rDNA序列测定,将其鉴定为侧孢短芽孢杆菌。     

贵州省烟草黑胫病菌对烯酰吗啉的敏感性

为了明确烟草黑胫病菌对烯酰吗啉的敏感性现状,从贵州省各地采集了47株烟草黑胫病菌株,分别测定了菌丝生长和游动孢子囊形成对烯酰吗啉的敏感性.结果表明,烯酰吗啉对烟草黑胫病菌菌丝生长和孢子囊形成的抑制基本一致,对菌丝EC50范围为0.792 6～4.232 7μg/mL,平均为2.476 8μg/mL;对孢子囊的抑制作用略强,EC50范围为0.394 5～4.625 3μg/mL,平均值为2.040 0 μg/mL.统计分析表明,烟草黑胫病菌47个菌株菌丝生长和孢子萌发对烯酰吗啉的敏感性符合正态分布,不同菌株对烯酰吗啉的敏感性有显著差异,而且菌株对烯酰吗啉的敏感性与其地理来源有一定的相关性.     

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     

安徽省烟草黑胫病菌的交配型及其地理分布研究

 Total 69 isolates of Phytophthora nicotianae var. nicotianae were isolated and identified from tobacco black shank samples collected from different areas of Anhui province, and mating types of the oomycete were investigated by the method of partnership culture in vitro on L-tryptophan medium with β-sitosterol. The results showed that A2 mating type was the predominant in isolates of P. nicotianae var. nicotianae, next was A₀ mating type, A₁ and other mating types were not observed. It was suggested that sexual reproduction of P. nicotianae var. nicotianae might not take place frequently, and Anhui province might not be the original center of P. nicotianae var. nicotianae. The results indicated further that mating type geographical distribution of P. nicotianae var. nicotianae in Anhui province was in line with that in China. In addition, the significance of the test results and the possible reason for A₁ mating type being absent were discussed.     

Pseudomonas sp.20#-5防治烟草黑胫病田间试验初报

20^#-5菌株是本实验室从土壤中分离保存的1株假单胞菌,2006年对其发酵液防治烟草黑胫病进行了田间防效试验。结果显示,不同发病时期20^#-5菌发酵液的防治效果在64.39%～79.70%,高于对照药剂,防治效果较好。不同施药时期调查结果表明,发病前施用20^#-5菌发酵液的防治效果要好于发病后施用,说明20^#-5菌发酵液对烟草黑胫病具有有效的预防作用。     

抗菌肽和几丁质酶基因提高烟草对黑胫病菌和赤星病菌的抗性研究

 通过农杆菌介导法将加信号肽修饰的人工合成杂合抗菌肽CEMA基因(SPCEMA),苜蓿防御素基因(AFP),苦瓜几丁质酶基因(CHI)以及SPCEMA-CHI、AFP-CHI、AFP-SPCEMA双价基因导入本明烟(Nicotiana benthamiana),并对转基因烟草T₀和T₁代进行了抗病性检测,比较了不同转基因植株的抗病效果。研究结果表明,转基因烟草对黑胫病菌(Phytophthora parasitica var.nicotianae)、赤星病菌(Alternaria alternata)的抗性均强于非转基因烟草,病情指数差异达极显著水平,其中转AFP-CHI双价基因烟草具有较强的抗性,与单价转基因烟草的抗性差异达显著水平。但各单价转基因以及双价转基因烟草之间对上述病菌并未表现出显著的抗病性差异。结果表明植物源的抗菌肽基因与几丁质酶基因在抗植物真菌病害中具有协同增效作用。     

Purification, Elicitor Activity, and Cell Wall Localization of a Glycoprotein from Phytophthora parasitica var. nicotianae, a Fungal Pathogen of Tobacco

ABSTRACT A glycoprotein of 34 kDa (GP 34) was solubilized at acidic pH from the mycelium of Phytophthora parasitica var. nicotianae and was purified by ion exchange and gel permeation chromatography. Whole tobacco plants treated with GP 34 through their roots showed an enhanced lipoxygenase activity as well as hydroxyproline-rich glycoprotein accumulation, indicating that this molecule had elicitor properties. An antiserum raised against the pure glycoprotein allowed localization of GP 34 by immunogold-labeling on the cell surface of the mycelium when the fungus was grown in vitro. In the wall-less zoospores, GP 34 was limited to the flagellum surface. It was then abundantly synthesized at the onset of encystment. During infection of tobacco plants, labeling was very faint at early stages of colonization, particularly in the susceptible host cultivar. It appeared earlier in the resistant host cultivar and was restricted to the living fungus, declining with mycelium cell death.     

烟草疫霉拮抗菌株P 72 10的鉴定及其拮抗代谢 产物 初步分析

 为探讨烟草根际生防细菌的生防机制,从重庆地区连作烟田健康烟株根际土壤中分离筛选到1株对烟草疫霉具有较强拮抗作用和对黑胫病具有良好防效的细菌菌株P-72-10。根据培养性状、形态特征、生理生化特性、基因组DNA的(G+C)mol%含量测定以及16S rDNA基因序列分析确定该菌株的分类地位。该菌株菌落乳白色,能产生水溶性荧光色素,革兰氏染色反应阴性,菌体杆状、大小(8.1~16.2)μm×(1.8~4.8)μm,单端生鞭毛,不形成芽孢。The BIOLOG GN2 结果显示该菌株属于假单胞菌属Pseudomonas。该菌株基因组DNA的(G+C)mol%含量为 60.72 mol%。16S rDNA基因序列分析显示该菌株与假单胞菌属荧光假单胞杆菌多个菌株的序列同源性达到99%,GenBank上的登录号为：HQ888871。结合其形态特征和生理生化特性,将菌株P-72-10鉴定为荧光假单胞杆菌P. fluorescens。平板检测拮抗代谢产物结果表明：该菌株具有分解纤维素、蛋白质和结合Fe ³⁺的能力,但不能分解几丁质。     

Thermal Inactivation of Phytophthora nicotianae

ABSTRACT Phytophthora nicotianae was added to pasteurized soil at the rate of 500 laboratory-produced chlamydospores per gram of soil and exposed to temperatures ranging from 35 to 53 degrees C for 20 days. The time required to reduce soil populations to residual levels (0.2 propagule per gram of soil or less) decreased with increasing temperatures. Addition of cabbage residue to the soil reduced the time required to inactivate chlamydospores. Temperature regimes were established to simulate daily temperature changes observed in the field, with a high temperature of 47 degrees C for 3 h/day, and were good estimators of the efficacy of soil solarization for the control of P. nicotianae in soil. Cabbage amendment reduced the time required to inactivate chlamydospores of P. nicotianae and its effect was more pronounced at lower temperature regimes.     

茄子绵疫病的发生与防治

茄子绵疫病是茄子的重要病害之一，在我国各地均有发生，茄子各生育期皆可受害，一般损火200％-30％，发病严重损失超过50％。此病除茄子外，还可侵染番茄、西瓜、辣椒、黄瓜以及马铃薯等蔬菜。     

Protection of Citrus Rootstocks Against Phytophthora spp. with a Hypovirulent Isolate of Phytophthora nicotianae

ABSTRACT Phytophthora root rot of citrus in Florida is caused by Phytophthora nicotianae and P. palmivora. A naturally occurring isolate of P. nicotianae (Pn117) was characterized as hypovirulent on citrus roots. Pn117 infected and colonized fibrous roots, but caused significantly less disease than the virulent isolates P. nicotianae Pn198 and P. palmivora Pp99. Coincident inoculation of rootstock seedlings of Cleopatra mandarin (Citrus reticulata) or Swingle citrumelo (C. paradisi x Poncirus trifoliata) with the hypovirulent Pn117 and the virulent isolates Pn198 and Pp99 did not reduce the severity of disease caused by the virulent Phytophthora spp. When either rootstock was inoculated with the hypovirulent Pn117 for 3 days prior to inoculation with virulent isolates, preinoculated seedlings had significantly less disease and greater root weight compared with seedlings inoculated with the virulent isolates alone. Recovery of the different colony types of Phytophthora spp. from roots of sweet orange (C. sinensis) or Swingle citrumelo was evaluated on semiselective medium after sequential inoculations with the hypovirulent Pn117 and virulent Pp99. Pn117 was isolated from roots at the same level as the Pp99 at 3 days post inoculation. Preinoculation of Pn117 for 3 days followed by inoculation with Pp99 resulted in greater recovery of the hypovirulent isolate and lower recovery of the virulent compared with coincident inoculation.     

致病疫霉拮抗细菌筛选及J-28菌株鉴定

为明确从马铃薯根际土壤中分离获得的细菌对致病疫霉的抑制作用,采用平板划线分离法分离马铃薯根际细菌,以对峙培养法和打孔法分别测试细菌菌株、菌液及无菌体发酵液对致病疫霉菌丝生长的抑制作用,并对其中最具优势的菌株通过培养特征、形态、生理生化特性及16S rDNA序列进行鉴定。结果显示,在分离的60株细菌中,有16株细菌对致病疫霉有明显拮抗作用,有6株抑菌率达60%以上。其中,J-5和J-26的抑菌率最高(90.24%和91.54%),其次为J-28,抑菌率87.80%;菌液以J-5和J-33的抑菌率最高(95.34%和92.93%);J-28菌液抑菌率为81.36%:无菌体发酵液以J-28和J-26的抑菌率最高(82.92%和75.60%)。综合评价以J-28菌株的抑菌作用最为稳定。经鉴定J-28菌株为一株巨大芽孢杆菌(Bacillus megaterium)。该菌株在马铃薯晚疫病防治中有较大潜力。