吲哚诱导对茶树抗茶小绿叶蝉的影响

为明确吲哚处理对茶树抗茶小绿叶蝉Empoasca onukii的影响,以龙井43为材料,使用吲哚诱导茶树作为处理,以未处理茶树作为对照,于室内测定茶小绿叶蝉对不同茶树的选择趋向,利用昆虫刺探电位图谱分析茶小绿叶蝉的取食行为,通过气相色谱-质谱联用仪分析吲哚处理对茶树挥发物的影响,并利用实时荧光定量PCR技术测定茶树防御相关基因的表达量。结果表明,在所有时间段茶小绿叶蝉成虫更趋向于取食对照组茶树,至48 h时对照组茶树上的成虫数量是处理组的2.45倍;茶小绿叶蝉3龄幼虫在吲哚处理组茶树上的非取食波（NP波）持续时间占总取食时间的72.15%,在对照组茶树上的非取食波（NP波）持续时间占比则为33.07%,且在对照组茶树上的主要取食波E2波的持续时间是处理组茶树上的3.58倍。相较于对照组茶树,吲哚处理组茶树释放出的挥发物总量显著增加,其中α-法尼烯可以趋避害虫并对天敌有吸引力,且吲哚处理组茶树的防御基因表达量显著升高。表明吲哚处理茶树后,茶小绿叶蝉对其的取食量下降,茶树的挥发物释放量增加且抗性防御基因表达量提高,增强了茶树对茶小绿叶蝉的抗性。     

玫瑰黄链霉菌活性代谢产物RM诱导黄瓜抗病性的信号转导途径

为明确玫瑰黄链霉菌Streptomyces roseoflavus菌株men-myco-93-63活性代谢产物roflamycoin和men-myco-A(简称RM)诱导黄瓜产生抗病性的信号转导途径,采用高效液相色谱法、分光光度法和实时荧光定量PCR法,测定喷施玫瑰黄链霉菌代谢产物RM后黄瓜叶片中水杨酸含量和脂氧合酶活性,以及水杨酸信号转导途径和乙烯茉莉酸信号转导途径的标志基因——NPR1、PR-1a、CTR1的表达量。结果表明,喷施玫瑰黄链霉菌活性代谢产物RM后120 h,黄瓜叶片内水杨酸含量达到最高值,为7.22μg/g,是对照的1.75倍;喷施玫瑰黄链霉菌活性代谢产物RM后12 h,黄瓜叶片内脂氧合酶活性达到最高值,为52.69 U/g,是对照的1.32倍,并且在120 h内整体活性均高于对照;喷施玫瑰黄链霉菌代谢产物RM后,NPR1和PR-1a基因的相对表达量上调,其最大值分别为0.99和1.35,分别是对照的2.24倍和12.09倍,但抑制CTR1基因的表达。推测玫瑰黄链霉菌代谢产物RM通过水杨酸通路、乙烯通路和茉莉酸通路共同诱导黄瓜抗病性信号转导途径。     

心叶烟NPR1基因全长cDNA序列的克隆及表达分析

 NPR1(non-expressor of pathogenesis-related gene 1)基因在拟南芥系统获得抗性中起着关键作用,可调控拟南芥植株广谱抗性的发生。本文报道了从心叶烟中克隆NPR1同源基因(NgNPR1)及其表达特性的研究结果。NgNPR1 cDNA全长2253 bp,编码588个氨基酸。将NgNPR1基因组全长与cDNA序列进行比对发现,NgNPR1基因组DNA含有4个外显子和3个内含子。Southern杂交分析表明,在心叶烟基因组中NgNPR1为单拷贝基因。采用绿色荧光蛋白在洋葱表皮瞬时表达的试验,证明了NgNPR1蛋白在水杨酸诱导时会从细胞质转运到细胞核中。Northern杂交分析发现,NgNPR1基因可以被与植物抗病相关的信号分子如水杨酸、茉莉酸甲酯、过氧化氢和乙烯所诱导。进一步研究发现,植物病原物如赤星病菌、青枯病菌和烟草花叶病毒对心叶烟植株的侵染也会使NgNPR1表达量增加。这些结果表明,NgNPR1基因在心叶烟植株抵御病原物侵染过程中可能起着重要作用。     

基于16S rDNA序列的小贯小绿叶蝉共生细菌多样性研究

利用Illumina HiSeq技术,对赤壁、大悟、武汉、咸安和英山5个茶园小贯小绿叶蝉地理种群的成虫共生细菌16SrDNA-V4变异区进行测序,应用Uparse和RDP Classifier等软件统计和分析样本的物种组成、丰度和多样性。5个地区小贯小绿叶蝉成虫的16SrDNA基因序列文库共获得239 645条有效tags,在97%相似阈值下将其聚类为3 403个OTUs。共注释到41个门,116个纲,197个目,272个科,372个属,105个种。5个样本的共生菌在不同分类水平上的组成有所不同,其中在门水平上,主要优势菌为变形菌门Proteobacteria(相对丰度60.6%~97.1%);在纲水平上,相对丰度排名前5位中共有的优势菌为γ-变形菌纲Gammaproteobacteria和α-变形菌纲Alphaproteobacteria;在属水平上,排名前10位中5个样本共有的优势菌为盐单胞菌属Halomonas、希瓦氏菌属Shewanella和沃尔巴克氏体属Wolbachia。小贯小绿叶蝉成虫细菌的Chao指数、Ace指数、Shannon指数和Simpson指数分别为1 065.55~2 841.89,1 130.76~2 914.90,1.07~8.63和0.18~0.99。茶园小贯小绿叶蝉成虫共生细菌多样性比较丰富,不同地理种群的小贯小绿叶蝉细菌群落结构和多样性有差异。本研究结果为进一步研究细菌对小贯小绿叶蝉种群生物学的影响奠定了基础。     

荔枝采后炭疽病的发生情况及对贮藏效果的影响

为了明确广东、海南荔枝采后主要病害及其发生和危害情况，从两省荔枝主产区取果，在常温贮藏条件下观察荔枝采后病害的发生情况，测定荔枝果皮上炭疽病菌潜伏侵染率及其对荔枝采后生理变化和贮藏效果的影响。结果表明：荔枝采收后在28℃下贮藏，炭疽病、霜疫病和酸腐病的发病率分别为95．3％、6．5％和8．2％。炭疽病在采后3—4天开始表现症状，7天后发病率为60．8％～100％，9天后发病率为83．1％～100％，该病害是引起荔枝采后腐烂的主要病害，其病原菌主要来自采前潜伏侵染。从幼果果皮上可分离出潜伏侵染的炭疽病菌，随着果实的生长，果皮上的潜伏侵染率不断上升，到采收时炭疽病菌潜伏侵染率达到90％左右。炭疽病菌潜伏侵染率较高的荔枝果实。其采后呼吸速率、乙烯释放量、果皮丙二醛含量均显著升高，褐变腐烂较快，贮藏效果较差，即使在采后用杀菌剂处理，防治效果也不明显。采收时荔枝果皮上炭疽病菌潜伏侵染率越低，贮藏效果越好。在荔枝果实生长期间喷雾2—3次杀菌剂可显著地降低炭疽病菌潜伏侵染率，减轻荔枝采收后炭疽病的发生严重程度，提高荔枝贮藏效果。     

植保无人飞机低容量喷雾在山东设施茶园防治半翅目害虫的应用研究

利用四旋翼植保无人飞机在山东设施茶园防治主要害虫, 研究两种类型的植保无人飞机和传统背负式电动喷雾器施药在不同冠层茶树叶片上雾滴密度?沉积量和覆盖率分布情况以及对茶树主要半翅目害虫的防治效果?结果表明, Ⅰ型植保无人飞机?Ⅱ型植保无人飞机和背负式喷雾器在茶树叶片上平均雾滴密度分别为102.72?50.58个/cm2和29.83个/cm2, 沉积总量分别为0.175?0.371 μL/cm2和53.562 μL/cm2, 雾滴覆盖率均值分别为1.95%?2.52%和19.42%?不同施药器械对茶树各层的雾滴密度?沉积量和覆盖率有显著影响?两种类型植保无人飞机施药雾滴密度总体为茶树树冠上层>中层>底层, 正面>背面?背负式电动喷雾器施药的雾滴沉积量和覆盖率显著高于植保无人飞机, 叶片正面沉积量和覆盖率明显高于叶背面?Ⅰ型植保无人飞机喷施22%氟啶虫胺腈悬浮剂240 mg/L 3 d和5 d后对黑刺粉虱的防治效果为82.20%和71.73%, 该处理对防治茶树黑刺粉虱具有速效性且持效性较好?Ⅰ型植保无人飞机喷施22%氟啶虫胺腈悬浮剂240 mg/L 3 d和5 d后对小贯小绿叶蝉的防效为84.44%和77.25%; Ⅱ型植保无人飞机喷施25 g/L溴氰菊酯乳油60 mg/L 3 d后对小贯小绿叶蝉的防效为98.18%, 具有较好的速效性?植保无人飞机施药对黑刺粉虱的防效优于背负式喷雾器, 但对叶蝉的防效与背负式喷雾器相当?不同施药器械不同药剂处理中瓢虫的数量没有差异?     

外源水杨酸诱导大麦对条纹病的抗性研究

 条纹病是影响大麦生产的主要病害之一,降低大麦条纹病的发生对大麦稳产具有重要意义。水杨酸作为植物内源激素,可以通过激活植物过敏反应和调节植物病程相关蛋白基因表达从而诱导植物产生系统性抗性。本研究以感病大麦品种Alexis为材料,分别采用0、5、10、15和20 mmol·L^-1浓度的水杨酸浸种后接种大麦条纹病菌(Pyrenophora graminea),三叶期调查Alexis的发病率、病情指数及生长状况;以10 mmol·L^-1浓度的水杨酸处理Alexis种子后人工接种P. graminea,测定不同侵染时间Alexis胚芽的过氧化物酶(POD)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)及苯丙氨酸解氨酶(PAL)活性,并检测侵染8 d后Alexis抗病相关基因的相对表达量,旨在明确不同水杨酸浓度对Alexis抗病性的影响及水杨酸诱导Alexis产生抗性的机制。结果表明:0、5、10、15和20 mmol·L^-1的水杨酸处理下,Alexis的发病率呈先下降后上升的趋势,病情指数与发病率的变化趋势一致;10 mmol·L^-1的水杨酸处理下Alexis的发病率及病情指数较其他浓度处理显著降低;10 mmol·L^-1的水杨酸处理下,Alexis的株高、根长、鲜质量及干质量变化显著,分别是0 mmol·L^-1水杨酸处理的1.27、1.33、1.27和1.47倍;Alexis萌发2~8 d内,接菌后Alexis胚芽的POD、CAT及SOD活性均高于不接菌处理,PAL活性于第2、4和8 d 时高于不接菌处理;水杨酸接菌处理下Alexis胚芽的POD、CAT、SOD及PAL活性均高于接菌处理;第8 d 水杨酸接菌处理下Alexis抗病相关基因HORVU2Hr1G116090(TGA)和HORVU2Hr1G033620(PR)的相对表达量较接菌处理显著升高,可能参与了Alexis对条纹病菌抗病调控。研究结果可为应用水杨酸防治大麦条纹病提供理论依据。     

转基因水稻对病原菌侵染的反应和防卫基因的表达分析

 为了阐明OsBTF3基因在水稻对白叶枯病菌(Xanthomonas oryzae pv. oryzae, Xoo)和细菌性条斑病菌(X. oryzae pv. oryzicola, Xoc)侵染抗/感反应中的功能,本研究以OsBTF3过量表达和RNAi转基因株系为材料,比较分析了T1代株系对病菌侵染的抗感病反应,定量测定了防卫基因的表达动态。结果表明,与野生型对照株相比,无论过量表达株系,还是RNAi株系对Xoo和Xoc侵染均表现为更加感病的反应; 经Xoo接种后,过量表达和RNAi株系OE37和RNAi株系RI30防卫基因的表达发生了不同程度的变化。因此,OsBTF3增量/减量表达对水稻抗/感病性具有重要的调控作用,这种作用可能并不依赖于水杨酸(SA)和茉莉酸(JA)介导的信号途径。     

河南省小麦全蚀病菌的鉴定、进化分析及标志基因筛选

为了解河南省郑州市中牟市小麦全蚀病菌的变种类型及进化情况,筛选全蚀病菌侵染小麦的分子标记,采用形态学观察、科赫氏法则验证、ITS序列分析和系统进化树构建等方法对分离的小麦全蚀病菌进行鉴定,并对其侵染后小麦病原相关蛋白(pathogen-related protein,PR)基因的表达进行实时定量PCR分析。结果表明,显微形态观察初步确定分离的小麦全蚀病菌为禾顶囊壳Gaeumannomyces graminis(Sacc.)v. Arx.Olivier,经科赫氏法则验证、ITS序列比对及系统进化树分析进一步证明该病菌均为禾顶囊壳。本研究分离的小麦全蚀病菌与地域相距较远的英国、美国的小麦全蚀病菌间的同源性比与来自中国陕西省的小麦全蚀病菌的同源性更高。采用禾顶囊壳4个变种的特异性引物进行PCR扩增,所有分离病菌均扩增出了小麦变种的特异性条带,证实分离菌株为禾顶囊壳小麦变种G. graminis var. tritici。实时定量PCR分析发现,病原相关蛋白基因PR4a、PR4b、PR2、PR10受到小麦全蚀病菌侵染后表达量均显著上调,在侵染后第5～6天达到最高峰,表明这些基因可作为小麦全蚀病菌侵染小麦的标志性基因。     

Effects of pre‐ and post‐treatment with ethephon on gum formation of peach gummosis caused by Lasiodiplodia theobromae

Peach gummosis, caused by Botryosphaeria spp. fungi, is the process of gum accumulation and exudation in plants. Ethephon (2‐chloroethylphosphonic acid) has profound effects on plants, including enhanced production of secondary metabolites and regulation of plant diseases. This study investigates the effects of application of ethephon before and after inoculation with Lasiodiplodia theobromae on gum formation. Gum formation was promoted by ethephon treatment prior to pathogen inoculation, but inhibited by ethephon applied after the pathogen. The inhibitory effect was counteracted by 1‐methylcyclopropane, which is an ethylene signal inhibitor. 1‐methylcyclopropane also promoted gum formation. Exposure of three isolates of Botryosphaeria to ethephon inhibited mycelial growth. Both treatment methods increased the sugar content at 12 and 24 h post‐inoculation (hpi). However, the sucrose, glucose and fructose contents were significantly higher in shoots with ethephon post‐treatment (application of ethephon after the pathogen inoculation) than those in shoots with ethephon pre‐treatment (application of ethephon prior to pathogen inoculation) at 48 and 72 hpi. The expression of two putative senescence‐related genes, SEN2 and SEN4, were significantly enhanced in pre‐ and post‐treated shoots with ethephon at 24, 48 and 72 hpi. Ethephon application also up‐regulated expression of the pathogenesis‐related protein PR4 while down‐regulating PR1a and PR10. The results show that ethephon has a dual function in regulating gum formation by affecting both the peach shoots and the pathogen.     

The oligopeptide elicitor Pep-13 induces salicylic acid-dependent and -independent defense reactions in potato

The Phytophthora-derived oligopeptide elicitor, Pep-13, originally identified as an inducer of plant defense in the nonhost–pathogen interaction of parsley and Phytophthora sojae, triggers defense responses in potato. In cultured potato cells, Pep-13 treatment results in an oxidative burst and activation of defense genes. Infiltration of Pep-13 into leaves of potato plants induces the accumulation of hydrogen peroxide, defense gene expression and the accumulation of jasmonic and salicylic acids. Derivatives of Pep-13 show similar elicitor activity in parsley and potato, suggesting a receptor-mediated induction of defense response in potato similar to that observed in parsley. However, unlike in parsley, infiltration of Pep-13 into leaves leads to the development of hypersensitive response-like cell death in potato. Interestingly, Pep-13-induced necrosis formation, hydrogen peroxide formation and accumulation of jasmonic acid, but not activation of a subset of defense genes, is dependent on salicylic acid, as shown by infiltration of Pep-13 into leaves of potato plants unable to accumulate salicylic acid. Thus, in a host plant of Phytophthora infestans, Pep-13 is able to elicit salicylic acid-dependent and -independent defense responses.     

Expression of δ-endotoxin Cry1EC from an inducible promoter confers insect protection in peanut (Arachis hypogaea L.) plants

BACKGROUND: Spodoptera litura (F.) is a polyphagous foliage insect and a major pest on peanut (Arachis hypogaea L.). Constitutive expression of δ‐endotoxin Cry1EC gives protection against S. litura, as reported earlier. In this study, insect bites and salicylic acid induced high‐level expression of Cry1EC was achieved in peanut. In order to achieve this, the expression of pathogenesis responsive promoter PR‐1a was enhanced by placing it downstream of the CaMV35S promoter in the pCAMBIA 1300 backbone. The resultant promoter CaMV35S(r)PR‐1a expressed a high level of insecticidal δ‐endotoxin Cry1EC. The Gus expression under the control of CaMV35S(r)PR‐1a served as a convenient marker for evaluation of promoter response to different treatments. RESULTS: Transgenic events that showed a very low level of uninduced expression and no expression in seeds were selected. The Cry1EC expression in leaves increased nearly eightfold in the selected event, following induction by salicylic acid. Both the salicylic‐acid‐treated and the S. litura‐bitten leaves showed the highest expression after 2 days. Leaves from salicylic‐acid‐induced transgenic plants caused 100% mortality of S. litura at all stages of larval development. CONCLUSION: The results suggest that high expression of inducible promoters provides a good strategy for the development of safer transgenic food and feed crops. Copyright © 2010 Society of Chemical Industry     

Role of methyl jasmonate in the expression of mycorrhizal induced resistance against Fusarium oxysporum in tomato plants

Arbuscular mycorrhiza (AM) colonization led to a decrease in the severity of fusarium wilt disease caused by Fusarium oxysporum f. sp. lycopersici in tomato plants. The involvement of two plant defense hormones, namely methyl jasmonate (MeJA) and salicylic acid (SA), in the expression of mycorrhiza induced resistance (MIR) against this vascular pathogen was studied in the AM colonized and non-colonized (controls) plants. Activity of lipoxygenase (LOX), which plays a role in jasmonic acid (JA) biosynthesis, as well as levels of methyl jasmonate (MeJA) increased in AM colonized plants as compared to controls, but did not show any further changes in response to F. oxysporum inoculation. On the other hand, activity of phenylalanine ammonia lyase (PAL), which is an enzyme from salicylic acid (SA) biosynthetic pathway, as well as SA levels, increased in both controls and AM colonized plants in response to application of F. oxysporum spores. Hence the JA and not the SA signalling pathway appeared to play a role in the expression of MIR against this vascular pathogen. The resistance observed in AM colonized plants was completely compromised when plants were treated with the JA biosynthesis inhibitor salicylhydroxamic acid (SHAM). This confirmed that the AM-induced increase in JA levels was involved in the expression of resistance toward F. oxysporum. The SA response gene pathogenesis-related 1 (PR1) showed an increased expression in response to F. oxysporum infection in SHAM treated AM colonized plants as compared to plants that were not treated with this JA inhibitor. This suggested the possibility that JA inhibited SA responses, at least in the roots. AM colonization therefore appeared to prime plants for improved tolerance against the vascular pathogen F. oxysporum, which was mediated through the JA signalling pathway.