β-氨基丁酸诱导烟草产生PR蛋白及对TMV的抑制作用

 本文研究了β-氨基丁酸(BABA)诱导系统侵染寄主烟草NC89产生PR蛋白及其对TMV的抑制作用。用5mmol/LBABA对抗性烟和非抗性烟进行叶面喷施处理均可以抑制烟草叶片中TMV的产生,平均抑制率分别达到59%和49%。BABA和SA处理均可以诱导2种烟草叶片中PR1、PR2和PR5基因的表达,其中对PR1的诱导作用相对较强。分别用BABA和SA处理非抗性烟草,在处理后的不同时间内2种药剂对PR1基因的诱导规律基本相同。用BABA和SA分别预处理非抗性烟草2d后接种TMV,2种药剂对烟草叶片中PR1基因的影响是完全不同的,其中,BABA处理后接种TMV抑制PR1基因的表达,在接种后第72h已经检测不到PR1基因的存在,SA处理后接种TMV对PR1基因的影响呈现弱-强-弱的变化趋势,在接种后第24hPR1的表达量达到最强,以后逐渐减弱。试验结果表明:BABA能够提高非抗性烟草对TMV的抗性,但是BABA诱导烟草产生抗TMV的作用机制可能是一种不同于目前普遍公认的SA的作用机制。     

瞬时表达靶向TMV外壳蛋白基因的siRNA能干扰病毒侵染

 RNA干扰(RNA interference,RNAi)是与内源性mRNA编码区某段序列同源的双链RNA导入细胞后,该mRNA发生特异性降解,从而导致该基因表达沉默的现象。小干扰RNA(small interfering RNA,siRNA)作为RNAi途径的重要中介,已被广泛应用于动、植物抗病毒治疗研究。本文以烟草花叶病毒(Tobacco mosaic virus,TMV)外壳蛋白基因为靶位,设计合成表达小干扰RNA的寡核苷酸,亚克隆到植物双元表达载体pBI121中,直接转化根癌农杆菌。通过根癌农杆菌介导的瞬时表达法,研究了同源于TMV外壳蛋白的siRNA对TMV侵染的干扰作用。结果表明,瞬时表达的siRNA能够特异性干扰TMV侵染。含有重组表达载体pBI121/siRNA的根癌农杆菌渗入普通烟植株,在TMV接种后14d其上部叶片没有表现典型的花叶症状。对这些叶片进行Northern杂交试验也没有检测到TMV病毒的RNA积累或仅有很少量的积累。在枯斑寄主心叶烟上,siRNA的瞬时表达可使TMV侵染后的枯斑数明显减少,甚至不产生枯斑。此外,同源于TMV外壳蛋白的siRNA瞬时表达对非同源的黄瓜花叶病毒(Cucumber mosaic virus,CMV)没有抑制作用,表明siRNA的干扰作用具有高度的同源依赖性。     

油菜花叶病毒15号和菸花叶病毒间干扰作用的研究

 TMV与YMV₁₅在系统感染寄主——菸(Nicotiana tabacum)上的相互干扰作用是很微弱的。同时接种二病毒时,在感染的早期(接种后2-3天) YMV₁₅占优势,在此时间以后TMV的浓度就超过YMV₁₅。二病毒以相同的侵染力同时接种心叶菸(Nicotiana glutinosa)时,则大部分的局部斑点是由YMV₁₅形成的。上述结果表明,YMV₁₅竞争侵染点的能力较TMV为大。试验并证明完整的YMV₁₅及其蛋白都能干扰TMV侵染菜豆(Phaseoluss vulgaris),因而这种干扰似决定于病毒蛋白质部分对于侵染点附着的特异性。     

一种新的90 kD胞外蛋白激发子诱导烟草系统获得抗性研究

 就棉疫病菌(Phytophthora boehmeriae Saw.)培养滤液中提纯获得的90 kD胞外蛋白激发子诱发烟草系统获得抗性进行了研究。以10 nmol/L激发子溶液注射处理Samsun NN烟草叶片24 h后,在处理叶片及其上、下各2片叶片接种TMV,结果是处理叶及其上、下各2片叶片上的枯斑数显著少于对照,诱抗防效达40.9%~53.1%;接种TMV7d后处理叶片的枯斑平均直径为1.23mm,显著小于对照叶片上的枯斑直径2.97mm,但是处理叶片的上、下叶片上的枯斑平均直径与对照没有显著差别。以10 nmol/L激发子溶液注射处理Samsun NN烟草叶片分别立即接种或于1、2、4、7、15 d接种TMV,结果证明该激发子诱导烟草对TMV的抗性以处理后第1d至第4 d接种为较好,防效达30.2%~5 0.4%;处理后立即接种和第7d接种TMV诱抗防效仅4%左右,处理叶片上的枯斑数与对照没有显著差别,表明较强的诱导抗性可持续时间<7d。用不同浓度激发子处理烟叶,所测定的0.5~100 nmol/L各浓度均可显著地诱发烟草对TMV产生获得抗性,诱导抗性效果为34.9%~5 8.2%,诱导抗性效果随浓度的降低呈下降趋势。以10 nmol/L激发子溶液注射处理W38烟草叶片,2 d后分别注射接种烟草野火病菌(Pseudomonas syringae pv.tabaci)菌液或喷雾接种烟草赤星病菌(Alternaria alternata)分生孢子,结果是,注射接种烟草野火病菌5d后处理叶片及其上、下叶片上的野火病病斑均显著小于对照;处理叶片上的赤星病病斑数及病斑面积明显小于对照,表明该激发子可诱导烟草对野火病和赤星病产生抗性。上述结果表明,90kD蛋白激发子诱导烟草产生的获得抗性是一种典型的系统获得抗性,该系统获得抗性对病原菌具广谱抗性。     

壳寡糖季铵盐衍生物纳米银的合成及其诱导烟株抗烟草花叶病毒活性

为开发符合高效、低毒且环境友好的新型烟草花叶病毒(TMV)抑制剂,以具有季铵盐结构的新型壳寡糖季铵盐衍生物为还原剂和稳定剂,合成了壳寡糖季铵盐衍生物纳米银溶液,并研究了不同浓度该纳米银溶液诱导烟株产生抗TMV的活性。结果表明:所合成的壳寡糖季铵盐衍生物纳米银溶液在透射电镜(TEM)下显示分布较均匀,粒径主要集中在7~12 nm。在珊西烟上以25μg/m L的壳寡糖季铵盐衍生物纳米银溶液对枯斑的抑制效果最好,抑制率为74.0%,比50μg/m L壳寡糖溶液和2%宁南霉素水剂分别高41.5%和24.4%;对于普通烟K326,壳寡糖季铵盐衍生物纳米银溶液可缓解被病毒侵染的烟草叶绿素含量的下降幅度,提高叶片中超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(PPO)的活性,降低丙二醛(MDA)的含量,提高可溶性蛋白含量。表明壳寡糖季铵盐纳米银溶液可提高烟株对TMV的抗病性。     

高温激活RNA沉默介导的心叶烟抗病毒防御反应

 在真核细胞内,RNA沉默是一种保守的抵抗病毒及外源基因等寄生分子的防御机制。温度可以明显地影响植物体与病毒之间的相互作用。在常温状态下,常常会伴随病毒病的爆发,但在较高温度下,症状则会减轻,甚至有些感病植株新生叶片可以较快地恢复健康。目前这类现象的内在机制还不完全清楚。本文通过定量分析不同温度下马铃薯X病毒(Potato virus X,PVX)和马铃薯Y病毒(Potato virus Y,PVY)侵染的心叶烟(Nicotiana glutinosa)植株体内病毒蛋白含量,结合症状观察,确定30℃左右是PVX和PVY在心叶烟上引起症状消失即发生系统性RNA沉默的临界温度,此时的感病植株上部新生展开叶片呈现无症状态,下部已发病叶片症状不变。在常温时,由病毒激发的RNA沉默会受到抑制,病毒来源的小分子干涉RNA (small interfering RNAs,siRNAs)含量极少或根本检测不到,植株对于病毒的敏感性较强。当温度达到30或32℃时,感病植株下部症状保持叶片中,病毒起源的siRNAs含量明显增加,而上部新生健康叶片中却检测不到siRNAs的存在。这说明高温激活了RNA沉默介导的植物抗病毒防御反应,导致病毒症状减轻甚至消失。     

Elicitin诱导烟草微敏反应和防卫基因的表达

 研究了一种α型elicitin-parasiticein对烟草微敏反应(microscopic hypersensitive response, micro-HR)和防卫反应分子表征基因表达的诱导作用。用150 nmol/L的parasiticein喷洒烟草叶片12 h后, 经曲利本蓝(trypan blue)染色, 光镜下可观察到有细胞坏死, 说明parasiticein引起了micro-HR, 而水和低浓度的parasiticein不能引起micro-HR。用parasiticein注射叶片可引起肉眼可见的HR, 注射用parasiticein浓度远比喷洒引起micro-HR的浓度低。Parasiticein还可诱导对TMV的抗性, 浓度在30 nmol/L时比150 nmol/L的效果好。用parasiticein注射烟草30 min, HR表征基因hsr203J和hin1开始表达, 在9 h内逐渐降低, 12~16 h肉眼可观察到HR。Parasiticein喷洒烟草后, 病程相关蛋白(pathogenesis-related, PR)基因PR-1b在诱导的第1 d到第5 d都有一定量的表达。可见, parasiticein能同步诱导过敏反 应和抗病 性及其分 子表征基 因的表达 。     

TMV侵染烟草诱导寄主产生细胞自噬

 细胞自噬(autophagy)—自体吞噬,是真核生物中一种高度保守并普遍存在的物质循环利用过程。为明确烟草花叶病毒(Tobacco mosaic virus,TMV)侵染烟草是否引起细胞自噬,本研究利用TMV-U1和普通烟(Nicotiana tabacum cv. Blight yellow)为主要研究材料,利用透射电子显微镜观察、单丹磺酰尸胺(MDC)荧光染色技术、自噬标记分子ATG8f-ECFP荧光观察和荧光定量RT-PCR(qRT-PCR)分别检测TMV侵染烟草后的烟草细胞内自噬结构的形成、酸性细胞器的存在和细胞自噬相关基因(Autophagy related gene,ATG)ATG3、ATG4、ATG5、ATG6、ATG7、ATG8a、ATG18a的表达特征。结果表明,TMV侵染72 h后,在液泡边际和液泡内发现大量疑似自噬体结构。利用MDC荧光染色和ATG8f-ECFP瞬时表达技术,通过激光共聚焦显微镜观察,在TMV侵染72 h后发现点状荧光信号。TMV侵染48 h后,除ATG5外,烟草ATG3、ATG4、ATG6、ATG7、ATG8a、ATG18a表达均上调并在72 h达到顶峰。表明TMV侵染后感病烟草均可检测到细胞自噬现象,推测TMV侵染普通烟可诱导寄主产生细胞自噬反应。     

云南、福建、湖南烟区烟草花叶病主要病毒种类检测及黄瓜花叶病毒亚组鉴定

 采用抗原直接包被和双抗体夹心酶联免疫吸附测定法(ELISA)对采自云南、福建、湖南烟区烟草花叶病样品进行了病毒种类检测,利用三抗体夹心ELISA对黄瓜花叶病毒(Cucumber mosaic virus,CMV)的亚组类型进行了鉴定。在云南采集的520个花叶病样品中,烟草花叶病毒(Tobacco mosaic virus,TMV)、CMV和马铃薯Y病毒(Potato virus Y,PVY)总检出率分别为71.74%、55.01%和6.35%;在福建采集的150个花叶病样品中,TMV、CMV和PVY的总检出率分别为94%、24.66%和8.00%;在湖南采集的74个花叶病样品中,TMV、CMV和PVY的总检出率分别为58.11%、51.35%和2.70%。部分样品为2种以上病毒复合侵染。云南、福建和湖南采集的64个CMV阳性样品中,属亚组Ⅰ的样品为57个,占89.1%;属亚组Ⅱ的样品为10个,占15.6%;其中3个样品为亚组Ⅰ和亚组Ⅱ的复合侵染。     

一种细菌蛋白CZ控制烟草花叶病毒(TMV)的作用机理初探

以活性蛋白CZ处理NC89烟(Nicotianatabacumvar.NC89)12 h后,摩擦接种TMV,1、5、8、12、16 d分别取各处理叶片测过氧化物酶(POD)和苯丙氨酸解氨酶(PAL)的活性及丙二醛(MDA)和叶绿素的含量。结果表明,POD和PAL2种酶的活性均比对照明显提高;CZ处理使NC89烟叶片内MDA的含量降低,而叶绿素的含量增高。电镜下观察到活性蛋白能打破TMV粒体的规则排列,与TMV粒体不可逆的结合,从而降低侵染力。     

Effect of salicylic acid treatment on tomato plant physiology and tolerance to potato virus X infection

Salicylic acid (SA) is an inducer of systemic acquired resistance (SAR) and could be a potential candidate in the control of plant virus diseases. In this study we assayed under controlled conditions the potential effect of three doses of exogenous SA treatment on tomato plants infected with Potato virus X (PVX) and measured their effects on: different physiological parameters (gas exchange, stable isotopes, chlorophyll content), the activation of secondary metabolism, viral accumulation and induction of the expression of pathogenesis-related proteins (PRs) such as ß-1, 3-glucanase (PR2) and chitinase (PR3). SA treatment increased the expression of PR2, the activity of phenylalanine ammonia lyase (PAL) and the concentration of antioxidant compounds at 7 days post-treatment. Earlier expression of PR3 compared to PR2 was observed. SA treatment delayed the detection of PVX by ELISA in uninoculated leaves of mechanically infected tomato plants. Although the effect of PVX infection on physiological parameters was weak, moderate SA treatments showed enhanced photosynthesis, particularly for infected plants. The results obtained confirm that SA promotes major changes in the induction of resistance in tomato plants and suggest that treatment with exogenous SA could be considered to reduce the infections caused by PVX.     

Virus-induced changes in the response of faba bean to infection by Botrytis

Prior infection of faba bean with the viruses bean yellow mosaic and bean leaf roll increased host susceptibility to subsequent infection by Botrytis fabae and B. cinerea. Cell necrosis beneath inoculum droplets, rate and extent of lesion spread and sporulation of B. fabae were all increased on detached leaves from virus-infected compared with healthy plants. Changes were most marked in young leaves showing conspicuous symptoms of systemic virus infection and in plants virus-infected for at least 2-4 weeks. Localized lesions produced by B. cinerea or a low concentration of B. fabae conidia (10³ spores/ ml) showed increased cell necrosis but were not transformed into aggressive, spreading lesions on virus-infected leaves.     

外源茉莉酸甲酯诱导水稻抗瘟性相关防御酶和内源水杨酸的变化

 用外源茉莉酸甲酯(MeJA)处理抗稻瘟病近等基因系水稻CO39和C101LAC,显著减轻了稻瘟病的发生,而对稻瘟病菌孢子萌发及菌丝生长均无明显抑制作用,证实MeJA处理后稻瘟病病情指数的下降是由于MeJA提高了水稻幼苗的抗瘟性。对水稻抗瘟性重要防御酶的活性测定结果表明,苯丙氨酸解氨酶(PAL)、过氧化物酶(POD)、过氧化氢酶(CAT)和脂氧合酶(LOX)活性均在MeJA处理早期上升,与亲和性互作水稻相比,高度非亲和性互作水稻中的诱导活性增加明显,且速度快。对病程相关蛋白β-1,3-葡聚糖酶和几丁质酶的活性测定结果表明,高度非亲和性互作水稻PR蛋白活性的高峰期出现和强度也明显要早且高于亲和性互作水稻。对内源水杨酸(SA)的测定结果表明,不同亲和性互作水稻的SA含量均没有明显的变化,表明MeJA诱导的水稻信号通路可能与SA信号通路无关。     

Increased activities of ribonuclease and protease after challenge in tobacco plants with induced systemic resistance

Inoculation of 3-4 lower leaves of tobacco with tobacco mosaic virus (TMV) increased ribonuclease (RNase) and protease activities in inoculated leaves. Little or no increase in the activities were apparent in upper noninoculated leaves prior to challenge. After challenge with TMV or Peronospora tabacina, RNase activities increased more rapidly in the upper leaves of induced plants as compared to those of noninduced plants. Protease activities in the leaves challenged with P. tabacina or TMV also increased after challenge, but little or no differences in the activities were apparent between induced and noninduced plants. The incubation of purified TMV with leaf extracts obtained from induced challenged, noninduced challenged and noninduced unchallenged plants prior to inoculation did not affect the number of local lesions formed on tobacco plants.     

Effects of virus infection of faba bean on subsequent infection by Uromyces viciae-fabae

Prior infection of faba bean with bean yellow mosaic virus (BYMV) and bean leaf roll virus decreased pustule density on leaves subsequently infected by Uromyces viciae-fabae. Changes were most marked on young leaves showing conspicuous symptoms of systemic virus infection and in plants virus-infected for at least 2-4 weeks. Pustule density progressively decreased when inoculations were made with uredospore generations successively produced on and inoculated to BYMV-infected leaves.
Uredospore germination and germ tube length were similar on virus-free and BYMV-infected leaves and on agar seeded with spores produced for one or several generations on BYMV-infected or virus-free plants. Formation of appressoria was slightly reduced on BYMV-infected leaves but changes in the post-penetration development of the rust probably accounted for much of the decreased pustule production encountered.     

Biological and molecular analyses of the acibenzolar-S-methyl-induced systemic acquired resistance in flue-cured tobacco against Tomato spotted wilt virus

Tomato spotted wilt virus (TSWV) is an economically important virus of flue-cured tobacco. Activation of systemic acquired resistance (SAR) by acibenzolar-S-methyl (ASM) in flue-cured tobacco was studied under greenhouse conditions by challenge inoculation with a severe isolate of TSWV. ASM restricted virus replication and movement, and as a result reduced systemic infection. Activation of resistance was observed within 2 days after treatment with ASM and a high level of resistance was observed at 5 days onward. Expression of the pathogenesis-related (PR) protein gene, PR-3, and different classes of PR proteins such as PR-1, PR-3, and PR-5 were detected at 2 days post-ASM treatment which inversely correlated with the reduction in the number of local lesions caused by TSWV. Tobacco plants treated with increased quantities of ASM (0.25, 0.5, 1.0, 2.0, and 4.0 g a.i./7,000 plants) showed increased levels of SAR as indicated by the reduction of both local and systemic infections by TSWV. The highest level of resistance was at 4 g a.i., but this rate of ASM also caused phytotoxicity resulting in temporary foliar spotting and stunting of plants. An inverse correlation between the TSWV reduction and phytotoxicity was observed with the increase of ASM concentration. ASM at the rate of 1 to 2 g a.i./7,000 plants activated a high level of resistance and minimized the phytotoxicity. Use of gibberellic acid in combination with ASM reduced the stunting caused by ASM. Present findings together with previous field experiments demonstrate that ASM is a potential option for management of TSWV in flue-cured tobacco.     

巴西橡胶树品系对棒孢霉落叶病的抗病性鉴定

采用巴西橡胶树棒孢霉落叶病病原菌的分生孢子接种和其毒素接种分别测定了国内天然橡胶主产区9个主栽品系的抗病性,结果表明过滤灭菌毒素划伤接种橡胶离体小叶片可以产生与分生孢子接种相似的症状,而不划伤接种则不产生病害症状;121℃、20 min湿热灭菌毒素接种无论划伤与否均不产生病害症状;分生孢子和病原菌毒素两种接种方法所测橡胶品系的抗病性之间有很好的一致性,说明毒素接种可以用来代替病原菌接种测定橡胶品系的抗病性。抗病性测定结果显示,供试9个巴西橡胶树品系中,PB86、热研88-13、南华1、PR107、热研7-33-97对病原菌表现出高抗,而RRIM600、南强1-97、热研44-9则表现高感,GT1则表现中感。     

A Variant of Cucumber mosaic virus Is Restricted to Local Lesions in Inoculated Tobacco Leaves with a Hypersensitive Response

A variant of Cucumber mosaic virus, CMV(Y/GM2), was isolated from a tobacco plant with mild green mosaic symptoms that was regenerated in vitro from a yellow strain of CMV [CMV(Y)]-infected tobacco leaves by tissue culture. CMV(Y/GM2) has two amino acid substitutions at 36 and 111 positions in the coat protein encoded on RNA3. CMV, assembled by mixing in vitro transcribed CMV(Y) RNA1 and RNA2 plus infectious RNA3 transcribed in vitro from cDNA to RNA3 of CMV(Y/GM2), was prepared and designated as CMV(Y/GM2)tr. When tobacco (Nicotiana tabacum cv. Xanthi nc) plants were inoculated with CMV(Y/GM2)tr, large necrotic local lesions in which the virus was localized, developed on the inoculated leaves. This host response unique to CMV(Y/GM2)tr was similar to the hypersensitive response (HR), which is a common resistance response to avirulent pathogens and was observed in five cultivars of Nicotiana tabacum and eight Nicotiana species. The revertant virus, however, accumulated to quite different levels in the various hosts. CMV(Y/GM2)tr induced pathogenesis-related 1 (PR-1) protein accumulation and systemic acquired resistance (SAR) which were generally observed in the HR. However, when tobaccos were inoculated with CMV(S36P)tr and CMV(V111I)tr, which have an amino acid substitution at either the 36 or 111 position in the coat protein of CMV(Y), respectively, CMV(S36P)tr was restricted to the primary infection site without necrotic local lesion formation and PR-1 protein and SAR induction. CMV(V111I)tr, however, systemically spread and induced mild green mosaic symptoms, while the host had the HR to CMV(Y/GM2)tr. The localization of CMV(Y/GM2)tr at the primary infection site may not only be caused by the HR, but also by the restriction of virus systemic movement resulting from the amino acid substitution at position 36 in the coat protein of CMV(Y). Received 15 December 1999/ Accepted in revised form 18 April 2000     

京郊大蒜病毒病的研究及其鳞茎中病毒的脱除

 京郊大蒜病毒病发生普遍,主要症状为条纹花叶、矮化和叶片扭曲畸形。病体细胞中含大量线状病毒粒体和风轮状内含物。线状病毒粒体长度范围为250-1875nm,以长度550-800nm粒体居多。其中长700-800nm的粒体被鉴定为大蒜花叶病毒(GMV),回接脱毒大蒜叶片产生条纹花叶症状。血清学鉴定表明GMV与洋葱黄矮病毒(OYDV)有近缘关系。长度500-600nm的粒体可能为大蒜潜隐病毒(GLV),回接脱毒大蒜不产生花叶症状。下述两种病毒为京郊大蒜的主要病毒。此外,个别标样含烟草花叶病毒,但不是大蒜的主要病毒。对800nm以下线状病毒粒体归属尚待研究。
应用营养茎尖、生殖茎尖和根尖分生组织培养技术,可以脱去上述主要病毒,获得脱毒大蒜。     

水稻锯齿叶矮缩病毒抗血清的制备及其应用

 采用人工接种发病的水稻锯齿叶矮缩病株茎叶的榨出液,以低速离心(4000rpm)结合聚乙二醇(PEG)的方法所得部分提纯的病毒,进行家兔免疫注射制备抗血清,结果以注射后3~4周的效价最高,可达1:4096,α-最适比值为1:13。应用这种方法制备成的水稻锯齿叶矮缩病毒(RRSV)的抗血清,进行了水稻矮缩病毒(RDV)病株与RRSV病株的鉴别诊断和RRSV毒源寄主的检测。结果证明,具卷叶、缺刻的RDV病株确非RRSV复合感染所致。毒源寄主测定表明,在供试的7种田间常见杂草中,有5种表现为阳性反应;经生物学回接证实,5种中有蟋蟀草、水蜈蚣和游草3种能成功地将RRSV传给水稻而引起发病。