花生HyPGIP基因克隆及抗叶腐病表达分析

本文采用基因克隆和生物信息学方法研究了花生Hy PGIP基因及编码蛋白的基本性质和生物学功能,并通过测定接种叶腐病菌后花生植株内PG酶的活性变化和PGIP蛋白的生成量变化,以及Hy PGIP基因瞬时表达分析,探讨了PGIP蛋白与寄主抗病性关系。结果表明,受叶腐病菌侵染的花生植株中,抗病品种的PG酶活性都明显低于感病品种,而生成的PGIP蛋白量都比感病品种显著多,暗示PGIP蛋白与花生叶腐病抗性有关。经基因克隆和测序获得HyPGIP基因全长序列为1 029 bp,编码342个氨基酸,无内含子,终止密码子为TGA,与已报道的五个花生PGIP序列的同源性都很高;结构预测发现该序列存在8个LRR结构域,存在信号肽,疏水性较强,定位于细胞壁上;RT-PCR显示,经接种叶腐病菌处理后,花生植株中的PGIP基因表达量明显增加。     

油菜miR6030通过靶标两个编码CC-NBS-LRR蛋白的基因调控对核盘菌的抗性

miRNAs对油菜抗核盘菌(Sclerotinia sclerotiorum)的调控作用尚不明确。本研究分析了油菜miR6030在菌核病抗性调控中的功能和机制。结果表明，miR6030在油菜不同组织中表达水平有差异，以根和茎中较高，花中最低。miR6030瞬时超表达和沉默分析以及基因沉默转基因油菜植株分析结果均显示，miR6030负调控油菜对核盘菌的抗性。降解组分析和GUS染色分析显示，miR6030靶标2个产物具有CC-NBS-LRR(CNL)结构的基因BnaCnng64090D和BnaA09g10520D。这2个基因均正调控油菜对核盘菌的抗性。综上，油菜miR6030降解编码CNL蛋白的BnaCnng64090D和BnaA09g10520D基因转录本，从而负调控对菌核病的抗性。该研究结果增进了对miR6030生物学功能以及油菜对核盘菌抗性分子机制的理解，并为油菜抗菌核病分子育种提供新思路。     

进境百合种球南芥菜花叶病毒的检测及分子鉴定

本研究根据南芥菜花叶病毒(Arabis mosaic virus,ArMV)CP基因序列,设计并合成了特异性巢式PCR检测引物,通过第1轮RT-PCR和第2轮PCR扩增得到预期的930bp和260bp的条带,扩增序列与GenBank中登录的外壳蛋白基因存在86%～97%的同源性.结果表明百合上分离到的病毒为ArMV,进一步研究也证明巢式PCR灵敏度与普通RT-PCR相比高出了103倍.     

利用栽培措施控制油菜菌核病的综合研究

通过以3个甘蓝型油菜品种作为材料,采用田间自然鉴定方法,研究了不同播期和不同密度等栽培措施对控制油菜茵核病的作用.结果表明,在油菜生育期间,无任何药剂防治的条件下,品种和播期对油菜菌核病的影响均达显著水平,而密度对菌核病无明显影响,要与其他栽培条件,特别是氮肥的用量结合起来,才具有显著作用.因此,选用抗病性的品种和适当的晚播可有效控制或避开油菜菌核病的发生,合理密植可提高产量.     

进境荷兰郁金香种苗中南芥菜花叶病毒的鉴定

为了防止南芥菜花叶病毒(Arabis mosaic virus,ArMV)传入我国,采用血清学、分子生物学、电镜观察及生物学接种等方法对从荷兰进口的郁金香种苗进行了ArMV检测。结果表明:ArMV血清学反应为强阳性;RT-PCR反应扩增出370bp的特异性目标条带;RT-PCR产物与已报道的3个ArMV部分外壳蛋白基因的核苷酸序列同源性为91.00%～93.24%,氨基酸序列同源性为99%～100%;免疫电镜观察到叶片病汁液中含有直径约30nm的球状病毒粒体;该病毒在黄花烟、白肋烟、矮牵牛和昆诺藜等鉴别寄主上引起坏死和褪绿等典型症状,经DAS-ELISA检测,这些接种寄主均呈ArMV血清学阳性反应。故将该病毒鉴定为南芥菜花叶病毒。     

浙江柑橘黄龙病病原β-操纵子核糖体蛋白基因的PCR检测及其序列比对

长期的观察发现,不同柑橘品种感染黄龙病后出现的病症也不尽相同,针对这种现象本文检测了浙江柑橘黄龙病病原β-操纵子核糖体蛋白基因并进行了序列比对,BLAST比对结果说明所扩增的基因序列之间差异性为0,与基因库(NCBI)中黄龙病亚洲韧皮杆菌DNA序列相似性为100％,由此说明浙江柑橘黄龙病病原β-操纵子核糖体蛋白基因并未发生变异,出现上述现象可能是其他基因发生变异或者与品种的抗病性有关。     

周口地区油菜白粉病病原的鉴定

本研究通过扫描电镜观察以及分子鉴定方法,对引起河南周口地区油菜白粉病的病原进行了鉴定。结果显示:在扫描电镜下该病原菌分生孢子为椭圆或圆柱形;采用真菌核糖体DNA转录间隔区(ITS)通用引物,扩增病菌核糖体基因ITS区,并对产物进行克隆、测序及进化树分析,其ITS序列与Erysiphe cruciferarum(韩国)的ITS序列同源性为100%,而聚在一个进化枝上。上述结果表明,周口地区油菜白粉病菌属于E.cruciferarum,与韩国甘蓝型油菜白粉病菌亲缘关系最近。     

禾谷镰孢菌γ-微管蛋白基因克隆及其与多菌灵抗药性关系分析

根据禾谷镰孢菌参考菌株NRRL31084(PH-1)的γ-微管蛋白基因核苷酸序列设计5对引物,采用PCR方法从禾谷镰孢菌(Fusarium graminearum)对多菌灵(MBC)的敏感菌株、室内诱导及田间多菌灵抗药性菌株中分段扩增测序,获得了γ-微管蛋白基因全序列.该基因全长1 868 bp,含有5个内元,编码一含493aa的多肽;与PH-1的γ-微管蛋白基因核苷酸序列同源性99%,存在10个差异核苷酸,与所编码的氨基酸序列同源性100%;与其它7种真菌的γ-微管蛋白基因所编码的氨基酸序列同源性为31%～72%.中国的2个敏感菌株和4个抗药菌株的γ-微管蛋白基因序列完全相同,认为多菌灵抗药性与该微管蛋白变异无关.     

禾谷镰孢菌γ-微管蛋白基因克隆及其与多菌灵抗药性关系分析

 根据禾谷镰孢菌参考菌株NRRL31084(PH-1)的γ-微管蛋白基因核苷酸序列设计5对引物,采用PCR方法从禾谷镰孢菌(Fusarium graminearum)对多菌灵(MBC)的敏感菌株、室内诱导及田间多菌灵抗药性菌株中分段扩增测序,获得了γ-微管蛋白基因全序列。该基因全长1 868bp,含有5个内元,编码一含493aa的多肽;与PH 1的γ-微管蛋白基因核苷酸序列同源性99%,存在10个差异核苷酸,与所编码的氨基酸序列同源性100%;与其它7种真菌的γ-微管蛋白基因所编码的氨基酸序列同源性为31%~72%。中国的2个敏感菌株和4个抗药菌株的γ-微管蛋白基因序列完全相同,认为多菌灵抗药性与该微管蛋白变异无关。     

草酸对拟南芥的毒性及其在筛选拟南芥突变体中的应用

菌核病菌[Sclerotinia sclerotiorum(Lib．)de Bary]引致75科408种和42个亚种的植物发生菌核病。该病是世界粮油作物、蔬菜等农作物生产上的分布最广、为害最严重的病害之一。我国油菜、大豆和保护地蔬菜的菌核病问题十分突出,严重时会导致毁灭性的损失。该病寄主范围极广,不具典型的基因对基因关系,给研究其致病和抗病分子机制带来了极大的困难,由此带来的抗原缺乏是防治该病的最大困难。有关油菜抗菌核病的筛选研究表明,尽管不同品种间抗病性有差异,但未发现高抗和免疫的材料;组织培养、诱变能创造一些新的类型,而且还可以不同程度地提高抗性,但多为中抗材料。所以更广泛、更全面地发现和创造抗原材料是今后的一个研究方向。     

BTH诱导花椰菜对菌核病的抗性研究

 利用苯并噻二唑BTH处理菌核病抗性不同的花椰菜品种幼苗, 采用营养生长期活体叶片菌丝块接种鉴定法评价菌核病抗性诱导效果,结果表明经BTH处理的植株菌核病病情指数明显下降, 对感病品种和抗病品种的诱抗效果分别达到81.5%和63.8%。对于花椰菜重要的防御酶活性变化研究结果表明,BTH诱导处理的花椰菜植株过氧化物酶（POD）、抗坏血酸酶( SOD )、过氧化氢酶（CAT）、苯丙氨酸解氨酶（PAL）和多酚氧化酶( PPO)的活性均有所提高。同时病程相关蛋白几丁质酶和β-1,3-葡聚糖酶的活性也增加。 利用半定量RT-PCR方法检测防御反应基因表达,结果表明BTH诱导首先激发了植株 PR-1等基因参与的水杨酸信号传导防御反应途径的发生,同时PDF1.2 基因的上调表达说明BTH诱导也影响了茉莉酸信号传导途径。     

Reaction of Brassica juncea (Indian Mustard) Lines to Australian Isolates of Leptosphaeria maculans under Glasshouse and Field Conditions

Brassica juncea (Indian mustard) lines from diverse geographical locations around the world and from Australian breeding programs were screened for resistance to the blackleg fungus, Leptosphaeria maculans, in both glasshouse and field trials. The five Australian L. maculans isolates used in glasshouse trials could be classified into two groups; those that attacked all B. juncea lines, and those that attacked none. All these isolates caused lesions on cotyledons of B. napus cultivars including Westar, Glacier and Quinta, suggesting that they are in Pathogenicity Group 4 as described by Koch et al. (1991). The two isolates that attacked B. juncea also attacked B. napus lines to a similar extent, but did not attack the two B. carinata lines tested. Brassica lines were sown in a blackleg disease nursery at Lake Bolac, Victoria, Australia, and five indicators of blackleg disease were measured (survival rate, disease rating, disease incidence, external and internal lesion length). All 92 B. juncea lines developed blackleg symptoms. Although they displayed a high disease incidence in the field, almost all of the B. juncea lines were more blackleg-resistant than a B. napus cultivar, Dunkeld, which is amongst the most resistant cultivars in commercial production in Australia. Four B. carinata lines were more resistant than any of the B. juncea lines, suggesting that this species may be a useful source of blackleg resistance in B. napus breeding programs.     

Pathogenicity of Leptosphaeria maculans Isolates on a Brassica napus-B. juncea Recombinant Line

ABSTRACT The Brassica napus-B. juncea recombinant line (MX), resistant to Leptosphaeria maculans, was produced by interspecific crosses and bears one gene (Jlm1) from the B. juncea B genome. We investigated whether this new resistance was race specific by characterizing protection against a large sample of L. maculans isolates. The pathogenicity of 119 isolates of L. maculans comprising 105 A-group isolates and 14 B-group isolates was studied at the cotyledon stage under controlled conditions using the MX line, the susceptible B. napus cultivar Westar, and the resistant B. juncea cultivar Picra. All but one of the isolates were pathogenic on 'Westar'. Only 3 of the 105 A-group isolates caused very mild symptoms on 'Picra'. Two of these strains were isolated from the MX line and the other from Sinapis arvensis. The other 102 strains caused hypersensitive-type responses. Most B-group isolates were pathogenic on 'Picra'. There were differences in pathogenicity among A-group isolates tested on the MX line, whereas all B-group isolates were pathogenic on this line. A-group isolates obtained from the MX line were more frequently pathogenic on the MX line than those obtained from B. napus cultivars. One isolate from S. arvensis infected the MX line. These results suggest that the resistance of the MX line is unlikely to be durable. Thus, the new resistance gene Jlm1 should probably be used in association with other sources of resistance, in plant breeding schemes, to prevent the breakdown of this resistance.     

不同核盘菌菌株及其近缘种的RAPD分析

 本文利用随机引物扩增多态性DNA (RAPD)技术分析了7个生物学性状差异较大核盘菌(Sclerotinia sclerotiorum)的遗传多样性,并同三叶草核盘菌(S.trifoliorum)、小核盘菌(S.minor)的代表性菌株和莴苣上的一种产菌核病原菌的代表菌株Let-19进行了比较。结果表明40个引物中8个引物能稳定地从供试菌株中扩增出多态性DNA片段。通过分析这些多态性片段可以看出7个供试核盘菌菌株之间的遗传相似系数变化幅度为0.505 2~0.793 1,而核盘菌、三叶草核盘菌,小核盘菌和Let-19之间的遗传相似系数的变幅则为0.194 2~0.385 3。莴苣上的菌株Let-19的RA PD图谱同供试其它种的菌株既存在明显差异的DNA片段电泳带,又显示出一些位置一致的DNA片段电泳带。因而Let-19同核盘菌属真菌的亲缘关系较近。供试的引物中OPL14既能介导从供试的7个核盘菌菌株和3个近缘种的3个菌株的DNA样品中扩增出相同的DNA片段,又能扩增出种或菌株特异性DNA片段。因而RAPD技术适于研究核盘菌的遗传多样性及分析核盘菌属真菌的亲缘关系。     

Polygalacturonase inhibiting proteins fromAllium porrumL. and their role in plant tissue against fungal endo-polygalacturonases

Five endo-polygalacturonases (PGs), three produced in culture filtrate byFusarium moniliforme, Sclerotium cepivorumandBotrytis aclada,respectively, and two (one acidic and one basic isoform) obtained fromSclerotinia sclerotiorumsoybean infected hypocotyls, were purified in order to characterize the activity of polygalacturonase inhibitor(s) (PGIP(s)) from leek stalk tissue (Allium porrumL.). Three apparently different PGIPs (PGIP-I, PGIP-II and PGIP-III) were purified from the leek tissue. The two more abundant PGIPs (PGIP-I and PGIP-III), although possessing similar pIs of about 6.5, differed in chromatographic behaviour, their molecular mass (39 and 42 kDa, respectively), and specific activity when assayed with the fungal endo-PGs. In addition, PGIP-I was solubilized from tissue homogenate with a low-salt buffer whilst PGIP-III needed a high-salt buffer for extraction (behaving as an ionically wall-bound protein). PGIP-II had very similar properties to PGIP-I, but was extracted using the high-salt buffer. The purified PGIPs and the crude leek extract showed similar inhibition activity patterns against the five fungal endo-PGs. The maximum inhibition activity was observed against the basic endo-PG fromS. sclerotiorum,followed by the acidic endo-PG ofS. sclerotiorumand the endo-PG fromB. aclada.In contrast, no inhibition of endo-PGs fromS. cepivorumandF. moniliformewas observed. Four different concentrations of the five fungal endo-PGs were incubated separately with slices of leek stalk, and the galacturonides released in the incubation mixture were measured. At every level used the endo-PGs ofF. moniliformeandS. cepivorumshowed the maximum activity in uronide releasing. The endo-PGs ofS. sclerotiorum(acidic PG) andB. acladawere active only when high levels were used while the basic endo-PG ofS. sclerotiorumwas not active in combustion with any level of PGIP. These results indicate that a close relationship exists between PGIP activityin vitroand the ability of PGIP to protect leek tissue from endo-PG degradation.     

Detection and quantification of airborne inoculum of Sclerotinia sclerotiorum using quantitative PCR

This paper reports the development of a new specific diagnostic technique to accurately quantify airborne inoculum of Sclerotinia sclerotiorum and discusses its potential use in disease-forecasting schemes, using examples of three contrasting epidemic seasons: 2007, when there was a severe epidemic of sclerotinia stem rot (SSR) in England and high numbers of airborne ascospores were trapped at Rothamsted, and, in contrast, 2003 and 2004, when the incidence of SSR in England was low and low numbers of airborne ascospores were trapped at Rothamsted. DNA was extracted from wax-coated plastic tapes, such as those used in Burkard (Hirst-type) spore traps and rotating-arm traps. A SYBR-green quantitative PCR (qPCR) method produced a linear relationship between ascospore numbers and S. sclerotiorum DNA (mean 0·35 pg DNA per spore) and was able to detect DNA representing as few as two ascospores. The technique was insensitive to DNA of the host plant, Brassica napus , and other plant pathogens, including Sclerotinia minor , S. trifoliorum and Botrytis cinerea , and common airborne fungal genera such as Cladosporium and Penicillium . There was no relationship between rainfall and numbers of airborne ascospores of S. sclerotiorum present at Rothamsted during the period of infection in the severe SSR season (2007).     

柑橘内生细菌YS45的鉴定、抗菌物质分析及其对油菜菌核病的防治作用

 从柑橘枝条中分离到26株对油菜菌核病菌(Sclerotinia sclerotiorum)具有拮抗作用的内生细菌,其中YS45菌株的拮抗活性最强。16S rRNA基因序列分析及形态学和生理生化鉴定结果表明YS45菌株为枯草芽孢杆菌(Bacillus subtilis)。高效液相色谱及质谱分析结果显示其主要的抑菌活性物质为一组fengycin同系物,包括fengycins A、fengycins B和一种稀少fengycin类型化合物。油菜离体叶片接种试验中,YS45菌株发酵液对油菜菌核病的防效在70%以上,与五氯硝基苯相当;田间小区接种试验表明,YS45菌株发酵液对油菜菌核病的防效也在50%以上。     

盾壳霉控制油菜菌核病菌再侵染及其叶面存活动态的研究

 本文评估了施于油菜(Brassica napus)叶片上的盾壳霉(Coniothyrium minitans)控制油菜菌核病菌再侵染能力,探讨了其作用机理,并测定了盾壳霉分生孢子在油菜叶面上的存活动态。结果如下:叶面上的盾壳霉对油菜菌核病菌的初侵染影响较小,但在高剂量(> 10⁶孢子/ml)时可以控制病斑的扩展。所有供试剂量的盾壳霉均可不同程度地控制再侵染。盾壳霉分生孢子可在叶面病部迅速萌发,48 h和72 h时孢子萌发率分别为51%和95%,而在健康叶面上6 d未能检测到萌发的孢子。自携带盾壳霉的叶面病部不能分离到核盘菌,表明叶面上的盾壳霉已寄生并破坏了核盘菌再侵染菌丝。自油菜叶面上分离到的盾壳霉菌落数随时间延长而降低,但其分生孢子至少可以在叶面上存活28 d。这即表明,在叶面上适时适量地添加盾壳霉可以控制油菜菌核病的为害。     

油菜菌核病内生拮抗细菌的筛选及防病作用研究

以油菜菌核病菌Sclerotinia sclerotiorum为靶标菌,测定了63株内生细菌对其菌丝生长、菌核形成及萌发的抑制作用。结果表明:在所有供试菌株中,除1株未表现出抑菌活性外,其余菌株均有抑菌作用,其中有6.3％的菌株抑菌带大于10 mm,7株细菌可完全抑制病原菌的菌核形成,1株细菌可完全抑制菌核萌发;此外,结合生物测定结果,从中筛选到1株对油菜菌核病的高效生防菌株Em7,其无菌培养滤液对温室苗期油菜菌核病的防治效果可达97.5％。通过形态特征、生理生化特性及16S rDNA序列分析,认为该菌株属于枯草芽孢杆菌Bacillus subtilis。于接种病菌前后不同时间喷施菌株Em7无菌培养滤液,对油菜菌核病均有防治效果,但防效之间差异显著,以接种病菌前24 h喷施防效最高。原液及不同稀释度无菌培养滤液对菌丝生长、菌核萌发及病害防治的效果明显不同,随着稀释度的增加,其抑菌效果降低。显微观察结果表明,菌株Em7对油菜菌核病菌菌丝的生长与发育会产生明显影响,可致使菌丝畸形、皱缩及细胞质外渗。