Flagellin from an incompatible strain of Pseudomonas avenae induces a resistance response in cultured rice cells

The host range of Pseudomonas avenae is wide among monocotyledonous plants, but individual strains can infect only one or a few host species. The resistance response of rice cells to pathogens has been previously shown to be induced by a rice-incompatible strain, N1141, but not by a rice-compatible strain, H8301. To clarify the molecular mechanism of the host specificity in P. avenae, a strain-specific antibody that was raised against N1141 cells and then absorbed with H8301 cells was prepared. When a cell extract of strain N1141 was separated by SDS-polyacrylamide gel electrophoresis and immunostained with the N1141 strain-specific antibody, only a flagellin protein was detected. Purified N1141 flagellin induced the hypersensitive cell death in cultured rice cells within 6 h of treatment, whereas the H8301 flagellin did not. The hypersensitive cell death could be blocked by pretreatment with anti-N1141 flagellin antibody. Furthermore, a flagellin-deficient N1141 strain lost not only the induction ability of hypersensitive cell death but also the expression ability of the EL2 gene, which is thought to be one of the defense-related genes. These results demonstrated that the resistance response in cultured rice cells is induced by the flagellin existing in the incompatible strain of P. avenae but not in the flagellin of the compatible strain.     

Flagellin from an incompatible strain of Acidovorax avenae mediates H2O2 generation accompanying hypersensitive cell death and expression of PAL,Cht-1, and PBZ1, but not of Lox in rice

Acidovorax avenae causes a brown stripe disease in monocot plants. We recently reported that a rice-incompatible strain of A. avenae caused hypersensitive cell death in rice and that the flagellin of the incompatible strain was involved in this response. The incompatible strain induced the rapid generation of H2O2 accompanying hypersensitive cell death and the expression of defense genes such as PAL, Cht-1, PBZ1, and LOX, whereas the compatible strain did not. The purified incompatible flagellin also induced the expression of PAL, Cht-1, and PBZ1, but LOX expression was not induced by the incompatible flagellin. PAL and LOX enzymatic activities were increased by inoculation with the incompatible strain, whereas only PAL activity was increased by the incompatible flagellin. Interestingly, the flagellin-deficient incompatible strain lost the ability to generate H2O2 and induce hypersensitive cell death, but PAL, Cht-1, and PBZ1 expression still were induced by inoculation with the deficient strain, suggesting that induction of these genes is regulated not only by flagellin but also by some other signal. Thus, the incompatible flagellin of A. avenae is a specific elicitor in rice, but it is not the only factor capable of inducing the rice defense system.     

Reclassification of subspecies of Acidovorax avenae as A. Avenae (Manns 1905) emend., A. cattleyae (Pavarino, 1911) comb. nov., A. citrulli Schaad et al., 1978) comb. nov., and proposal of A. oryzae sp. nov

The bacterium Acidovorax avenae causes disease in a wide range of economically important monocotyledonous and dicotyledonous plants, including corn, rice, watermelon, anthurium, and orchids. Genotypic and phenotypic relatedness among strains of phytopathogenic A. avenae subsp. avenae, A. avenae subsp. citrulli, A. avenae subsp. cattleyae and A. konjaci, as well as all other Acidovorax species, including A. facilis, the type strain of Acidovorax, was determined. The 16s rDNA sequencing confirmed previous studies showing the environmental species to be very distant from the phytopathogenic species. DNA/DNA reassociation assays on the different strains of A. avenae revealed four (A, B, C, and D) distinct genotypes. Taxon A included six A. avenae subsp. avenae strains from corn that had a mean reciprocal similarity of 81%; taxon B included six A. avenae subsp. avenae strains from rice that had a mean reciprocal similarity of 97%; taxon C contained 11 A. avenae subsp. citrulli strains from cucurbits (cantaloupe, watermelon, and pumpkin) that had a mean reciprocal similarity of 88%, and taxon D contained four A. avenae subsp. cattleyae strains from orchids that had a mean similarity of 98%. The mean reciprocal relatedness between taxa A, B, C, and D was less than 70%. Sequence analysis of 16S rDNA and the 16S-23S rDNA internally transcribed spacer region, as well as AFLP analysis, revealed the same four taxa. All four were easily differentiated phenotypically from each other and from all other recognized Acidovorax species. Strains of A. avenae did not contain 3-hydroxyoctanoic acid, which was found in all other species. On the basis of these and previous genetic and phenotypic results, we propose an emendation of the species A. avenae. A. avenae subsp. citrulli (C strains) and A. avenae subsp. cattleyae (D strains) should be elevated to species rank as A. citrulli and A. cattleyae, respectively. We further propose a new taxon for the B strains, A. oryzae sp. nov. with FC-143T = ICPB 30003T = ICMP 3960T = ATCC 19882T as the type strain.     

Genetic organization of the hrp gene cluster in Acidovorax avenae strain N1141 and a novel effector protein that elicits immune responses in rice (Oryza sativa L.)

The immune system of plants consists of two main arms, pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI). The multiple effectors that trigger ETI are translocated into plant cells by the type III secretion system (T3SS) of pathogenic bacteria. The rice-avirulent N1141 strain of Acidovorax avenae causes ETI in rice, including hypersensitive response (HR) cell death. Sequence analysis indicated that the N1141 genome contains the hrp gene cluster (35.3 kb), including genes encoding the T3SS apparatus. The T3SS-defective N1141 mutant (NΔT3SS) did not cause HR cell death, suggesting that ETI is caused by translocation of effector proteins into rice cells via T3SS. Computational sequence analysis predicted that Lrp, HrpW, and HrpY are secreted by T3SS. The hrpY deletion mutant (NΔhrpY) did not cause ETI, suggesting that HrpY is an important effector of ETI in the interaction between A. avenae N1141 and rice.     

Volatile emission in wheat and parasitism by Aphidius avenae after exogenous application of salivary enzymes of Sitobion avenae

Enzymes in the saliva of the English green aphid, Sitobion avenae (F.) (Homoptera: Aphididae), may be responsible for inducing indirect defence in wheat. In this study, we examined the effects of exogenous application of three salivary enzymes of S. avenae , namely, polyphenol oxidase, pectinase, and cellulase, on the release of volatiles from wheat and the attractiveness of these volatiles to the parasitoid Aphidius avenae Haliday (Hymenoptera: Aphidiidae). In greenhouse experiments, parasitism of S. avenae on plants surrounded by pectinase-treated plants and aphid-damaged plants was significantly higher than parasitism of aphids on plants surrounded by cellulase- and polyphenol oxidase-treated plants. In Y-tube olfactometer tests, female A. avenae significantly preferred the volatiles collected from pectinase-treated plants and aphid-damaged plants. Gas chromatography-mass spectrometry analysis showed that two kinds of semiochemicals, sulcatol and sulcatone, were identified only in pectinase-treated plants and aphid-damaged plants. In field experiments, parasitism of S. avenae by A. avenae was significantly higher on plants sprayed with a mixture of sulcatol and sulcatone than parasitism on plants sprayed with volatiles collected from cellulase- and polyphenol oxidase-treated plants. These results show that pectinase in S. avenae watery saliva can trigger wheat plant indirect defence, and play a crucial role in interaction between wheat, wheat aphid, and A. avenae .     

Analysis of flagellin perception mediated by flg22 receptor OsFLS2 in rice

Plants have sensitive perception systems that recognize various pathogen-derived molecules. We previously reported that rice detects flagellin from a rice-incompatible strain of gram-negative phytopathogenic bacterium, Acidovorax avenae, which induces subsequent immune responses involving cell death. The mechanism of flagellin perception in rice, however, has remained obscure. In this study, we found that flg22, a peptide derived from the flagellin N-terminus, induced weak immune responses without cell death in cultured rice cells. To elucidate the mechanism by which flg22 induced signaling in rice, we characterized OsFLS2, the rice ortholog of AtFLS2, which mediates flg22 perception. Heterologous expression of OsFLS2 functions in Arabidopsis, showing the conservation of the flg22 signaling pathway across divergent plant taxa. OsFLS2-overexpressing rice cultured cells generated stronger immune responses with the induction of cell death following stimulation with flg22 and flagellin. However, examination of the growth rate of the compatible strain in inoculated OsFLS2-overexpressing rice could not confirm bacterial growth suppression compared with wild-type rice. These results suggest that rice possesses a conserved flagellin perception system utilizing the FLS2 receptor which, when upregulated, hardly affects resistance against compatible A. avenae.     

不同营养层次挥发物对燕麦蚜茧蜂寄主搜寻行为的影响

“Ｙ”型管嗅觉计及风洞测定试验明,小麦植株,麦长管蚜（Sitobion avenae),禾谷缢管蚜（Rhopalosiphum padi)对燕麦蚜蜂（Aphidius avenae)雌蜂的吸引作用较小,而有蚜植株及蚜害植株对其吸引作用较大,并以麦长管蚜有蚜植株的吸引作用最强,尽管该蜂对禾谷缢管蚜的寄生率极你,工作量 由其危害诱导产生的挥发性信息化僵物对该蜂仍具有较强的吸引作用,GC－MS鉴定结果表明,麦蚜取食诱导的挥发性信息化合物主要是2－莰烯,6－甲基－5－已烯－2－酮,顺－3－已酰酸酯有水杨酸甲酯,其中6－甲基－5－已烯－2－酮和6－甲基－5－已烯－2－醇对燕麦蚜划蜂的吸引作用最强,水杨酸酯无明显吸引作用。     

麦长管蚜和禾谷缢管蚜对吡虫啉敏感性的比较研究

采用麦穗浸渍法在室内测定了麦长管蚜Macrosiphumavenae(Fabricius)和禾谷缢管蚜Rhopalosiphumpadi(L .)对吡虫啉可湿性粉剂和乳油的敏感度。结果表明 ,禾谷缢管蚜对吡虫啉的敏感度是麦长管蚜的 3～ 4倍 (基于LC50 的比较 )。在北京、河南、江苏、湖北、四川等地同浓度的吡虫啉对禾谷缢管蚜的防治效果要好于麦长管蚜 (特别是在低浓度时尤为明显 )。依据我国不同省份小麦穗蚜的优势种不同 ,建议用吡虫啉防治小麦穗蚜时 ,在南方麦区 ,禾谷缢管蚜为优势种的麦田 ,吡虫啉用药量为 15～ 3 0g(a.i) hm2 ;北方麦区 ,以麦长管蚜为优势种的麦田 ,吡虫啉的用药量应大于 3 0g(a.i) hm2 。     

麦长管蚜抗吡虫啉品系和敏感品系的生殖力比较

通过建立抗性品系和敏感品系的种群生命表,比较麦长管蚜Sitobion avenae(Fabricius)抗吡虫啉品系和敏感品系的生殖力差异,结果表明,抗性品系适合度显著下降,世代历期延长,成蚜寿命缩短;在繁殖上表现为产仔量下降,生殖期缩短。以净生殖率(R0)和内禀增长率(rm)来评价抗性品系的相对生物适合度,抗性品系分别是敏感品系的0.5200和0.7481。因此,麦长管蚜对吡虫啉的抗性品系存在显著的生殖劣势。     

小麦互益素对麦长管蚜及其天敌的影响

研究了在田间使用小麦互益素--水杨酸甲酯和6-甲基-5-庚烯-2-酮对麦长管蚜及其主要天敌--异色瓢虫和燕麦蚜茧蜂的影响.结果表明,小麦互益素虽然没有明显改变麦长管蚜的田间种群变动趋势,但显著降低了麦长管蚜的种群数量.使用小麦互益素能够恶化麦长管蚜的生存环境,使有翅蚜数量明显增加.尽管小麦互益素使天敌物种丰富度有所下降,天敌群落多样性和均匀度指数下降,但燕麦蚜茧蜂、异色瓢虫等优势天敌的种群数量不仅没有减少反而有所增加.因此,田间使用小麦互益素对控制麦长管蚜的危害具有重要作用.     

我国小麦抗麦长管蚜(Sitobion avenae)研究概况

麦长管蚜是我国小麦的重要害虫之一。选育和种植抗虫品种是防治麦长管蚜的理想方法。本文就我国小麦抗麦长管蚜种质资源筛选、抗性机制和小麦品种对麦蚜种群动态影响等方面的研究进展进行了概括,同时指出今后应加强小麦抗麦长管蚜基因的研究。     

模拟风雨对麦长管蚜自然种群发展的干扰作用

麦长管蚜Macrosiphum avenae（Fabricius）是我国小麦生产上的重大害虫,给小麦生产造成严重的威胁。本研究采用人工模拟风雨气象因子的方法,研究了吹风和喷水对麦长管蚜种群数量的干扰作用,解析了麦长管蚜在种群不同发展阶段受模拟风雨干扰后种群变化的特征,明确了麦长管蚜受模拟风雨作用影响其种群生长的关键时期。结果表明,在小麦田进行模拟风雨试验,处理强度越大,防治效果越好;有目标的对靶喷施处理的防治效果明显高于非目标喷施处理;确定人工喷水或吹风处理的最佳时期为小麦灌浆初期,即在该阶段进行一次喷水处理,可以获得最佳的防治效果和保产作用。     

Preliminary Evidence for Phytoalexin Production by Eucalyptus urophylla Leaves as a Response to Inoculation with the Incompatible Pathogen Acidovorax avenae pv. avenae

Leaves of eucalyptus ( Eucalyptus urophylla ) were infiltrated with a cell suspension of the incompatible pathogen Acidovorax avenae pv. avenae and showed a typical hypersensitive response within 24h. Necrotic leaf areas were excised, vacuum infiltrated with 40% ethanol and left under continuous agitation at room temperature for 24h. The diffusate was concentrated, partitioned with ethyl-acetate, concentrated to dryness and resuspended in a small volume of methanol. The biological activity of extracts was evaluated by an agar diffusion method against noncompatible bacteria ( A. avenae pv. avenae and Xanthomonas axonopodis pv. manihotis ) and fungi ( Penicillium sp. and Aspergillus sp. ). Inhibition haloes, when present, were always larger in extracts from leaves infiltrated with the incompatible bacterium than the water control. Thin layer chromatography resolution of crude extracts from leaves infiltrated with both incompatible pathogen cell suspension and water, followed by bioautography with Thieleviopsis paradoxa, consistently rendered, in both situations, a large, diffuse inhibition halo near the origin, assumed to be due to preformed antimicrobial substances. However, extracts from leaves infiltrated with the living cells of the incompatible pathogen gave rise to a smaller, second inhibition halo, near the front, that was interpreted as being one or several phytoalexins.     

影响麦长管蚜体色变化的主导因素

研究麦长管蚜Macrosiphum avenae(Fabricius)体色变化生态主导因素,田间红色麦长管蚜种群对不同抗蚜性寄主的反应和自然条件下,不同体色麦长管蚜的生殖力以及后代种群体色变化情况。结果显示:在实验温度范围内,麦长管蚜种群中红体色蚜虫所占比例随温度升高而增加,在28,29,30,31℃时,红体色蚜虫所占比例分别为6.66%,38.30%,70.60%和65.24%。麦长管蚜体色变化过程中,温度起到重要的作用,而与光周期和寄主营养的关系甚微。红体色麦长管蚜在不同抗蚜性的品种上其种群消长情况存在差异。田间红绿体色麦长管蚜经2代观测,平均蚜量比值分别为9.96和15.85,生殖力差异不显著。在小麦抽穗期到乳熟期红体色麦长管蚜个体比例随着田间条件的改变逐代升高(分别由第1代的17.55%和14.70%增至第2代的29.80%和42.2%)。     

不同地理种群和红色及绿色麦长管蚜遗传多样性分析

麦长管蚜Sitobion avenae(Fabricius)体色变异(红色和绿色)是一种常见的生物学现象,揭示其体色的转换机制对该类害虫的防控和测报具有潜在意义。本文利用微卫星标记技术,对麦长管蚜种群进行聚类分析和分子方差分析,从群体遗传学角度研究了不同地理种群和不同色型之间遗传多样性差异。研究结果表明,麦长管蚜的遗传分化主要以种群内的变异为主,种群间的变异占总变异的比例很小。此外,研究发现,红、绿体色间的遗传分化并不显著,且存在频繁的基因交流。这说明不同区域的不同体色麦长管蚜种群各自之间具有相似的遗传背景。     

Stagonospora avenae secretes multiple enzymes that hydrolyze oat leaf saponins

The phytopathogenic fungus Stagonospora avenae is able to infect oat leaves despite the presence of avenacoside saponins in the leaf tissue. In response to pathogen attack, avenacosides are converted into 26-desglucoavenacosides (26-DGAs), which possess antifungal activity. These molecules are comprised of a steroidal backbone linked to a branched sugar chain consisting of one alpha-L-rhamnose and two (avenacoside A) or three (avenacoside B) beta-D-glucose residues. Isolates of the fungus that are pathogenic to oats are capable of sequential hydrolysis of the sugar residues from the 26-DGAs. Degradation is initiated by removal of the L-rhamnose, which abolishes antifungal activity. The D-glucose residues are then hydrolyzed by beta-glucosidase activity. A comprehensive analysis of saponin-hydrolyzing activities was undertaken, and it was established that S. avenae isolate WAC1293 secretes three enzymes, one alpha-rhamnosidase and two beta-glucosidases, that carry out this hydrolysis. The major beta-glucosidase was purified and the gene encoding the enzyme cloned. The protein is similar to saponin-hydrolyzing enzymes produced by three other phytopathogenic fungi, Gaeumannomyces graminis, Septoria lycopersici, and Botrytis cinerea, and is a family 3 beta-glucosidase. The gene encoding the beta-glucosidase is expressed during infection of oat leaves but is not essential for pathogenicity.     

Biochemical and Morphological Features of Rice Cell Death Induced by Pseudomonas avenae

Pseudomonas avenae is a Gram-negative phytopathogenie bacteriumthat causes the symptom of a brown stripe in infected susceptibleplants. The host range of P. avenae is wide among the monocotyledonousplants, however, individual strains can infect only one or afew host species. A rice-incompatible strain, N1141, causedrapid cell death in sheath sections and in cultured rice cells.A rice-compatible strain, H8301, also induced cell death, however,this cell death in a compatible interaction was delayed comparedto the cell death induced by the N1141 incompatible strain.Inoculation of N1141 strain induced expression of EL2 gene whichis thought to be one of the defense-related gene. Terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) of culturedrice cells showed that DNA cleavage occurred only in N1141-inoculatedrice cells. N1141 strain caused cytoplasmic condensation, shrinkage,and plasma membrane blebbing, all of which are important morphologicalcharacteristics of programmed cell death (PCD). In contrast,H8301 strain inoculated rice cells appeared to show weakeningof the cell wall instead of cytoplasm condensation, shrinkageand membrane blebbing. These results suggest that the rapidcell death of rice induced by the incompatible strain is characterizedas PCD. (Received May 22, 1999; Accepted July 22, 1999)     

小麦间作大蒜或油菜对麦长管蚜及其主要天敌种群动态的影响

麦长管蚜是我国冬小麦产区的重要害虫.试验探索了不同间作方式对麦长管蚜及其主要天敌的影响.结果表明,在调查期内,麦-油(油菜)间作田和麦-蒜(大蒜)间作田中麦长管蚜无翅蚜的种群密度多显著低于单作田;麦-油间作田中有较高的瓢虫种群密度和瓢蚜比;5月2日前,麦-油间作田中蚜茧蜂的种群密度高于单作田和麦-蒜间作田;5月5日后,麦-油间作田的僵蚜率和蜂/蚜比也显著高于单作田和麦-蒜间作田;麦-蒜间作田有翅蚜的种群数量较高,但瓢虫和蚜茧蜂的种群数量无明显变化.麦-油间作和麦-蒜间作均能对麦田中麦长管蚜起到较好的控制作用.     

不同小麦品种对麦长管蚜的抗性

在大田对18个品种98-10-34、PI-矮、千斤早、XZ1三代、AmigoF3、98-10-30、98-10-35、Tm44、98-10-9、Amigo、XZ13三代、98-10-32、AmigoF2、AmigoF4、PI-高、XZ13母本、陕229及Largo进行耐害性测定,对小麦的感蚜量做了系统调查,分别分析各品种耐害补偿功能及自然感蚜特征之间的关系。结果显示:品种Tm44、AmigoF3、Amigo、AmigoF2、AmigoF4和98-10-9虽然自然感蚜率比较高,但与同类相比减产率却不高;品种Largo、98-10-32、陕229、98-10-35、XZ13母本、PI-高和XZ1三代自然感蚜率比较低,而且其减产率也相对较低;品种PI-矮不仅自然感蚜率高,减产率也排名第二;98-10-34为明显感虫品种,还有98-10-30、XZ13三代和千斤早因为虽然感蚜率最小,但减产率相对较高,所以也是感虫品种。采用室内网罩饲养实验系统研究4个小麦品种陕229、98-10-9、Tm44和largo对麦长管蚜Macrosiphum avenae(Fabricius)生命周期及生殖情况等的影响,同时通过组建不用品种上麦长管蚜繁殖特征生命表,从量的角度揭示小麦品种抗性与麦长管蚜增长之间的关系。结果表明:不同小麦品种对麦长管蚜的存活率,发育历期和净增值率的影响明显不同,品种间抗蚜性存在显著差异。