温度对烟草黑胫病菌致病力及代谢表型的影响

为揭示温度对烟草黑胫病菌Phytophthora nicotianae致病力及代谢表型的影响,采用菌丝生长速率法和离体叶片法分别测定不同温度下烟草黑胫病菌的生长速率和致病力,同时采用Biolog代谢表型技术测定其在20、27、30和35℃下的渗透压和pH代谢表型。结果表明,烟草黑胫病菌在21~35℃范围内生长良好且具致病力,30℃时生长速率最快,为17.72 mm/d,30℃和35℃时致病力较强,叶片接种烟草黑胫病菌后24 h内便出现病斑,随温度升高病害的潜伏期缩短。温度影响烟草黑胫病菌的渗透压和pH适应性;27℃和30℃时烟草黑胫病菌的渗透压适应范围最广,其次为21℃,35℃时烟草黑胫病菌渗透压适应范围最窄;在20、27和30℃时,烟草黑胫病菌在pH 5.0~10.0范围内可正常代谢,在35℃时pH代谢范围为5.5~10.0。     

温度对烟草赤星病菌生物学特性及代谢表型的影响

为了解温度对烟草赤星病菌致病力及代谢表型的影响, 本研究采用菌丝生长速率法和离体叶片法, 分别测定不同温度下烟草赤星病菌的生长速率和致病力, 同时采用Biolog代谢表型技术测定了其在22?25?30℃和35℃下不同碳源?渗透压和pH下的代谢表型?结果表明, 烟草赤星病菌在15~35℃下均可生长, 30℃时菌丝生长最快?致病力和产孢能力最强, 35℃时孢子萌发率最高?在22?25?30℃和35℃时, 赤星病菌均可代谢Biolog FF代谢板上的95种碳源, 且随着温度升高对碳源的代谢能力逐渐增强?22℃和25℃时烟草赤星病菌对渗透压的适应范围最广, 其次为30℃, 35℃时适应范围最窄?在22?25?30℃和35℃时, 赤星病菌在pH 3.5~10范围内均可正常代谢, 在22?25℃和30℃下, 赤星病菌表现出强脱羧酶活性和弱脱氨酶活性, 在35℃下, 其脱羧酶和脱氨酶活性均相对较弱?研究结果揭示了烟草赤星病菌在不同温度下的适应能力, 为赤星病菌与环境互作研究提供了参考?     

烟草青枯病菌与其拮抗菌解淀粉芽胞杆菌的代谢表型差异分析

为探究解淀粉芽胞杆菌X60作为烟草青枯病生防菌剂的潜力,采用Biolog代谢表型技术比较了2种细菌的不同代谢表型。结果表明,烟草青枯病菌和解淀粉芽胞杆菌分别能代谢19%、41%的碳源,43%、77%的氮源,95%、86%的磷源以及100%、69%的硫源,分别有94、91种生物合成途径,49、95种渗透压表型以及19、94种pH代谢表型;解淀粉芽胞杆菌比烟草青枯病菌代谢显著的碳源有L-果胶糖、D-甘露糖等34种,氮源有腺苷、胞苷等29种;烟草青枯病菌比解淀粉芽胞杆菌代谢显著的碳源有D-糖二酸、半乳糖醇等9种,氮源有缩二脲、葡萄糖苷酸等11种;解淀粉芽胞杆菌的渗透压和pH环境适应力比烟草青枯病菌强;解淀粉芽胞杆菌具有脱羧酶和脱胺酶的活性。研究表明,2种细菌的代谢表型间存在较大差异,解淀粉芽胞杆菌的碳源、氮源、渗透压及pH代谢表型较烟草青枯病菌的丰富,烟草青枯病菌的磷源、硫源和生物合成途径代谢表型较解淀粉芽胞杆菌的丰富。     

烟草赤星病菌嘧菌酯敏感与抗性菌株的代谢表型差异分析

 为了解烟草赤星病菌(Alternaria alternata)嘧菌酯抗性菌株与敏感菌株在代谢表型上的差异,采用Biolog代谢表型技术比较了抗性菌株(6-5和6-11)和敏感菌株(J6)的950种代谢表型。结果表明,抗性菌株和敏感菌株在代谢表型上基本一致,2个抗性菌株均不能代谢糖酵解中的氮源D-甘露糖胺,抗性菌株6-11还不能代谢尿素循环中的氮源L-鸟氨酸。烟草赤星病菌能代谢24.74%、85.26%、97.14%、89.83%的供试碳、氮、硫、磷源;具有广泛渗透压和pH适应能力;具有脱羧酶活性而无脱氨酶活性;能在高达10%氯化钠、6%氯化钾、5%硫酸钠、20%乙二醇、6%甲酸钠、6%尿素、12%乳酸钠、200 mmol·L^-1 磷酸钠、100 mmol·L^-1硫酸铵、100 mmol·L^-1硝酸钠和20 mmol·L^-1亚硝酸钠的渗透液中正常代谢,不能在20~200 mmol·L^-1的苯甲酸钠渗透液中代谢;其pH适应范围为3.5~10.0,最适约为6.0。研究结果有助于了解烟草赤星病菌的营养需求特性、渗透压和pH环境适应力,同时从代谢表型上揭示了赤星病菌对嘧菌酯抗性的潜在机理。     

两种植物粗提液对烟草青枯病菌致病力的影响

洋葱和大蒜对多种病原微生物具有较好地抑制作用,能有效降低病原菌的致病力。用洋葱和大蒜植物粗提液5种浓度对烟草青枯病菌做室内抑制测定,采用TTC培养基检测其抑制效果。结果表明,洋葱和大蒜粗提液均可以减弱烟草青枯病菌的致病力。在5种浓度下,随着植物粗提液浓度的升高,菌落在TTC培养基上表现的红斑范围越大,颜色越深。烟草青枯菌的致病力强弱与红斑大小、颜色深浅呈反比。因此,洋葱和大蒜粗提液可抑制烟草青枯病菌的致病力,是良好的植物源生物药剂。     

利用表型芯片技术筛选利于枯草芽胞杆菌Bacillussubtilis BAB-1生长与芽胞形成的营养物质

为获得枯草芽胞杆菌Bacillus subtilis BAB-1菌株的芽胞高效率生产技术,采用Biolog微生物细胞表型芯片(phenotype microarray,PM)技术分析了适合该菌株细胞生长的营养物质、渗透压和pH等生长环境条件;通过单因素试验分析了5种碳源及5种氮源对该菌株生长及芽胞形成的影响。结果表明,BAB-1菌株能够利用114种碳源、228种氮源、29种磷源及29种硫源作为其营养物质,其中52种碳源、66种氮源、3种硫源及1种磷源能够明显促进该菌株的细胞生长;BAB-1菌株对渗透压的忍受能力较强,在1%～10%氯化钠、3%～6%氯化钾、2%～5%硫酸钠、5%～20%乙二醇、1%～6%甲酸钠、2%～7%尿素、1%～12%乳酸钠、20～200 mmol/L磷酸钠、10～100 mmol/L硝酸钠、10～100 mmol/L硫酸铵中均能较好的生长;BAB-1菌株对pH的耐受范围较广,在4.5≤pH≤10.0条件下均能够生长。葡萄糖与氯化铵分别是最适宜BAB-1菌株生长的碳源与氮源,在不同培养时间其菌体总量均最高,48 h时该菌株的菌体总量分别达到6.90×10~8CFU/mL与6.87×10~8CFU/mL;培养12 h时,D-木糖、D-核糖、L-阿拉伯糖及熊果苷能够显著提高BAB-1菌株的芽胞形成率,其中L-阿拉伯糖与熊果苷的芽胞形成率较高,分别为61.94%与66.92%;培养12～36 h,L-脯氨酸、L-谷氨酰胺及L-天冬酰胺能够显著提高BAB-1菌株的芽胞形成率,芽胞形成率介于27.88%～82.37%之间。表明表型芯片技术可以应用于枯草芽胞杆菌生长及芽胞形成营养物质的高通量分析及培养工艺的优化中。     

大蒜根系分泌物对烟草青枯病的影响

为探讨大蒜根系分泌物对烟草青枯病菌的抑菌活性,采用抑菌圈法和盆栽试验研究了大蒜根系分泌物及其主要成分对烟草青枯病的影响。结果表明：大蒜根系分泌物浓度为1 g/mL时,对烟草青枯病菌抑制效果最好,其抑菌率为53.67%。大蒜根系分泌物4种成分的抑菌效果由强到弱依次为：2,6-二异丙基苯酚＞二烯丙基二硫＞2,6-二叔丁基对甲酚＞邻苯二甲酸二丁酯。其中,2,6-二异丙基酚对烟草青枯病菌的抑制作用最强,在1和5 mmol/L时的抑菌率分别为99.66%和100.00%;在盆栽试验中也具有较好的防治效果,接种后7 d和14 d后,其防效分别为34.75%和31.35%。因此,大蒜根系分泌物及其成分均对烟草青枯病有明显的抑制作用。本研究揭示了大蒜作为轮作或间作作物对烟草青枯病的防控机理,以及大蒜根系分泌物和2,6-二异丙基酚作为烟草青枯病防治剂的潜力。     

青枯病菌噬菌体P3株系的生物学特性及其应用研究

作物青枯病是一种土传细菌性维管束病害，由于作物病害的发生和严重度与土壤中的病原菌数量呈正相关，因而快速准确测定土壤中的病原菌数量是病害预测及有效防控的前提。本文对筛选获得的一株具有生防潜力的噬菌体P3株系进行了生物学特性测定，并探讨其在检测烟草根际土壤中青枯病菌数量的可行性，为烟草根际土壤青枯病菌的检测提供一种新方法。研究结果表明，噬菌体P3株系由一个二十面体的头部和非常短的尾部构成；裂解谱广，可裂解烟草、番茄、辣椒、马铃薯和甘薯的青枯病菌；最佳感染复数为0.0001；潜伏期60 min，裂解期80 min，裂解量179；对1%氯仿不敏感；55℃以下及pH 4~12的环境中保持稳定；对紫外线敏感，照射18 min失活；20℃~28℃存放一个月效价稳定。同等条件下，检测烟草根际土壤中青枯病菌数量，平板检测法的实测值仅为1.79×10⁴CFU/g，而噬菌体检测法实测值为2.83×10⁵CFU/g，说明噬菌体检测法的灵敏度较平板检测法高10倍。本研究通过噬菌体检测法检测了烟草根际土壤的青枯病菌数量，可为今后烟草青枯病的预测预报提供技术支持。     

青枯菌无致病力菌株FJAT-1458与强致病力株FJAT-91的竞争生长作用

为了明确青枯菌无致病力菌株FJAT-1458的生防竞争机制，本研究采用体外共培养方法研究了碳源、氮源和培养时间对菌株FJAT-1458和青枯菌强致病力菌株FJAT-91竞争生长的影响；同时，研究了菌株FJAT-1458和FJAT-91在番茄植株体内的竞争生长。结果表明，碳源含量低于20%时，菌株FJAT-1458不能生长，而菌株FJAT-91可生长；无氮源条件下，FJAT-1458和FJAT-91均不能生长；碳源和氮源含量达100%时，FJAT-1458的菌体浓度（2.16×10⁹ cfu/mL）显著低于FJAT-91的菌体浓度（3.24×10⁹ cfu/mL）。混合培养24 h前，FJAT-1458生长量大于FJAT-91，24 h后则相反。在单独接种或混合接种5 d后，FJAT-1458和FJAT-91均在番茄植株体内出现最大定殖量，随后迅速减少；FJAT-91在单独接种15 d时，定殖数量为0；两种菌株单独接种的定殖数量均大于混合接种（接种15 d除外）。先接种FJAT-1458，3 d后接种FJAT-91对番茄青枯病的防效（100%）显著高于同时接种FJAT-1458和FJAT-91（74.67%）。本研究表明，在体外无致病力菌株FJAT-1458对碳源和氮源的营养竞争能力弱于强致病力菌株FJAT-91；在体内无致病力菌株FJAT-1458会抑制强致病力菌株FJAT-91的生长；因此，FJAT-1458的生防竞争机制中，营养竞争起非主导作用，而位点竞争可能是主导因素。     

烟草赤星病菌致病力分化与弱毒株抗性诱导作用的研究

对我国不同地区烟草赤星病菌(Alternaria alternata)20个菌株致病力的测定结果表明,病菌不同菌株致病力有明显的差异。菌株致病力的强弱与其生长量和产孢量有一定的关系。致病力强的菌株菌落生长慢、气生菌丝生长好、产孢量低,致病力弱的菌株则相反。据此选出11个弱毒株对烟草幼苗作诱导接种,其中菌株 TBA16可明显诱导烟苗对赤星病的抗性,当用菌株 TBA28和 TBA19挑战接种时,抗性诱导效应分别为50%—61%和60%—71.4%。     

Specific Detection of Biovars of Ralstonia solanacearum in Plant Tissues by Nested-PCR-RFLP

A sensitive and specific assay, based on a Nested-PCR-RFLP protocol, was developed for the detection of biovars of Ralstonia solanacearum, the causal agent of bacterial wilt. Oligonucleotide primer pairs were selected within the hrp gene region. Specific amplification of the hrp fragments was obtained for all R. solanacearum strains and also for two closely related species, Pseudomonas syzygii and the blood disease bacterium. No amplification was observed for a wide range of other bacterial species, including R. pickettii and Burkholderia cepacia. Digestion with HindII provided four distinct restriction profiles specific to biovars or groups of biovars of R. solanacearum: one for biovar 1 strains originating from the Southern part of Africa, one for American biovar 1 and biovars 2 and N2 strains, one for biovars 3 and 4 strains, and one for biovar 5 strains. When applied to either pure culture or infected plant tissues, Nested-PCR allowed detection as low as 10³cfu ml^–1, which corresponds to 1cfu per reaction. Amplification was partially or completely inhibited by compounds contained in plant extracts (potato plant and potato tuber, tomato, tobacco, eggplant, pepper and Pelargonium asperum). A combined PVPP/BSA treatment prior to amplification permitted reliable Nested-PCR detection of R. solanacearum strains in plant samples. Nested-PCR-RFLP, assessed with isolates from Reunion Island but also applicable to any R. solanacearum strain, provides a wide range of possible uses for identification, detection and epidemiological investigations.     

Factors affecting the virulence of Ralstonia solanacearum and its colonization on tobacco roots

Tobacco bacterial wilt caused by Ralstonia solanacearum is a serious disease affecting tobacco cultivation in southwest China. The response surface methodology was employed to evaluate the optimal conditions of tobacco bacterial wilt, and green fluorescent protein gene (gfp) labelling was applied to monitor the location and survival dynamics of R. solanacearum (Rs::gfp) on tobacco roots and in soil under these optimal conditions. The results showed that the highest wilt incidence was 91.13%, which occurred when the population reached 6.6 × 10⁶ CFU/g soil, the temperature was 30.55 °C, and the humidity was >81.42%. The Rs::gfp densely colonized the root tips and root hairs, and cells of Rs::gfp were observed intermittently in the elongation zone or at the point of the emerging lateral roots. The Rs::gfp number in the rhizosphere soil was 10.75‐, 73.13‐ and 74.86‐times higher than that in the bulk soil at 10, 15 and 20 days after transplantation, respectively. Increased colonization by Rs::gfp was related to the population of the pathogen, the environmental temperature and the humidity in the soil. These three conditions determined whether R. solanacearum would induce tobacco wilt. This is the first study to investigate factors affecting the virulence of a tobacco wilt bacterial pathogen, which is important for conducting field diagnosis and biocontrol of tobacco bacterial wilt.     

我国长江流域和南方地区花生青枯菌遗传多样性分析

为明确不同青枯菌的遗传多样性和其在花生植株上的致病力差异,采用国际上新的青枯菌演化型分类模式,对从我国长江流域和南方地区9个花生种植区分离的95株花生青枯菌Ralstonia solanacearum菌株进行遗传多样性分析,基于内源葡聚糖酶基因egl对青枯菌进行系统发育研究,并对供试青枯菌的致病力进行测定。结果表明,所有95株菌株均属于青枯菌演化型I型,即亚洲分支类型。在序列变种分类上,所检测的9个花生种植区中有8个种植区的花生青枯菌菌株属于序列变种14,仅有1个种植区(广西壮族自治区贺州市)的花生青枯菌菌株属于序列变种48,表明我国长江流域和南方地区花生青枯菌群体遗传多样性水平较低。青枯菌致病力测定结果表明,来自赣州市的菌株GZ-1、贺州市的菌株HZ-2和宜昌市的菌株YC接种到花生植株14 d后,花生的病情指数分别为43.8、75.0和87.5,而来自其它6个花生种植区的菌株接种花生后,其病情指数均为100.0,表明菌株GZ-1和HZ-2的致病力较弱,而其它7个花生种植区代表性菌株的致病力均较强。     

青枯菌无致病力菌株对烟草青枯病的控病作用初步研究

从茄子、番茄、辣椒、烟草青枯病株中分离出116株无致病力青枯菌,室内平板喷雾法拈抗试验结果表明,有21株菌在NA培养基上可明显抑制青枯菌TbRs的生长;烟草MSK326品种温室盆栽控病试验表明,Tnljdl-3和Aujd8—2—1两株菌具有较好控病效果,20d后的相对防效分别为58．4％和97％。     

Molecular identification of some African strains of Ralstonia solanacearum from eucalypt and potato

Ralstonia solanacearum is a known bacterial pathogen of eucalypt and potato plants in Africa. A survey was undertaken to detect this pathogen in eucalypt plantations in South Africa, the Democratic Republic of Congo, and Uganda. Numerous bacterial strains were isolated from trees with symptoms typical of bacterial wilt, but only seven were positively identified as R. solanacearum. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique, based on the hrp (hypersensitive response and pathogenicity) gene region was used to determine and group the biovars of these R. solanacearum strains. The eucalypt isolates and one potato isolate formed a biovar 3 cluster, whereas the two other potato isolates formed a cluster that corresponded to biovar 2. Amplified fragment length polymorphism (AFLP) analysis confirmed these clusters. Therefore, PCR-RFLP can be used as a reliable diagnostic technique to enable researchers to rapidly identify the pathogen.     

Role of organic acids in the mechanisms of biological soil disinfestation (BSD)

Biological soil disinfestation (BSD), or reductive soil disinfestation, achieved by amendment with organic materials such as wheat bran followed by flooding and covering the soil surface, has been used to control some soilborne diseases including Fusarium wilt and bacterial wilt of tomato. During a BSD treatment, accumulation of acetic acid and/or butyric acid was detected with high-performance liquid chromatography. Survival of Fusarium oxysporum f. sp. lycopersici or Ralstonia solanacearum was suppressed by these organic acids. Amendment of these organic acids into soil suppressed the survival of R. solanacearum at lower concentrations than the maximum detected in BSD treatment, indicating that production of these organic acids is one of the mechanisms of control. However, F. oxysporum f. sp. lycopersici in soil survived with the maximum concentrations of these organic acids achieved by BSD; thus, involvement of factors other than organic acids may be involved.     

Bacterial wilt of bellflower caused by Ralstonia solanacearum in Japan

A sudden wilt of bellflower (Campanula lactiflora) was observed in Japan in 1997. A bacterium that formed white fluidal and mucoid colonies resembling those of Ralstonia solanacearum was isolated from the infected plants. The bacterium was bacteriologically identified as biovar 3 of R. solanacearum. This is the first report of R. solanacearum affecting a plant species of the Campanulaceae family.     

Implementation of an artificial reaction control in a TaqMan method for PCR detection of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> race 3 biovar 2

A previously published TaqMan PCR test for R. solanacearum race 3 biovar 2 was modified to enable both the validation of negative results and the confirmation of positive results in a closed-tube system. Negative results were validated through the use of a reaction control plasmid, designated pRB2C2, which was designed to generate a 94bp product using the same amplimers targeting the primary diagnostic 68bp sequence in R. solanacearum race 3 biovar 2 DNA. SYBR Green was included in the reaction mix to facilitate the identification of post-reaction products using melt peak analysis. The 94bp reaction control had a melt peak temperature of about 90°C, while the diagnostic target amplicon had a melt peak temperature of about 83°C; thus positive results could be easily confirmed and distinguished from the reaction control product. Addition of pRB2C2 at 100 copies per reaction had no effect on the sensitivity of the TaqMan assay for R. solanacearum race 3 biovar 2, and the modified assay successfully detected R. solanacearum race 3 biovar 2 in infected, asymptomatic tomato stems and leaves as well as in potato tubers and stems.