炭样小单孢菌产抗生素对水稻细菌性条斑病菌的抗菌作用及其机理

【背景】水稻细菌性条斑病菌为水稻细菌性条斑病的病原菌,土壤中分离到的一株具有广谱抗菌活性的炭样小单孢菌JXNU-1,其发酵产物(即抗生素JX)对植物病原菌具有较强的抑菌活性。【目的】研究抗生素JX对水稻细菌性条斑病菌的抗菌作用及其机理。【方法】采用杯碟法测定抑菌圈大小,二倍稀释法测定最低抑菌浓度、最低杀菌浓度,并且从菌体形态观察、电导率变化、培养液大分子漏出、蛋白质合成、核酸合成和膜电位变化6个方面探究其作用机理。【结果】抗生素JX对水稻细菌性条斑病菌的抑菌圈直径达18.84±0.28mm,最低抑菌浓度和最低杀菌浓度分别为1.39μg/m L和2.78μg/mL,且杀菌速度很快,作用12 h的杀菌率达100%。在抗生素JX作用下,水稻细菌性条斑病菌的细胞形态发生改变,培养液电导率、膜电位和大分子漏出量均随抗生素浓度增加而增大,但菌体蛋白质含量随着抗生素浓度增加而降低,同时,通过实时荧光定量PCR方法检测发现ef-p表达量下调。【结论】抗生素JX对水稻细菌性条斑病菌具有较强的抗菌作用,推测其抑菌机理是通过抑制菌体蛋白质的生物合成和影响细胞膜完整性而起作用。     

Synthesis of Optically Active N-(2-Pyridyloxiran-2-ylmethyl) benzenesulfonamide Derivatives and Their Herbicidal Activity

Novel herbicidally active sulfonamide compounds having a 2-arylsubstituted oxiranylmethyl structure are racemates due to a chiral carbon in the oxirane moiety. To clarify the stereochemical structure-activity relationship, we synthesized each enantiomer of 4-chloro-N-[2-(6-chloropyridin-2-yl)-2-oxiran-2-ylmethyl]-3,N-dimethylbenzenesulfonamide and N-[2-(6-chloropyridin-2-yl)-2-oxiran-2-ylmethyl]-N-methyl-5,6,7,8-tetrahydronaphthalene-2-sulfonamide by chemical methods including Sharpless asymmetric chlorohydroxylation. The results of herbicidal tests indicated that the (S)-isomers were the active forms.     

鸭跖草生防菌叶点霉F-3菌株生物学特性的研究

主要研究影响鸭跖草生防菌叶点霉菌株菌丝生长和孢子萌发的条件。研究表明,PDA培养基和鸭跖草培养基是该菌生长的适宜培养基,该菌生长的温度范围为20~35℃,最适温度为25℃,分生孢子萌发的温度范围为25~35℃,最适温度为30℃;pH为6时生长最快,偏酸偏碱有利于孢子的萌发,在pH为10时萌发最好。F-3菌株在供试的6种碳源中生长速度相差不大,在不同氮源中差异显著,以硝酸铵为氮源生长最好。     

水稻细菌性条斑病菌中受DSF 调控的鞭毛基因flgD、flgE 的功能分析

[目的]为了阐明可扩散性信号分子(diffusible signal factor,DSF)调控的鞭毛基因对水稻细菌性条斑病菌(Xanthomonas oryzae pv.oryzicola,Xoc)Rs105的致病性等方面的影响.[方法]采用PCR的方法克隆靶标基因flgDxoc和flgExoc,用同源重组法构建缺失突变体,测定突变体及其互补菌株的菌体形态、运动性、致病性及过敏性反应等表型,用反转录PCR(RT-PCR)的方法验证Rs105和ArpfFxoc(rpfFxoc基因的缺失突变体,不产生DSF)中flgDxoc、、flgExoc表达量的差异.[结果]从Rs105基因组中克隆到flgDxoc、flgExoc基因,并证实这两个基因在基因组中均为单拷贝.PCR和Southern杂交结果显示,flgDxoc、flgExoc基因被成功敲除.与野生型相比,突变体的鞭毛产生能力丧失,游动性和趋化性能力减弱,接种水稻叶片显示其致病性部分减弱,基因互补可使其恢复.生长能力和对烟草叶片的致敏性无明显改变.RT-PCR结果显示,flgDxoc、flgExoc基因在△rpfFxoc中的转录水平明显降低.[结论]本试验表明:FlgD、FlgE是水稻细菌性条斑病菌鞭毛形成所必需的因子;进一步证明了DSF通过调控flgDxoc、flgExoc基因表达,从而影响条斑病菌的致病性等表型.为深入认识DSF对细菌性条斑病菌鞭毛基因簇的调控提供了科学依据.     

水稻黄单胞菌黄色素合成相关基因的克隆与鉴定

用硫酸二乙酯(DES)诱变水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae,简称Xoo)和条斑病细菌(Xanthomonas oryzae pv. oryzicola, 简称Xooc),分别得到5株和13株黄色素缺失突变体,其中来自Xooc的M6和M12 还丧失了对水稻的致病性和在烟草上激发过敏反应的能力。以Xooc黄色素缺失突变体M51为受体菌交叉互补从Xoo JXOIII基因文库中筛选出一个黄色素合成相关的基因克隆pA341,以Xoo黄色素缺失突变体M1071为受体菌,从Xooc RS105基因文库中获得了一个黄色素合成相关的基因克隆pA270。功能互补显示,18株黄色素缺失突变体中的10株能分别被pA341和 pA270互补后正常产生黄色素,但这两个克隆不能同时互补同一株黄色素缺失突变体。能被pA341互补的黄色素缺失突变体M6没有恢复对水稻的致病性和在烟草上激发过敏反应,表明黄色素合成相关基因与hrp基因间不存在相关性。斑点杂交结果表明,pA270与pA341之间没有同源性。pA270亚克隆结果显示,与黄色素合成相关的基因约11.6kb大小,以基因簇的形式存在,不仅决定了黄色素的产生,还影响黄色素合成的数量和质量（吸收峰）。在紫外光条件下,黄色素能够提高菌体的存活率,提示黄色素对病原细菌有保护作用。     

非寄主抗病基因Rxo1介导的水稻对Xanthomonas oryzae pv.oryzicola的抗病反应

通过表型鉴定、反转录PCR和实时定量PCR方法,利用转基因和非转基因水稻植株,研究由Rxol基因介导的,水稻对细菌性条斑病菌的抗性反应。结果观察到3个涉及过敏性反应的基因由Rxol基因诱导表达,并对其进行了分析。这3个基因参与编码病程相关蛋白,在转基因水稻植株中呈上调表达,表明水杨酸信号转导途径在抗性反应中发挥重要的作用。     

Status of Streptomycin Resistance Development in Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola in China and their Resistance Characters

Rice leaves with bacterial blight or bacterial leaf streak symptoms were collected in southern China in 2007 and 2008. Five hundred and thirty‐four single‐colony isolates of Xanthomonas oryzae pv. oryzae and 827 single‐colony isolates of Xanthomonas oryzae pv. oryzicola were obtained and tested on plates for sensitivity to streptomycin. Four strains (0.75%) of X. oryzae pv. oryzae isolated from the same county of Province Yunnan were resistant to streptomycin, and the resistance factor (the ratio of the mean median effective concentration inhibiting growth of resistant isolates to that of sensitive isolates) was approximately 226. The resistant isolate also showed streptomycin resistance in vivo. In addition to resistant isolates, isolates of less sensitivity were also present in the population of X. oryzae pv. oryzae from Province Yunnan. However, no isolates with decreased streptomycin‐sensitivity were obtained from the population of X. oryzae pv. oryzicola. Mutations in the rpsL (encoding S12 protein) and rrs genes (encoding 16S rRNA) and the presence of the strA gene accounting for streptomycin resistance in other phytopathogens or animal and human pathogenic bacteria were examined on sensitive and resistant strains of X. oryzae pv. oryzae by polymerase chain reaction amplification and sequencing. Neither the presence of the strA gene nor mutations in the rpsL or rrs were found, suggesting that different resistance mechanisms are involved in the resistant isolates of X. oryzae pv. oryzae.     

水稻细菌性条斑病菌RpfCxoc/RpfGxoc双组分系统的功能

【目的】旨在阐明双组分系统RpfCxoc/RpfGxoc在水稻细菌性条斑病菌(Xanthomonas oryzae pv.oryzicola,Xoc)DSF(diffusible signal factor)合成、致病性等相关方面的生物学功能。【方法】以Xoc野生型菌株Rs105为母体,利用自杀载体pK18mobsacB缺失突变rpfCxoc、rpfGxoc和rpfGCxoc(rpfCxoc和rpfGxoc双基因),测定突变体及其互补菌株的DSF合成水平、对水稻的致病性、胞外多糖(extracellular polysaccharide,EPS)产量、菌体形态及群体结构。【结果】从Rs105基因组中克隆了rpfCxoc和rpfGxoc基因,并获得了相应的单基因或双基因缺失突变体。与Rs105相比,ΔrpfCxoc和ΔrpfGCxoc过量合成DSF信号分子,但是ΔrpfGxoc合成DSF的能力显著下降;rpfCxoc和rpfGxoc单基因或双基因的缺失突变均导致Xoc的致病性丧失,EPS合成水平下降34.1%-48.5%,形成菌体高度聚集的生物膜结构。【结论】RpfCxoc/RpfGxoc双组分系统调控Xoc的DSF生物合成、EPS产生和生物膜的驱散,是Xoc保持致病性所必需的因子。     

Isolation of an Antibacterial Substance from Mahonia fortunei and its Biological Activity against Xanthomonas oryzae pv. oryzicola

This study aimed to isolate the antibacterial substance from Mahonia fortunei and determine its antibacterial activity against Xanthomonas oryzae pv. oryzicola (Xoc). Bacterial leaf streak of rice, caused by Xoc, is an important rice disease and difficult to control. During a screening of antibacterial plants against plant pathogenic bacteria at an early stage, the extract from M. fortunei was found to have a strong inhibitory effect on Xoc. In this study, the chemical components of M. fortunei stems were extracted using methanol solvent, the antibacterial substance was isolated and purified by liquid–liquid partition and silica gel column chromatography and its structure was identified by nuclear magnetic resonance. The effect and mode of action of the antibacterial substance on bacterial leaf streak of rice were also detected under greenhouse conditions. Two compounds were identified, berberine and jatrorrhizine, which had a strong inhibitory effect on Xoc. The antibacterial activity of berberine was stronger, with a half‐maximal inhibitory concentration (IC₅₀) of 2.9008 mg/l. At the concentration of 0.5 g/l, its control efficacy on bacterial leaf streak of rice was more than 84%. Additionally, berberine could be absorbed by rice leaves and be translocated up and down in the rice plant, and the effective period was long, but its capability of lateral translocation inside the blade was poor.