First report on the whole genome sequence of Pseudomonas cichorii strain JBC1 and comparison with other Pseudomonas species

Pseudomonas cichorii, a plant pathogen that infects a wide range of host plants worldwide, causes several diseases in economically important vegetable crops. Availability of the genome sequences of pathogens can greatly enhance research necessary for the advancement of disease management programmes. Despite the significance of P. cichorii, its whole genome sequence has not been reported previously. The genome sequence of P. cichorii JBC1, described for the first time in this study, is 5 986 012 bp with an average GC content of 58·1% and has 5174 coding sequences (CDS). The genes related to virulence, transport mechanisms, phytotoxic compounds, and secondary metabolite products were analysed and the genome was compared to eight other Pseudomonas species to understand the diversity at species level. Despite the high similarity (up to 80·85%), significant diversity was found among the different Pseudomonas species at the genome level. A comparison of JBC1 pathogenicity island (PAI) regions indicated that the P. viridiflava UASWS0038 PAI has more similarity than the P. syringae PAI region, and the analysis revealed significant divergence at PAI regions among the Pseudomonas species, providing an insight into the differences in host specificity and degree of virulence. In addition, JBC1 encodes antibiotic resistance and tolerance to heavy metals, and two different prophage segments were inserted at three different regions. The genome sequence of JBC1, which was deposited into the NCBI GenBank (accession no. CP007039 ), will be a reference sequence for other P. cichorii strains and a useful resource for further research.     

The N-acetyltransferase gene-implicated iron acquisition contributes to host specificity of Pseudomonas cichorii strain SPC9018 and its virulence

A pathogenicity island within the genome of a multi-host plant bacterium, Pseudomonas cichorii strain SPC9018, comprises the hrp genes encoding a type III secretion system and the pat gene encoding an N-acetyltransferase proposed to play a role in virulence. However, the function of the N-acetyltransferase remains poorly characterized. Interestingly, limiting the iron condition using a phytosiderophore, mugineic acid, resulted in reduced virulence of strain SPC9018 on respective host plants, including eggplant, similar to the reduced virulence observed with a pat gene-deletion mutant. Spectroscopic analyses showed that the pat deletion reduced the concentration of pyoverdine, which is the main siderophore produced by strain SPC9018, leading to a reduction in pyoverdine-mediated iron acquisition. Furthermore, the pat gene deletion mutant showed enhanced expression of the fecA, pvdL, and pvdR genes, whose expression is induced under deficient siderophore-mediated iron uptake. The pat-deletion mutant showed a hyper-swarming phenotype, and the addition of iron decreased this swarming motility. The pat deletion also reduced the adhesion ability of the bacteria, similar to the effect of iron-limited conditions. Furthermore, deletion of the pat gene enhanced expression of the hrp genes. These findings suggested that the pat gene encoding the N-acetyltransferase may be implicated in iron acquisition, contributing to host specificity of P. cichorii strain SPC9018 and its virulence.     

水稻条斑病菌hrpB1基因决定寄主致病性和非寄主过敏反应的功能研究

水稻条斑病菌(Xanthomonas oryzaepv.oryzicola,Xoc)的hrp基因决定了病原菌在非寄主植物上的过敏反应(hypersensitive response,HR)和在寄主植物上的致病性(pathogenicity),基因产物形成Ⅲ型分泌系统(type-Ⅲ secretion system,T3SS)将致病性效应分子注入寄主细胞从而引起水稻产生抗病性或者感病性反应。以位于hrpB操纵单元的首个hr-pB1基因为对象,通过基因敲除方式对其进行了突变,发现hrpB1突变体丧失了在水稻上的致病性和在烟草上激发HR的能力,并且在水稻组织中的生长能力显著降低。RT-PCR测定结果表明,hrpB1的转录表达受HrpG和HrpX的正调控。免疫杂交结果显示,HrpB1蛋白可通过T3SS进行分泌。这些结果不仅明确了hrpB1基因在病原菌致病性中的功能,而且提示了hrp结构基因不仅仅局限于形成Ⅲ型分泌系统,部分hrp基因产物本身也通过Ⅲ型系统分泌到胞外,并且可能起到效应分子的功能。     

XopN-T3SS effector modulates in planta growth of Xanthomonas axonopodis pv. punicae and cell-wall-associated immune response to induce bacterial blight in pomegranate

Bacterial blight caused by Xanthomonas axonopodis pv. punicae (Xap) is an important disease of pomegranate. Most phytopathogenic strains in the Genera Xanthomonas secrete effector proteins by the type III secretion system (T3SS) to suppress pathogen-associated molecular pattern (PAMP)-triggered plant immunity (PTI). The T3SS effectors, referred to as Xanthomonas outer proteins (Xops), are known to be key factors required for bacterial growth and colonization in distinct eukaryotic hosts. Xap contains six Xop-effectors, namely XopC2, XopE1, XopL, XopN, XopQ and XopZ. In this study we analyzed xopN, a conserved effector in Xanthomonas, with reference to sequence identity and its role in governing bacterial growth, and virulence. The xopN of Xap shared maximum sequence identity (98.6%) with pathovar citri. Overlapping extension PCR and double crossing over based homologous recombination strategy was employed to generate a xopN null mutant (Xap ΔxopN) of Xap. A kanamycin gene was used to replace the xopN gene. XopN was required for maximal Xap pathogenicity in its natural host pomegranate. The detailed image analysis on blight lesions revealed 3 fold reduction in watersoaked areas on leaves infiltrated with mutant Xap ΔxopN compared to that of with wild Xap. The in planta population count of Xap ΔxopN was reduced approximately 32-fold relative to the wild strain indicating that xopN is required for maximal growth of Xap in pomegranate. In addition, the Xap ΔxopN mutant induced more callose deposition in infected pomegranate leaves. Taken together, the present study shows that XopN governs Xap growth and modulates cell-wall associated immune response in pomegranate.     

7种检疫性植物病原黄单胞菌III型分泌系统和效应蛋白编码基因的差异性分析

 γ-变形菌纲(γ-Proteobacteria)的黄单胞菌属(Xanthomonas)的大多数种类可引起植物病害,多数是我国检疫对象。与其他革兰氏阴性植物病原细菌一样,植物病原黄单胞菌可通过高度保守的III型分泌系统(type-III secretion system, T3SS)分泌效应蛋白(T3SS-secreted effectors, T3SEs)进入植物细胞,在非寄主植物和抗病寄主植物上产生过敏反应(hypersensitive response, HR)以及在感病寄主植物上具有致病性。尚不清楚哪些种类的黄单胞菌具有T3SS和缺少哪些T3SE是否可作为检疫的依据。搜集7种检疫性植物病原黄单胞菌,通过PCR和Southern杂交试验结果发现:香蕉细菌性青枯病菌(X. campestris pv. musacearum)的ICMP287和ATCC49084菌株、甘蔗流胶病菌(X. axonopodis pv. vasculorum)ATCC13901菌株、洋葱细菌性叶枯病菌(X. axonopodis pv. allii)的LMG576和LMG578菌株中不含有tale基因,并且ATCC13901菌株既不含有T3SS基因也不含有hpa1和xopQ基因;菜豆细菌性疫病菌(X. campestris pv. phaseoli)ATCC49119菌株不含有hpa1基因。相应地,推测含有2~12个tale基因的黄单胞菌有:大豆斑疹病菌(X. axonopodis pv. glycines)ICMP5732和ATCC43911菌株、豌豆细菌性疫病菌(X. axonopodis pv. vignicola)ATCC11648菌株、棉花细菌性角斑病菌(X. campestris pv. malvacearum)ATCC12131和(X. campestris pv. phaseoli)ATCC49119菌株。大豆细菌性斑疹病菌ATCC43911菌株尽管含有hpa1、xopQ和hrcC基因,但在非寄主烟草上不能激发HR反应;而甘蔗流胶病菌ATCC13901菌株不含有hpa1、xopQ和hrcC基因,却激发烟草产生HR反应。这些结果对于分析比较不同植物病原黄单胞菌的致病性因子和设计特定的植物检疫靶点提供了科学线索。     

Cry1Ba3、Cry1Ia8蛋白对Cry1Ac抗性小菜蛾的杀虫活性研究

利用2种苏云金芽胞杆菌原毒素Cry1Ba3、Cry1Ia8及其组合,分别对Cry1Ac抗性种群小菜蛾幼虫进行生物活性测定。结果表明Cry1Ba3、Cry1Ia8对2种目标试虫均有高毒力,LC50分别为0.2175、0.6706μg/mL;Cry1Ba3毒力3倍于Cry1Ia8。2种蛋白混配的结果也表现出高毒力,LC50为0.4375μg/mL,没有显著的协同增效作用,也不存在拮抗。敏感与抗性小菜蛾种群生测结果统计分析比较,结果表明这2种蛋白及其组合与Cry1Ac并无交互抗性。     

The Xanthomonas oryzae pv. oryzae type IV pilus alignment subcomplex protein PilN contributes to regulation of bacterial surface-associated behaviours and T3SS system

The gram-negative plant pathogen Xanthomonas oryzae pv. oryzae (Xoo) is able to infect the host rice and effectively colonize in vascular tissues. The type IV pilus (T4P) is one of the major virulence factors playing an important role in migration of Xoo through host vascular tissues. Here, we identified PilN, a T4P alignment subcomplex protein, which is involved in regulation of swimming motility, and analysed its contribution to bacterial surface-associated behaviours and virulence. We found that the pilN deletion mutant exhibited dramatically reduced twitching motility and scarcely detectable levels of T4P major pili PilA, as well as enhanced biofilm formation and exopolysaccharide (EPS) production. In addition, deletion of the pilN gene in Xoo resulted in impaired virulence in host rice and attenuated type III secretion system (T3SS) genes expression, which is independent of PilA assembly. Expression of the relevant pilN gene in trans was capable of restoring twitching motility and biofilm formation to the wild-type levels in the pilN mutant but partially recovering EPS production and virulence. Moreover, the expression of trh and xrvA genes, which encode the HrpG positive regulators, was decreased in the pilN mutant. Our results suggest that PilN executes versatile functions in bacterial virulence and cell surface-associated behaviours.     

假单胞茵YT3和枯草芽孢杆菌DZl的发酵罐生长条件优化

假单胞菌（Pseudomonas sp．）YT3和枯草芽孢杆菌（Bacillus subtilis）DZl是从植物根际土壤筛选出的具有应用潜力的植物根际促生细菌。研究发酵罐中接种量、转速、通气量等因素对YT3菌量以及对DZl芽孢率、菌量的影响。结果表明：不同的接种量、转速和通气量对Pseudomonas sp．YT3的活菌数、Bacillussubtilis DZl的活菌数及芽孢率均有显著影响。10％的接种量、2m3／ll的通气量对YT3和DZl菌株的作用效果与15％的接种量、3m3／h相当,但显著高于5％的接种量和1m3／h。当转速为200r／min时,YT3具有最高的活菌数,DZl具有最高的活菌数和芽孢率。综合分析认为,YT3和DZl发酵罐的最佳条件为接种量10％、转速200r／min和通气量2矗m。     

利用桥梁载体策略构建水稻条斑病菌T3SS基因诱导表达检测体系

 水稻条斑病菌(Xanthomonas oryzae pv. oryzicola, Xoc)侵染水稻,引起细菌性条斑病(bacterial leaf streak, BLS)。Xoc主要依靠hrp基因簇(T3SS基因)编码的III型分泌系统(type III secretion system, T3SS)将效应蛋白注入水稻细胞中,激发寄主水稻的感(抗)病性。为了准确在Xoc中进行T3SS基因表达调控的分析,本研究设计出桥梁载体策略,将目标基因的启动子或者功能片段构建在高拷贝的桥梁载体,获得融合表达元件,通过亚克隆的方式将融合元件转入骨架载体上,获得用于转录表达和蛋白表达分析的载体。为了验证该策略的可行性,选取hrpG、hrpX和hrcC基因构建了相应的启动子探针载体和蛋白表达载体,将这些载体导入毒性调控子基因trh、lrpX和zur的突变体中,GUS活性测定、荧光定量PCR以及蛋白免疫杂交结果显示:在trh、lrpX和zur的突变体中,hrpG、hrpX和hrcC的转录表达水平、mRNA水平和蛋白表达水平呈现一致。这表明这套桥梁载体策略能够有效应用于T3SS基因转录表达和蛋白表达的分析。综上所述,运用桥梁载体能够克服低拷贝载体DNA遗传操作效率低的缺陷,这一策略也适用于毒性相关基因调控机理的研究,这将为Xoc-水稻互作研究提供高效的工作系统。     

Development of a real‐time PCR assay for Pseudomonas cichorii,the causal agent of midrib rot in greenhouse‐grown lettuce,and its detection in irrigating water

Midrib rot is an emerging disease in greenhouse production of lettuce caused by Pseudomonas cichorii, and probably introduced through contaminated irrigation water. Concentrations of 100 CFU mL^?1 are enough to induce the typical midrib rot symptoms. A sensitive real‐time PCR assay was developed, based on a 90‐bp amplicon from the pathogenicity gene cluster hrcRST and a Taqman Minor Groove Binding probe. Specificity of the assay was tested with 39 P. cichorii strains, including the type strain, and 89 strains from 83 other Pseudomonas species. The relationship between detection signals and P. cichorii DNA concentrations was linear over 6‐logs. Detection threshold with excellent reproducibility was 500 fg of DNA or about 70 genome copies. Sample preparation and DNA isolation were optimized to allow detection in 1 L water samples. The assay was first evaluated with greenhouse irrigation water spiked with serial dilutions of P. cichorii. The calculated cell numbers obtained with real‐time PCR were 10‐fold lower than plate counts of actual spiked cells. However, the assay consistently detected 100 CFU per reaction, corresponding to the detection of 1 CFU mL^?1 of irrigation water, which is well below the concentration needed for midrib rot infection. Finally, the assay proved to be valuable for detecting infective P. cichorii concentrations in the irrigation water of a commercial lettuce production greenhouse.     

十字花科黑腐病菌IV型分泌系统的诱导表达与抗逆功能分析

 细菌通过IV型分泌系统(Type IV Secretion System, T4SS)的接合系统、效应物转运系统和释放/吸收系统3个子家族进行DNA、蛋白质及毒素的分泌和转运。十字花科黑腐病菌(Xanthomonas campestris pv. campestris, Xcc)是一种重要的植物病原菌,也是研究植物病原细菌与植物相互作用机理的模式细菌之一。本研究通过检测Xcc 8004野生型菌株和T4SS突变体在不同条件下的生长、诱导情况及对过敏反应的影响发现:T4SS不影响在培养基中的生长,与野生型菌株相比,T4SS突变体在非寄主辣椒ECW-10R上的过敏反应减弱;T4SS相关基因受基本培养基MMX诱导表达,且与Ni²⁺、H₂O₂、Phenol等抗逆相关。推测T4SS相关基因在该病原菌的接触、识别阶段起作用。     

Characterization and functional analysis of clpB gene from Acidovorax avenae subsp. avenae RS‐1

The type VI secretion system (T6SS) has been reported to be highly associated with various cellular activities in strain RS‐1 of Acidovorax avenae subsp. avenae (Aaa), the pathogen of bacterial brown stripe of rice. However, the role of the clpB gene that presents in the T6SS gene cluster in Aaa pathogenicity has not been clarified. The aim of the current study was to characterize the function of clpB and to investigate its contribution to bacterial pathogenesis using insertional deletion mutation and complementation approaches. The results indicated that mutation of clpB significantly affected bacterial growth, virulence, exopolysaccharide (EPS) production, biofilm formation and expression of 13 other T6SS genes of Aaa RS‐1. The reduction of virulence may be also partially due to the change in EPS composition, which was characterized by the Fourier transform infrared (FTIR) spectra. Furthermore, analysis of protein homology modelling showed that the structure of ClpB is different from those of the other T6SS components. In addition, structural difference was observed between ClpB and Type IV pili (TFP) as well as Type IV pilus biogenesis proteins (PilP), whose functions are similar to ClpB. Taken together, this study demonstrated that the clpB gene plays a key role in Aaa bacterial virulence.     

高温处理西花蓟马若虫对其雌成虫寿命、繁殖力及后代发育的影响

温度是影响昆虫生长发育的重要生态因素之一,高温逆境作为一种绿色防控手段可以有效控制温室害虫种群发展。为了探究高温处理西花蓟马若虫对其种群发展的影响,本试验用41℃和43℃两个温度分别处理西花蓟马初孵若虫2、6、12、24和36h后,观察并记录其雌成虫寿命、繁殖力及后代发育指标的变化情况。结果表明,随着处理时间的延长,两个高温处理后西花蓟马雌成虫寿命,子代若虫总数、成虫总数和总存活率(若虫发育到成虫的存活率)均显著下降,后代雌雄性比呈总体下降趋势。41℃处理后,性比从2.30∶1降低到2.13∶1;43℃处理后从2.25∶1降低到2.07∶1,均明显低于对照的2.69∶1。另外,两性生殖种群相比于孤雌生殖种群更易遭受高温处理的影响。     

Effect of calcined lime (CaO) on chlamydospores of the rice false smut pathogen,Villosiclava virens,and their root attachment and infection

Rice false smut caused by Villosiclava virens is a devastating disease. The smut balls contain chlamydospores, which fall onto paddy soils to become the primary inoculum. After transplanting, the chlamydospores subsequently germinate and infect rice roots. The application of a CaO-containing fertilizer to paddy soils can inhibit the development of rice false smut disease; however, the underlying mechanism is unknown. In this study, we evaluated the suppression of chlamydospore behaviour due to CaO. Specifically, we clarified the effects of the following on chlamydospore morphology and germination: (a) pH, (b) calcium (Ca) concentration, (c) Ca concentration (pH adjusted to 6.5), and (d) CaO-added soil solution. Germination was suppressed at pH 4 and 10, in contrast with the normal germination at pH 6, 7, and 8. Treatments with more than 10 mg/L Ca melted the outer layer of chlamydospores and suppressed germination regardless of whether the pH was adjusted to 6.5. A saturated CaO solution induced bursting of chlamydospores. Suppressed germination and a melted outer chlamydospore layer were also observed, even though the soil buffering effect initially prevented the CaO-mediated pH increase. Furthermore, the chlamydospores within 15 mm from the CaO small lump exhibited suppressed germination in soil. In addition, there was no effect of CaO treatment on chlamydospore attachment to rice roots and hyphal invasion of rice roots in in vitro inoculation tests. These results suggest that Ca concentration is an important factor for inhibiting the occurrence of rice false smut disease.     

裸仁美洲南瓜叶枯病病原菌鉴定及其寄主范围和侵染条件

近年来,在甘肃省武威地区裸仁美洲南瓜发生了一种叶枯病,采用常规组织分离法对罹病组织进行了病原菌分离培养,以及病原菌的形态、分离培养性状观察和致病性测定.结果表明,引起裸仁美洲南瓜叶枯病的致病菌为瓜链格孢Alternaria cucumerina(E11.et Ev.)E11iott.寄主范围测定表明,该病原菌可侵染所有供试葫芦科植物,其中南瓜、西葫芦和甜瓜发病最严重;丝瓜和苦瓜发病较轻;该病原菌还可引起白菜和菜豆发病.温度和保湿时间对病原菌的侵染影响较大,25～30℃温度范围内最易侵染,病害潜育期仅为2～3天;当保湿时间60h时,病叶率达到88%,而保湿时间为6h时,病叶率仅为8%.南瓜不同品种对叶枯病的抗性差异较大,其中二星、天然、光板1号、无壳(李建民)和金无壳为高抗品种;四星、粒赛金、金大地、郑峰光板、日本南瓜为高感品种.     

真菌病毒FpgMBV1中P3基因的原核表达及多克隆抗体的制备

 采用RT-PCR法从假禾谷镰孢菌(Fusarium pseudograminearum)弱毒菌株FC136-2A的菌丝中扩增出假禾谷镰孢菌巨型双链RNA病毒1(Fusarium pseudograminearum megabirnavirus 1,FpgMBV1)的P3蛋白编码序列。测序结果显示,FpgMBV1-P3蛋白的编码序列长2 700 bp,编码901个氨基酸残基。构建原核表达载体pET28a-FpgMBV1-P3并转化大肠杆菌BL21(DE3)菌株,在25℃以0.5 mmol·L^-1 IPTG诱导表达重组蛋白。SDS-PAGE分析表明,重组蛋白分子量约为90 kDa,与预期分子量大小相符。将该重组蛋白作为抗原免疫日本大耳白兔,获得抗血清,采用间接ELISA测定其效价达1∶128 000,能够特异性检测到假禾谷镰孢菌菌丝中的FpgMBV1-P3蛋白。研究结果可为探究FpgMBV1-P3的结构和功能,以及进一步探讨FpgMBV1在假禾谷镰孢菌弱毒菌株FC136-2A中的作用机制奠定基础。