西瓜噬酸菌Ⅲ型分泌系统hrcQ基因功能分析

Ⅲ型分泌系统(type Ⅲ secretion system,T3SS)是影响西瓜噬酸菌Acidovorax citrulli致病性的重要因素,hrcQ作为T3SS的保守基因,在组成和维持该系统功能方面扮演着重要角色,但hrcQ在西瓜噬酸菌中的具体生物学功能尚不明确。本试验以西瓜噬酸菌Aac5菌株为研究对象,通过基因敲除、致病力及相关表型测定等,解析了hrcQ基因的功能。结果表明:hrcQ缺失导致Aac5菌株对寄主的致病能力丧失,运动性及生物膜形成能力降低,并丧失了引起烟草过敏性反应能力;hrcJ和hrcR基因在hrcQ缺失突变株中的表达量下调,hrcQ对hrcJ和hrcR基因均为正调控。表明hrcQ在维持西瓜噬酸菌致病能力中起着不可或缺的作用。     

西瓜噬酸菌hflX基因功能分析

为明确GTP结合蛋白基因hflX在西瓜噬酸菌Acidovorax citrulli中的功能尤其是对该菌致病力的影响,以西瓜噬酸菌Aac5菌株为材料获得hflX基因的缺失突变菌株及互补菌株,通过对致病力等表型以及相关基因表达量的测定初步探究西瓜噬酸菌hflX基因的功能。结果显示,同野生型菌株Aac5相比,缺失突变菌株的致病力显著下降,III型分泌系统基因hrpG和hrpE的表达量显著下调,仅为野生型菌株表达量的54.99%和27.39%;hflX基因的缺失并未影响其引起烟草过敏性坏死反应的能力;但缺失突变菌株的运动能力显著下降,鞭毛基因fliR和fliC的表达量显著下调,仅分别为野生型菌株的37.04%和29.68%。此外,缺失突变菌株的生物膜形成能力相较于野生型菌株显著增强了26.32%,同时,其体外生长能力也有所增强。表明hflX基因可能通过调控III型分泌系统基因hrpG和hrpE、鞭毛基因fliR和fliC的表达以及运动能力来影响西瓜噬酸菌Aac5菌株的致病力。     

西瓜噬酸菌中C-di-GMP代谢相关基因Aave_2620的功能研究

 环鸟苷二磷酸(cyclic diguanylate,c-di-GMP)是一种广泛存在于植物病原细菌中的第二信使,调控细菌多种生物学功能,从而影响病原细菌的致病性,但其在西瓜噬酸菌中的研究还未见报道。本实验以Aac5菌株为研究对象,通过构建c-di-GMP代谢相关基因Aave_2620缺失突变株和互补菌株,从其致病能力及其生长能力两部分进行测定,探究此信号途径是否在西瓜噬酸菌中调控致病机制。结果表明,基因Aave_2620的缺失显著降低Aac5菌株致病力、生物膜形成能力和生长能力,减弱烟草过敏性反应以及运动能力,互补菌株表型基本得到恢复。基因Aave_2620的缺失显著影响三型分泌系统相关基因、毒性相关基因以及鞭毛及趋化性相关基因在西瓜噬酸菌中的表达量。以上结果表明c-di-GMP代谢相关基因Aave_2620与西瓜噬酸菌的致病密切相关。     

西瓜噬酸菌MarR家族转录因子Aave_3922的功能研究

 MarR(Multiple antibiotic resistance regulator)家族转录因子是一种广泛存在于细菌及古细菌中的转录抑制子。MarR可通过调控毒力因子的表达来影响病原细菌的致病性,其调控机制独特,在不同菌中存在不同的调控网络。为明确其在西瓜噬酸菌中的功能及信号通路,以西瓜噬酸菌Aac5菌株为研究对象,构建MarR转录因子Aave_3922的缺失突变株及互补菌株,通过对表型及转录水平的测定,初步探究西瓜噬酸菌中MarR转录因子的功能及可能的调控网络。结果显示,与野生型Aac5菌株相比,突变菌株致病能力、生物膜形成能力、体内生长能力均显著下降,互补菌株有所恢复。同时,基因Aave_3922的缺失会显著影响三型分泌系统关键基因hrpX及hrcJ、毒性相关基因trbC、鞭毛及趋化性相关基因fliC及cheA、生物膜相关基因Aave_2620在西瓜噬酸菌中的表达量;与WT-hrpXpGUS相比,ΔAave_3922-hrpXpGUS的启动子活性显著减弱。以上结果表明Aave_3922基因可能通过参与hrpX的转录调控,以及调控生物膜形成来影响西瓜噬酸菌的致病能力。     

利用GICA-PCR快速检测瓜类果斑病菌

瓜类果斑病菌(Acidovorax avenae subsp.citrulli,Aac)是瓜类作物上重要的病原细菌,为我国进境植物检疫性有害生物。胶体金免疫层析试纸条方便快捷,应用广泛。该方法使用不当会出现假阳性问题,仅适用于病原菌的初筛检测。本研究将Aac胶体金免疫层析方法(GICA)与PCR技术相结合,建立了GICA-PCR检测方法。检测结果表明,该方法在蛋白与核酸2个层面上从发病西瓜叶片上检测到瓜类果斑病菌,有效解决了试纸条检测的假阳性问题,提高了瓜类果斑病菌检测的准确性,值得推广应用。     

西瓜噬酸菌Ⅲ型分泌系统hrcS基因功能分析

为明确编码Ⅲ型分泌系统（typeⅢsecretion system,T3SS）中重要核心蛋白的hrcS基因对西瓜噬酸菌Acidovorax citrulli致病力的影响,以西瓜噬酸菌野生型菌株Aac5为对象,通过基因敲除构建hrcS基因缺失突变菌株ΔhrcS及其互补菌株ΔhrcS-comp,测定其致病力及致病相关基因表达量、烟草过敏性反应能力、运动能力及运动能力相关基因表达量、生物膜形成能力和离体生长能力。结果显示,与野生型菌株Aac5相比,hrcS基因缺失突变菌株丧失了对寄主的致病能力和引起非寄主烟草过敏性反应能力,运动能力显著增强了191.75%,生物膜形成能力显著下降了43.90%,离体生长能力在达到对数期后呈下降趋势。hrcS基因缺失使西瓜噬酸菌T3SS中hrpG、hrpX和hrpE基因以及鞭毛fliC基因表达量显著上调,分别为野生型菌株的2.27倍、2.38倍、1.26倍和1.77倍,hrcQ基因表达量显著下调,仅为野生型菌株的50.75%。表明hrcS基因在维持西瓜噬酸菌致病能力中发挥着重要作用。     

西瓜噬酸菌ftsH基因功能分析

 AAA ATPase (ATPase associated with diverse cellular activities) 在西瓜噬酸菌的多种生命过程中发挥重要作用。AAA家族中的FtsH蛋白(filamentation temperature-sensitive H)是由ftsH基因编码的,参与生长、致病、环境应激反应、膜内在蛋白质量控制等过程的蛋白,但其在西瓜噬酸菌中的作用尚未明确。本实验以西瓜噬酸菌Aac5菌株为研究对象,通过构建基因缺失突变体,测定致病力等各项表型以及突变株中致病相关基因和其他AAA ATPase基因的表达量,初步探索西瓜噬酸菌中ftsH的功能,为FtsH蛋白的功能研究奠定基础。结果表明,ftsH缺失显著降低菌株致病力、运动能力、生物膜形成能力、生长能力和环境胁迫耐受能力,不影响烟草过敏性反应,显著影响西瓜噬酸菌致病相关基因、AAA ATPase基因以及热激转录因子σ³²的表达量。这表明ftsH基因的功能与西瓜噬酸菌的致病性和环境耐受性密切相关。     

西瓜噬酸菌ftsH基因功能分析

 AAA ATPase (ATPase associated with diverse cellular activities) 在西瓜噬酸菌的多种生命过程中发挥重要作用。AAA家族中的FtsH蛋白(filamentation temperature-sensitive H)是由ftsH基因编码的,参与生长、致病、环境应激反应、膜内在蛋白质量控制等过程的蛋白,但其在西瓜噬酸菌中的作用尚未明确。本实验以西瓜噬酸菌Aac5菌株为研究对象,通过构建基因缺失突变体,测定致病力等各项表型以及突变株中致病相关基因和其他AAA ATPase基因的表达量,初步探索西瓜噬酸菌中ftsH的功能,为FtsH蛋白的功能研究奠定基础。结果表明,ftsH缺失显著降低菌株致病力、运动能力、生物膜形成能力、生长能力和环境胁迫耐受能力,不影响烟草过敏性反应,显著影响西瓜噬酸菌致病相关基因、AAA ATPase基因以及热激转录因子σ³²的表达量。这表明ftsH基因的功能与西瓜噬酸菌的致病性和环境耐受性密切相关。     

利用环介导等温扩增方法快速检测瓜类细菌性果斑病菌和番茄细菌性溃疡病菌

为建立简单、快速和灵敏地检测瓜类细菌性果斑病菌Acidovorax avenae subsp. citrulli(Aac)和番茄细菌性溃疡病菌Clavibacter michiganensis subsp. michiganensis(Cmm)的环介导等温扩增(loop-mediated isothermal amplification,LAMP)方法,以Aac的ugpB基因和Cmm的micA基因为靶标,分别设计、合成和筛选特异性引物,摸索和优化各项反应条件和反应体系,成功建立了以钙黄绿素颜色为指示且只需要金属浴恒温反应30～60 min的LAMP扩增体系。特异性分析表明该LAMP方法可以快速检出5株不同的Aac菌株和2株不同的Cmm菌株,其它对照菌株如燕麦嗜酸菌燕麦亚种A. avenae subsp. avenae、丁香假单胞菌Pseudomonas syringae、水稻黄单胞菌水稻致病变种Xanthomonas oryzae pv. oryzae和茄科雷尔氏菌Ralstonia solanacearum则呈现阴性反应;引物比目前报道的LAMP引物有更高的DNA样品检测灵敏度,Aac和Cmm的灵敏度分别为1.72×10~2fg/μL和1.26×10~2fg/μL。研究结果表明,所设计的Aac和Cmm引物特异性好、灵敏度高,更有利于从源头上控制这2种检疫性细菌病害的流行和传播。     

西瓜噬酸菌铁吸收调控因子furA的功能研究

 铁吸收调控因子(Ferric uptake regulator,Fur)是细菌中主要调控铁代谢的重要调控蛋白,也影响氧化应激和致病性。生物信息学分析显示,西瓜噬酸菌(Acidovorax citrulli,Ac)中存在2个Fur蛋白,其生物学功能未知。本文以其中之一的furA为研究对象,通过同源重组原理,构建了野生型菌株Aac5的furA缺失菌株ΔfurA和互补菌株ΔfurAcomp,并进行了致病力和其他相关生物学表型的测定。结果显示,furA缺失后,显著降低了Ac对西瓜、甜瓜的致病力、运动能力、产铁载体能力,提高了生物膜形成能力、以及对Fe³⁺、过氧化氢和链黑菌素的敏感性;基因表达量分析结果显示,furA影响与铁代谢、应激反应和致病性相关基因的表达。综上所述FurA在Ac致病过程、铁代谢和其他表型中起着重要的调控作用。     

瓜类细菌性果斑病菌hrcN基因的生物学功能分析

以分离自西瓜上的Aac5菌株为例,通过同源重组的方法,构建了hrc N基因插入缺失突变体,通过PCR方法和Southern blot验证突变菌株,对突变体进行致病性、致敏性、生长曲线和运动性测定。为明确hrc N基因与其他基因的关系,通过实时荧光定量PCR法定量检测了hrp A、hrc V、hrc U、Lux I、LuxR 5个基因的表达量。结果显示:与野生型相比,突变体致病力和致敏性明显减弱,致病时间延迟,群体感应信号减弱,生长能力明显下降,运动性减弱,互补菌株只能恢复部分功能;5个基因在突变体中的表达量均上调,hrc N基因与这5个基因之间均为负调控关系。说明hrc N基因在果斑病菌致病能力上发挥重要作用。     

黄芪根腐病生防放线菌筛选鉴定及其优化培养

为获得对黄芪根腐病具有强抗菌活性的生防菌株,采用平板对峙法、抑制菌丝生长速率法对分离于河西走廊盐碱土中的172株放线菌进行拮抗菌的筛选,获得优菌株DA4-4-1,根据其形态、培养特征、生理生化特性和16S rDNA基因序列进行系统发育学分析鉴定,并根据单因素试验与正交试验对培养基条件进行优化。结果表明,菌株DA4-4-1对黄芪根腐病的2种主要致病菌尖孢镰刀菌Fusarium oxysporum和茄腐镰刀菌F.solani均有较好的抑制作用,抑菌圈直径最大分别为20.16 mm和19.33 mm,抑菌率最大分别为34.62%和32.44%。结合16S rDNA基因序列的系统发育分析,将菌株DA4-4-1鉴定为链霉菌属蓝紫链霉菌Streptomyces tuirus。培养基最佳碳源为麦芽糖,氮源为(NH_4)_2SO_4,其优化条件为麦芽糖15 g/L、(NH_4)_2SO_4 0.5 g/L、NaCl 10 g/L、pH 7,在此条件下菌株DA4-4-1的抑菌率可达到86.72%。表明菌株DA4-4-1是1株具有较强抗菌活性的放线菌,具有良好的生物防治和开发应用潜力。     

群体感应系统对甜瓜果斑病菌MH21致病力的影响

 以N-乙酰高丝氨酸内酯（N-acyl-homoserine lactone, AHL）为信号分子的群体感应（quorum-sensing, QS）系统是很多病原细菌的重要致病性调控因子。本文自甜瓜果斑病菌——西瓜食酸菌（Acidovorax citrulli）菌株MH21中克隆到AHL信号合成基因luxI_MH21,并构建了其缺失突变体MΔluxI_MH21及转化有AHL信号降解酶编码基因aiiA和aidH的工程菌株MAiiA和MAidH。信号检测结果显示MΔluxI_MH21、MAiiA和MAidH菌株均无AHL信号产生,同时细菌的游动能力及在基本培养基中的生长速率均显著下降,但对细菌生物膜形成和在非寄主植物烟草上诱导过敏性坏死反应的能力没有影响。盆栽条件下,经低浓度（10⁴ CFU/mL）MΔluxI_MH21、MAiiA和MAidH菌株处理的甜瓜种子萌发后幼苗死亡率显著低于野生型MH21和luxI_MH21基因互补菌株MΔluxI_MH21HB的处理;而高浓度细菌（10⁸ CFU/mL）处理种子后,除MAidH菌株处理引起的死苗率明显低于野生型MH21处理,其他菌株与MH21没有显著差异。子叶注射试验也得到相似的结果,以低浓度细菌（10⁴ CFU/mL）注射甜瓜子叶后发现MΔluxI_MH21、 MAiiA和MAidH菌株甜瓜子叶中的繁殖速率及对子叶的致病力与野生型MH21相比均显著下降;而高浓度细菌（10⁸ CFU/mL）处理子叶时,MΔluxI_MH21和MAiiA菌株与野生菌MH21相比致病力无显著差异,仅有MAidH菌株的致病力明显下降。说明QS系统影响菌株MH21在低细菌浓度下对甜瓜幼苗的致病力,这种作用可能与影响细菌生长有关。     

Assessment of <Emphasis Type="Italic">Pichia anomala</Emphasis> (strain K) efficacy against blue mould of apples when applied pre- or post-harvest under laboratory conditions and in orchard trials

The yeast Pichia anomala strain K was selected in Belgium from the apple surface for its antagonistic activity against post-harvest diseases of apples. The efficacy of this strain against P. expansum was evaluated in the laboratory in three scenarios designed to mimic practical conditions, with different periods of incubation between biological treatment, wounding of fruit surface, and pathogen inoculation. Higher protection levels and higher final yeast densities were obtained when the applied initial concentration was 1 × 10⁸ cfu ml⁻¹ than when it was only 1 × 10⁵ cfu ml⁻¹. The protection level correlated positively with the yeast density determined in wounds and was influenced by apple surface wetness. In orchard trials spanning two successive years, biological treatment against P. expansum, based on a powder of P. anomala strain K (1 × 10⁷ cfu ml⁻¹), β-1,3-glucans (YGT 2 g l⁻¹), and CaCl₂.2H₂0 (20 g l⁻¹), was applied to apples pre- or post-harvest under practical conditions and its effect compared with standard chemical treatments. The first year, the highest reduction (95.2%) against blue decay was obtained by means of four successive fungicide treatments and the next-highest level (87.6%) with pre-harvest high-volume spraying of the three-component mixture 12 days before harvest. The second year, the best results were obtained with post-harvest Sumico (carbendazim 25% and diethofencarb 25%) treatment and post-harvest biological treatment, both by dipping the apples, 88.3 and 56.3% respectively. A density threshold of 1 × 10⁴ cfu cm⁻² of strain K on the apple surface seemed to be required just after harvest for high protective activity, whatever the method and time of application. In the case of pre-harvest biological treatments, variations in meteorological conditions between the 2 years may have considerably affected strain K population density and its efficacies.     

拮抗细菌菌株BC98-I对青椒枯萎病菌的抑制作用

拮抗细菌菌株BC98-I是从沤肥浸渍液中分离筛选到的一株对青椒枯萎病菌Fusarium oxysporum f.sp. vasinfectum具有强烈抑制作用的蜡质芽孢杆菌。菌含量为5.0×108 cfu/mL的该拮抗菌发酵液在平皿上对青椒枯萎病菌的抑制率为91.7% ±0.5%;该发酵液经高温高压灭活后对青椒枯萎病菌仍有抑制作用,抑制率为58.3% ±2.2%。经硫酸铵沉淀从发酵液中粗提到对青椒枯萎病菌具有抑制作用的拮抗蛋白粗提物,经孔碟法试验表明,浓度从5 ~60 mg/mL的粗提物对病原菌的抑菌圈直径达12 ~27 mm。显微观察显示:拮抗蛋白粗提物可造成青椒枯萎病菌菌丝异常;对孢子的抑制作用主要表现为孢子萌发产生的芽管短,部分芽管膨大形成大泡囊。该拮抗蛋白对热、蛋白酶、氯仿稳定;耐酸碱,对紫外线和离子强度部分敏感。     

Bacterial fruit blotch of melon: screens for disease tolerance and role of seed transmission in pathogenicity

Bacterial fruit blotch (BFB) of cucurbits, caused by Acidovorax avenae subsp. citrulli, is a serious threat to the watermelon and melon industries. To date, there are no commercial cultivars of cucurbit crops resistant to the disease. Here we assessed the level of tolerance to bacterial fruit blotch of various commercial cultivars as well as breeding and wild lines of melon, using seed-transmission assays and seedling-inoculation experiments. Selected cultivars were also tested in a greenhouse experiment with mature plants. All tested cultivars/lines were found to be susceptible to the pathogen, and most of them showed different responses (relative tolerance vs. susceptibility) in the different assays; however, some consistent trends were found: cv. ADIR339 was relatively tolerant in all tested assays, and cv. 6407 and wild lines BLB-B and EAD-B were relatively tolerant in seed-transmission assays. We also provide evidence supporting a strong correlation between the level of susceptibility of a cultivar/line and the ability of the pathogen to adhere to or penetrate the seed. To the best of our knowledge, this is the first attempt to assess melon cultivars/lines for bacterial fruit blotch response.     

Acidified peel of litchi fruits selects for postharvestPenicillium decay

Litchi fruits are fumigated after harvest with sulfur dioxide (SO₂) to prevent their rapid browning. SO₂ blocks enzymatic activity but bleaches the fruits and, if this process is followed by dipping the fruit in dilute hydrochloric acid, the appealing red color is regained. Hot water brushing (HWB) is among the alternative methods that were developed to replace the use of SO₂. HWB reduced fungal population size on the surface of the fruit peel after treatment but did not eliminate fruit infection after storage. Whereas untreated fruits were infected with a variety of fungal species,Penicillium sp. was the only fungus that developed on the pericarp after storage in fruits that had been dipped in 1.5M HCl. Fruit treated by HWB followed by handling and storage under sterile conditions suffered greater decay than fruit stored under non-sterile conditions but with more ventilation. APenicillium sp. isolated from litchi grew well in liquid medium acidified to the pH range reported for SO₂ and HCl-treated litchi fruits. Morphological analysis identified fungal isolates asP. aurantiogriseum. Internal transcribed spacer sequence analysis of five isolates suggested a sequence similarity toP. commune. Our data support the hypothesis that dipping litchi fruit in hydrochloric acid eliminates infection by common opportunistic fungi and selects forPenicillium species that tolerate low pH. http://www.phytoparasitica.org posting April 30, 2004.     

火龙果细菌性软腐病菌的分离与鉴定

<正>火龙果(Hylocereus undatus Britt.et Rose)属仙人掌科三角柱属植物,是集水果、花卉、蔬菜、保健为一体的热带亚热带果树,在中美洲、美国南部地区,以及以色列、越南、泰国和我国的台湾、海南、广东、福建等地均有种植。近年来,随着火龙果种植面积不断扩大,火龙果病害呈逐渐加重趋势,严重威胁火龙果的产业发展。火龙果细菌性病害主要由欧文氏菌(Erwinia sp.)、阴沟肠杆菌(En-     

The effect of nitrogen on the growth and development of giant witchweed,Striga hermonthica Benth.: effect on cultured germinated seedlings in host absence

For the first time in sterile nutrient media in the absence of the host plant, different forms and rates of nitrogen compounds were screened for their effect onS. hermonthica Benth. shoot development beyond seed germination. There was no shoot formation beyond the inoculation stages whenS. hermonthica germlings were grown in media devoid of nitrogen source. In culture media containing some nitrogen sources, healthy shoots were formed. Increasing concentrations of KNO₃, NaNO₃, Ca(NO₃)₂, Mg(NO₃)₂ and asparagine resulted in a significant increase inS. hermonthica shoot development. Inversely, increasing concentrations of (NH₄)₂SO₄, NH₄H₂PO₄, NH₄Cl and urea led to increasing significant reduction ofS. hermonthica shoot development. The amino acids, glycine and asparagine supported reduced shoot development ofS. hermonthica, indicating that organic nitrogen cannot replace inorganic nitrogen forStriga growth. The ammonium nitrogen compounds, (NH₄)₂SO₄ and NH₄H₂PO₄, suppressed further shoot elongation and total dry weight of 20 and 40 days oldStriga hermonthica plants, in sterile culture media. The organic compounds urea, allylthiourea and thiourea had an effect similar to ammonium compounds. Arginine and glycine on the other hand did not suppress the further development of the parasite. The suppressive effect of nitrogen however, was greater when the parasite was 20 days old than when it was 40 days old. This work provides data to show that some nitrogen compounds reduce the severity ofS. hermonthica attack by direct suppression ofStriga growth and development at the post-germination stage and after shoots have been formed.