Need for flagella for complete virulence of Pseudomonas syringae pv. tabaci: genetic analysis with flagella-defective mutants ?fliC and ?fliD in host tobacco plants

The flagellins purified from Pseudomonas syringae pv. tabaci induce a hypersensitive reaction in nonhost tomato cells. To investigate the role of flagella and flagellin in the compatible interaction, we generated two types of flagella-defective mutant. The fliC mutant lost the fliC gene that encodes flagellin protein, whereas the fliD mutant lost the fliD gene that encodes HAP2-capping protein. The two mutants had markedly reduced ability to cause disease symptoms in tobacco leaves. Furthermore, propagation of the mutants in tobacco leaves was less than that in wild-type pv. tabaci. Compared to the inoculation with wild-type pv. tabaci, inoculation with the two mutants did not markedly induce the expression of typical defense response-related genes such as PAL and hsr203J. Complementation of each fliC and fliD gene to the corresponding deficient mutant restored motility and virulence. These results indicate that flagella of P. syringae pv. tabaci are indispensable organelles for complete virulence on host tobacco plants.     

Defense responses of Arabidopsis thaliana inoculated with Pseudomonas syringae pv. tabaci wild type and defective mutants for flagellin (ΔfliC) and flagellin-glycosylation (Δorf1)

Flagellin, an essential component of the bacterial flagellar filament, is capable of inducing a hypersensitive response (HR), including cell death, in a nonhost plant. A flagellin-defective mutant (ΔfliC) of Pseudomonas syringae pv. tabaci lacks both the flagellar filament and motility, whereas a flagellin-glycosylation-defective mutant (Δorf1) retains the flagellar filament but lacks the glycosyl modification of flagellin protein. To investigate the role of flagellin protein and its glycosylation in the interaction with its nonhost Arabidopsis thaliana, we analyzed plant responses after inoculation with these bacteria. Inoculation with wild-type P. syringae pv. tabaci induced HR, with the generation of reactive oxygen species and cell death. In contrast, inoculation with either ΔfliC or Δorf1 mutant induced a low level of HR, and inoculated leaves developed a disease-like yellowing. These mutant bacteria multiplied better than the wild-type bacteria in A. thaliana. These results indicate that A. thaliana expresses a defense reaction in response to the bacterial flagellin with its glycosyl structure.     

Erwinia amylovora hrpN mutants,blocked in harpin synthesis,express a reduced virulence on host plants and elicit variable hypersensitive reactions on tobacco

Erwinia amylovora is the bacterium responsible for fire blight, a necrotic disease affecting many rosaceous plants and especially pear tree and apple tree. A protein named harpin, secreted through the Hrp secretion pathway and able to elicit an hypersensitive reaction (HR) on tobacco has recently been isolated. Mutants inhrpN, the gene encoding harpin were described as non pathogenic on immature pear fruit and unable to elicit an HR on tobacco [Weiet al., 1992; Wei and Beer, 1993]. In this paper, the phenotype on plant ofhrpN mutants was carefully determined.hrpN mutants expressed a weak but significant virulence on host plants. Furthermore, when infiltrated into tobacco leaf mesophyll, thehrpN mutants elicited varied responses that fluctuated from null reaction to full necrosis of the infiltrated area. These results show that harpin is not absolutely required neither for pathogenicity on host plant nor for elicitation of an hypersensitive reaction on tobacco. Furthermore, in all the tests performed, mutant blocked in harpin secretion remained non pathogenic and unable to elicit an HR on tobacco. This suggests that factor(s), different from harpin, involved both in pathogenicity and HR eliciting ability is (are) secreted through the Hrp secretion pathway.Abbreviations HR hypersensitive reaction - NSI necrosis severity index - CFU colonie forming units     

Hpa1 secretion via type III secretion system in <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

In many Gram-negative plant pathogenic bacteria the type III secretion system (TTSS), encoded by hrp genes, is essential for pathogenicity in the host and induction of a hypersensitive reaction (HR) in nonhost plants. The expression of hrp genes has been suggested to be repressed in complex media, whereas it is induced in planta and under certain in vitro conditions. We recently reported that XOM2 medium allows efficient hrp expression by Xanthomonas oryzae pv. oryzae. In this study, we investigated hrp-dependent secretion of proteins by the bacteria in vitro. Using modified XOM2, in which bovine serum albumin was added and the pH was lowered to 6.0, we detected at least 10 secreted proteins and identified one as Hpa1. This is the first evidence of protein secretion via TTSS in X. oryzae pv. oryzae.     

Identification of dspEF, hrpW, and hrpN loci and characterization of the hrpN Ep gene in Erwinia pyrifoliae

Erwinia pyrifoliae, the causal pathogen of shoot blight in the Asian pear tree (Pyrus pyrifolia cv. Singo), is host-specific and endemic to Korea. To identify the genes associated with the hypersensitive response (HR) and pathogenicity, a genomic library of E. pyrifoliae WT3 was constructed, and the cosmid clone Escherichia coli (pCEP33) was selected. Sequence analysis of 19.7-kb pCEP33 determined disease-specific (dsp) region homolog and approximately 40% of the hrp genes, which included hrpW, hrpN_Ep, hrpV, hrpT, hrcC, hrpG, hrpF, and partial hrpE homologs, with respect to the cluster of Erwinia amylovora. Additionally, two open reading frames, ORFD and ORFE, were found downstream of the dspEF region. The results of the sequence analysis showed that the pCEP33 did not contain any hrp regulatory genes or most of the genes encoding components of the Hrp protein secretion system. The hrpN_Ep gene of E. pyrifoliae contained five intergenic nucleotide fragment insertions (INFIs) and produced the HR elicitor protein harpin_Ep, with a molecular mass of approximately 44kDa. The purified HrpN_Ep protein elicited faster and stronger HR when infiltrated into tobacco leaves than did HrpN_Ea from E. amylovora. To observe the role of the hrpL gene in the expression of HrpN_Ep, the pEL2 containing hrpL was used to transform E. coli (pCEP33). Expression of HrpN_Ep in E. coli (pCEP33 + pEPL2) was detected with an immunoblot using antiserum raised against HrpN_Ep, indicating a role of hrpL gene in enhancing the expression of HrpN_Ep.     

Flagella-mediated motility is required for biofilm formation by Erwinia carotovora subsp. carotovora

To elucidate the role of flagella in biofilm formation by Erwinia carotovora subsp. carotovora EC1, we used a nonflagellate, nonmotile mutant (ΔfliC) and a flagellate, nonmotile mutant (ΔmotA). A biofilm-inducing medium, which contains the yeast peptone (YP) medium plus the salts of M-63 minimal medium, supported biofilm formation to a greater extent than either the YP or Luria Bertani (LB) medium alone. We demonstrated that both the ΔfliC and ΔmotA mutants greatly reduced their ability to form a biofilm on the surface of the wells of polyvinyl chloride (PVC) microtiter plates. The inability of both mutants to form biofilm on the PVC surface was further confirmed with phase-contrast microscopy. Both aflagellate (ΔfliC) and flagellate (ΔmotA) nonmotile mutants were equally defective in attachment to the PVC surface. The treatment of bacteria with the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), which inhibits the motility of this organism, reduced greatly the biofilm formation. Based on these results, flagella-mediated motility may play an important role in biofilm formation of E. carotovora subsp. carotovora EC1.     

烟粉虱传播的番茄褪绿病毒和番茄黄化曲叶病毒对不同番茄品种的复合侵染

为明确烟粉虱传播的番茄褪绿病毒(Tomato chlorosis virus,ToCV)与番茄黄化曲叶病毒(Tomato yellow leaf curl virus,TYLCV)对不同番茄品种的复合侵染情况,于2015年11月在山东省寿光市温室内采集13个番茄品种共390份疑似发病植株叶片,对不同番茄品种的TYLCV抗性和2种病毒的复合侵染以及温室内发病番茄植株上烟粉虱成虫的带毒率进行检测。结果表明,采集的13个番茄品种经分子标记检测鉴定均为TYLCV杂合抗性;不同番茄品种ToCV与TYLCV的复合侵染率存在明显差异,大果番茄粉宴和贝瑞上复合侵染率最高可达73.3%,而樱桃番茄八喜上未检测到这2种病毒的复合侵染。此外,在发病番茄植株上采集的烟粉虱成虫体内可检测到2种病毒,其中烟粉虱ToCV带毒率为90.7%,TYLCV带毒率为80.0%,同时检测到ToCV与TYLCV的概率为71.3%。表明ToCV和TYLCV的复合侵染在山东省番茄生产中普遍发生,烟粉虱可同时携带这2种病毒并广泛传播。     

N-terminal domain including conserved flg22 is required for flagellin-induced hypersensitive cell death in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Flagellin in Pseudomonas syringae is a potent elicitor of defense responses including hypersensitive cell death in dicot plants. The oligopeptides flg22 consisting of 22 conserved amino acids near the N-terminus of flagellins is reported to induce plant defense responses. Because glycosylation of the central domain of flagellin affects its elicitor activity, we investigated whether any peptide sequence in addition to flg22 is required for flagellin-induced hypersensitive reaction. A study of recombinant flagellin polypeptides indicated that the N-terminal domain including the conserved flg22 is required for flagellin-induced hypersensitive cell death in Arabidopsis thaliana.     

Motility-mediated regulation of virulence in Pseudomonas syringae

We have investigated Pseudomonas syringae pv. tabaci–plant interactions using a large variety of virulence-related mutants. A flagellin-defective mutant, ΔfliC, lost flagellar motility and the ability to produce N-acyl homoserine lactones; it had reduced ability to cause disease symptoms, but the expression of genes encoding a multidrug efflux pump transporter, mexEFoprN, was activated. A type IV pili (T4P)-defective mutant, ΔpilA, lost swarming motility, had reduced expression of hrp-related genes and virulence toward the host tobacco plant, but expression of the genes encoding another multidrug efflux pump transporter, mexABoprM, was activated. These results suggest that the genes regulating flagella- and T4P-mediated motilities also regulate expression of other virulence-related genes.     

繁育寄主植物对浅黄恩蚜小蜂生防潜能的影响

为明确以雪莲果Smallanthus sonchifolius为寄主植物繁育的浅黄恩蚜小蜂Encarsia sophia的生防潜能,测定雪莲果繁育的浅黄恩蚜小蜂个体大小以及其对烟粉虱Bemisia tabaci MED隐种和温室白粉虱Trialeurodes vaporariorum的致死能力,并解析其寄生2种粉虱若虫后的子代发育情况。结果表明,雪莲果繁育的浅黄恩蚜小蜂雌雄蜂体长、头宽及后足胫节长度均显著高于番茄繁育的浅黄恩蚜小蜂。雪莲果繁育的浅黄恩蚜小蜂对烟粉虱和温室白粉虱的平均致死数量分别为24.7头和25.0头,显著高于番茄繁育的21.4头和21.0头。相对于番茄,雪莲果繁育的浅黄恩蚜小蜂寄生烟粉虱和温室白粉虱若虫后其子代发育时间更短,平均分别为13.2 d和12.5 d;而且子代羽化率也显著高于番茄繁育的子代羽化率,分别为84.1%和86.9%。表明与番茄相比,雪莲果为寄主植物繁育的浅黄恩蚜小蜂对烟粉虱和温室白粉虱具有更强的生防潜能。     

山东寿光地区Q型烟粉虱对番茄褪绿病毒的传播

为明确山东寿光地区Q型烟粉虱对番茄褪绿病毒(Tomato chlorosis virus,To CV)感病流行的影响及其传毒特性,于2014年调查了该地区设施番茄上烟粉虱种群动态与To CV发病情况,利用特异引物对烟粉虱体内To CV进行了RT-PCR检测;并在室内测定了带毒Q型烟粉虱取食时间和种群数量对To CV感病株率的影响。结果表明,在番茄发病植株上采集的烟粉虱种群体内可检测到To CV;春茬番茄To CV发病株率随烟粉虱种群数量增加而逐渐升高,4—6月是To CV发生高峰期,6月22日发病株率达100%;秋茬番茄烟粉虱种群数量从10月下旬明显下降,而To CV发病株率升高,11月12日发病株率达100%;室内试验表明,To CV感病株率随着带毒Q型烟粉虱数量与取食时间的增加而明显升高。研究表明,Q型烟粉虱能有效传播To CV,且其种群数量对To CV发病株率存在显著影响,可通过防控烟粉虱以控制To CV的危害。     

Hypersensitive response suppression by type III effectors of plant pathogenic bacteria

The suppressor activity of four representative avirulence (avr) genes from the Pseudomonas syringae group against elicitors of a general hypersensitive response (HR) was examined in tobacco leaves inoculated with double transformants of Pseudomonas fluorescens containing both a cosmid plasmid (pHIR11) carrying the hrp gene cluster and a plasmid carrying each avr gene. The double transformants Pf (pHIR11) containing avrB, avrRps4, or avrPto failed to induce an HR, but that carrying avrRpt2 did induce an HR as Pf (pHIR11 + empty vector) did. Thus, some Avr proteins seem to have suppressor activity against a general HR and should promote aggressiveness of the pathogens.     

普通烟对烟粉虱取食及其与TYLCCNV共侵染的生理生化反应

为明确普通烟遭受烟粉虱Bemisia tabaci取食及与中国番茄黄化曲叶病毒(Tomato yellow leaf curl China virus,TYLCCNV)共侵染时的响应机制,采用常规生理生化指标测定法分析比较MED隐种烟粉虱取食及其与TYLCCNV共侵染处理对普通烟植株中营养组分、防御物质积累及主要氧化酶活性的影响。结果显示,与对照植株相比,烟粉虱取食及其与TYLCCNV共侵染均可导致普通烟植株中可溶性糖和可溶性蛋白含量明显降低,多酚含量显著升高,过氧化氢酶(CAT)活性显著升高,超氧化物歧化酶(SOD)和过氧化物酶(POD)活性呈现先升后降的变化趋势。与烟粉虱取食植株相比,烟粉虱与TYLCCNV共侵染后1 d时普通烟植株中可溶糖含量显著降低了21.05%,3、7和9 d时可溶性蛋白含量分别显著降低了27.40%、26.84%和20.90%;共侵染后的1、3、5和9 d时多酚含量分别显著降低了34.60%、14.30%、3.07%和15.19%;共侵染后的1、3、5和7 d时CAT活性分别提高了3.04倍、1.42倍、1.68倍和1.96倍;共侵染后7 d时POD活性显著降低,共侵染后9 d时SOD活性显著升高。表明普通烟可通过改变其营养组分及积累防御物质或提高氧化酶活性来防御烟粉虱取食及其与TYLCCNV的共侵染,而烟粉虱与TYLCCNV共侵染可诱导植株内产生较单独取食时更少的多酚类防御物质,以提高其在宿主植物上的适应性。     

Differentiation of three whitefly-transmitted geminiviruses from the Republic of Yemen

Three viruses collected in southern Yemen in 1990, infecting watermelon, tobacco and tomato were shown to be transmitted by the whiteflyBemisia tabaci and to have particle morphologies typical of geminiviruses. Colonies ofB. tabaci collected from different locations and from different hosts were used in virus transmission tests with the same host range of plants. Colonies established from both watermelon and cotton in the Yemen were identified as the squash silverleaf-inducing B biotype. The culture host of the colony did not influence virus acquisition and transmission efficiencies to and from other hosts. The tobacco and tomato geminiviruses had a similar host range, but differed in their severity in some hosts. Both these viruses differed from the watermelon geminivirus in host range and symptoms.Datura stramonium, an alternative host for all three viruses, could be co-infected by the watermelon and tobacco viruses.B. tabaci was able to acquire both viruses from the co-infectedD. stramonium and infect seedlings of either original host plant species with their respective viruses orD. stramonium with both. The viruses were identified as watermelon chlorotic stunt virus, tobacco leaf curl virus and tomato yellow leaf curl virus and were distinguished by cross hybridisation.     

中国部分农区作物上本地烟粉虱隐种的鉴定

烟粉虱Bemisia tabaci是一个至少包括36个不同隐种的物种复合体,为明确中国本地烟粉虱隐种的分布,采用线粒体COI基因(mt COI)分子标记法对2011—2012年在中国27个省(市)61个烟粉虱种群中获得的45个非B/Q隐种烟粉虱个体进行了隐种鉴定和分析,并利用邻接法基于mt COI序列构建了本地烟粉虱隐种的系统发育树。结果显示:45个非B/Q隐种烟粉虱对应的45条线粒体COI序列中共有10个单倍型,所有单倍型分别属于烟粉虱Asia II 2、Asia II 6、Asia II 7、Asia I与China1隐种,均为我国本地隐种。其中,在安徽省发现烟粉虱Asia II 2和China1隐种,在福建省发现Asia II 6和China1隐种,在海南省发现Asia II和Asia I隐种,在广东省发现Asia II 7隐种,在江西省发现China1隐种。表明中国部分农区作物上仍然存在少量的本地烟粉虱。     

Visualisation of <Emphasis Type="Italic">hrp</Emphasis> gene expression in <Emphasis Type="Italic">Xanthomonas euvesicatoria</Emphasis> in the tomato phyllosphere

The plasmid pUFZ75 conferred constitutive GFP expression on the bacterial pathogen Xanthomonas euvesicatoria (syn. X. campestris pv. vesicatoria). Colonisation of the tomato phyllosphere and invasion of tomato leaves by X. euvesicatoria was examined using both fluorescence and confocal laser scanning microscopy. Xanthomonas euvesicatoria established a limited population on the tomato leaf surface, primarily occupying the depressions between epidermal cells and around the stomata, prior to invasion of the leaf via the stomata and subsequent growth within the substomatal chamber and the leaf apoplast. Additionally, hrp-gfp fusions were used to report on the temporal and spatial expression of hrp genes during epiphytic colonisation and invasion. Xanthomonas euvesicatoria cells carrying hrpG- and hrpX-gfp reporter constructs were not fluorescent in vitro on non-hrp-inducing LB agar but did exhibit a low level of fluorescence on the leaf surface within 24 h of inoculation, particularly in the vicinity of stomata. Cells carrying hrpG- and hrpX-gfp fusions exhibited high levels of fluorescence 72 h after inoculation in the substomatal chamber and the leaf apoplast. Cells carrying the hrpF-gfp fusion were slightly fluorescent on LB agar and showed no further increase in fluorescence on the leaf surface by 24 h after inoculation, but did show a significant increase in fluorescence 72 h after inoculation in the substomatal chamber and apoplast. The apparent low-level induction of the regulators hrpG and hrpX on the tomato leaf surface may suggest that some of the genes of the X. euvesicatoria HrpG/HrpX regulon are up- or down-regulated prior to invasion of the stomata while still on the leaf surface.     

两种蚜小蜂对烟粉虱MED隐种的田间笼罩控效评价

为明确烟粉虱Bemisia tabaci MED隐种优势寄生蜂海氏桨角蚜小蜂Eretmocerus hayati ZolnerowichRose与浅黄恩蚜小蜂Encarsia sophia GiraultDodd对其控制效果的影响,在棉田尼龙纱网笼罩中释放烟粉虱之后,再分别单独释放海氏桨角蚜小蜂、浅黄恩蚜小蜂以及二者以不同比例组合(1∶1、1∶3、3∶1)释放,定期调查统计2种蚜小蜂对烟粉虱的寄生量和烟粉虱的种群动态。结果表明,相对于不放蜂对照,自首次放蜂后40 d开始,所有放蜂处理均能显著降低烟粉虱若虫种群密度,每100 cm~2叶片上均少于1.00头,但各处理间的烟粉虱种群密度无显著差异;海氏桨角蚜小蜂和浅黄恩蚜小蜂以3∶1比例组合释放的处理中对烟粉虱的寄生量最高,每100 cm~2棉花叶片上能达到4.25头。表明在棉田中对烟粉虱进行生物防治时,以初级寄生蜂海氏桨角蚜小蜂与复寄生蜂浅黄恩蚜小蜂为3∶1的比例释放,可以到达较好的控制效果。