人工microRNAs干扰At3A06基因增强了拟南芥对菌核病的敏感性

基于本研究室自主开发的油菜防御cDNA芯片分析结果,油菜基因Bn3A06受菌核病诱导后特异上调表达。序列比对发现,该基因与拟南芥基因At3A06高度同源,后者编码289个氨基酸,功能未知。本研究用花序浸染法将At3A06基因的人工微RNA(amiRNA)干扰载体(该载体包含一个除草剂草胺磷抗性基因)转化拟南芥。经草胺磷筛选获得3株T1代转基因植株,PCR检测均为阳性;荧光定量PCR检测表明,与野生型对照相比,3个转基因植株的At3A06基因的表达量均显著降低;对其T2代进行菌核病抗性鉴定,结果显示转基因株系对菌核病的敏感性均显著增强(P0.05)。推测At3A06基因可能参与了植物的防御反应,与植株对核盘菌抗性相关。     

苜蓿菌核病生防菌及化学药剂的筛选

通过毒力测定法比较11种化学药剂对苜蓿菌核病的抑菌效果,其中45%五氯·福、50%速克灵和55%甲硫·菌核净可湿性粉剂3000倍液均能抑制病原菌生长。温室生测表明45%五氯·福防治苜蓿菌核病效果最好,500倍液防效达73.9%。从江苏省句容地区苜蓿根围土壤中分离到415株细菌,采用平皿对峙法从中筛选到3株对菌核病具有拮抗效果的生防菌,代号S1和S2的菌株抑菌效果最好,抑菌带超过1.5cm。温室生测菌株S2对苜蓿菌核病的生防效果最好,接种病原菌10d后防效达67.5%,15d后为61.7%。     

几种向日葵菌核病早期抗性鉴定方法的比较

通过菌土盆栽法、菌丝块茎部贴接法、皮壳接种法和草酸浸根法四个试验,初步查明在向日葵菌核病早期抗性鉴定中以草酸浸根法和皮壳接种法效果显著,快速易行,可作为早期和苗期抗性鉴定的重要方法.菌土盆栽法虽需时较长,但属自然侵染,在抗性鉴定中也应予以考虑;而菌丝块茎部贴接法反应强烈,绝大部分接种植株死亡,不能较好地区分品种间的抗性差异.四种方法均为早期和苗期抗性试验,为得出更确切而全面的结果,应结合田间抗性试验,最好结合田间病圃鉴定,以便更准确地鉴定出整个生长期不同品种间的抗性水平.     

14个甘蓝型油菜品种对菌核病的抗性初报

【研究目的】为探明不同类型的甘蓝型油菜品种对油菜菌核病的抗性差异,提供筛选抗耐病性的材料的新来源;【方法】选用14个甘蓝型油菜品种为材料,利用人工接种方法和病圃自然发病鉴定,研究了其青角期茎秆接种后病斑的扩展情况和成熟期自然发病情况以及在大田生产中的侵染发病;[结果]研究结果表明,不同甘蓝型油菜品种抗病性存在有明显差异,自然鉴定冬油菜材料Wanyou14的抗耐病要优于其他材料,抗病指数为0.36(对照Zhongyou821抗病指数为0),人工接种春油菜材料Ranger具有较好的病害抗扩展性,抗病指数为0.64,中晚熟的材料抗耐病强于早熟材料;【结论】冬油菜材料的抗耐病要优于春油菜材料,春油菜类型也具有较好的抗耐病性,抗性资源通过转育可提高冬油菜的抗病性;生产中油菜类型的选择以中晚熟的冬油菜品种为主。     

Host‐induced gene silencing in the necrotrophic fungal pathogen Sclerotinia sclerotiorum

Sclerotinia sclerotiorum is a necrotrophic fungus that causes a devastating disease called white mould, infecting more than 450 plant species worldwide. Control of this disease with fungicides is limited, so host plant resistance is the preferred alternative for disease management. However, due to the nature of the disease, breeding programmes have had limited success. A potential alternative to developing necrotrophic fungal resistance is the use of host‐induced gene silencing (HIGS) methods, which involves host expression of dsRNA‐generating constructs directed against genes in the pathogen. In this study, the target gene chosen was chitin synthase (chs), which commands the synthesis of chitin, the polysaccharide that is a crucial structural component of the cell walls of many fungi. Tobacco plants were transformed with an interfering intron‐containing hairpin RNA construct for silencing the fungal chs gene. Seventy‐two hours after inoculation, five transgenic lines showed a reduction in disease severity ranging from 55·5 to 86·7% compared with the non‐transgenic lines. The lesion area did not show extensive progress over this time (up to 120 h). Disease resistance and silencing of the fungal chs gene was positively correlated with the presence of detectable siRNA in the transgenic lines. It was demonstrated that expression of endogenous genes from the very aggressive necrotrophic fungus S. sclerotiorum could be prevented by host induced silencing. HIGS of the fungal chitin synthase gene can generate white mould‐tolerant plants. From a biotechnological perspective, these results open new prospects for the development of transgenic plants resistant to necrotrophic fungal pathogens.     

The control of sclerotinia stem rot on oilseed rape (Brassica napus): current practices and future opportunities

Sclerotinia stem rot (SSR) caused by the phytopathogenic fungus Sclerotinia sclerotiorum is a major disease of oilseed rape (Brassica napus). During infection, large, white/grey lesions form on the stems of the host plant, perturbing seed development and decreasing yield. Due to its ability to produce long‐term storage structures called sclerotia, S. sclerotiorum inoculum can persist for long periods in the soil. Current SSR control relies heavily on cultural practices and fungicide treatments. Cultural control practices aim to reduce the number of sclerotia in the soil or create conditions that are unfavourable for disease development. These methods of control are under increased pressure in some regions, as rotations tighten and inoculum levels increase. Despite their ability to efficiently kill S. sclerotiorum, preventative fungicides remain an expensive gamble for SSR control, as their effectiveness is highly dependent on the ability to predict the establishment of microscopic infections in the crop. Failure to correctly time fungicide applications can result in a substantial cost to the grower. This review describes the scientific literature pertaining to current SSR control practices. Furthermore, it details recent advances in alternative SSR control methods including the generation of resistant varieties through genetic modification and traditional breeding, and biocontrol. The review concludes with a future directive for SSR control on oilseed rape.     

向日葵菌核病接种方法及品种抗病性鉴定

为建立有效的向日葵菌核病田间接种鉴定方法,以核盘菌菌丝体悬浮液和孢子悬浮液作为接种物,分别对抗、感向日葵品种在现蕾期、始花期和盛花期进行人工接种,并对接种后保湿材料和保湿时间进行比较。试验结果表明:两种接种物均可使向日葵抗、感品种产生盘腐症状。用菌丝体悬浮液和孢子悬浮液接种时,浓度分别为10.0~15.0g/L和200~500个/mL,始花期接种,牛皮纸袋保湿2~4d,即可区分出向日葵品种间抗感性差异。用此方法鉴定出13个对盘腐型菌核病表现抗病的向日葵品种。     

重寄生真菌盾壳霉胞外蛋白酶产生条件及酶活影响因子

重寄生真菌盾壳霉Coniothyrium minitans是核盘菌Sclerotinia sclerotiorum的重要生防菌。为了探讨盾壳霉胞外蛋白酶在寄生核盘菌过程中的作用,采用明胶平板法对盾壳霉寄生核盘菌菌核产生的蛋白酶活性进行了检测,并进一步采用福林酚法定量测定蛋白酶活性,研究盾壳霉产生胞外蛋白酶的培养条件及影响蛋白酶活性的因子。试验结果表明,在被盾壳霉寄生的核盘菌菌核中检测到蛋白酶活性,表明蛋白酶可能参与盾壳霉重寄生作用。发现核盘菌菌核浸出液培养基适合盾壳霉产生胞外蛋白酶,摇培(20℃、200r/min)5d时蛋白酶活性最高,达到0.22U/mL。盾壳霉胞外蛋白酶酶促反应的最适温度为60℃,最适pH7.0。当温度不高于40℃时,蛋白酶酶活较稳定。5mmol/L的金属离子Mg²⁺、Zn²⁺、Ca²⁺、Cu²⁺、Mn²⁺、Li⁺和K⁺等对蛋白酶酶活没有显著影响(P>0.05),而Fe²⁺(5mmol/L)显著(P<0.05)提高了蛋白酶活性。盾壳霉蛋白酶对苯甲基磺酰氟(PMSF)敏感,说明盾壳霉产生的胞外蛋白酶可能主要是丝氨酸蛋白酶。这些结果为盾壳霉胞外蛋白酶的分离纯化和功能研究奠定了基础。     

亚麻白腐病发生及综合防治技术研究

从亚麻白腐病的发生、发展、危害及病原菌生长发育规律到病害综合防治进行了系统研究,明确了该病的发生与品种、土壤类型及气象因素等诸方面的关系;也明确了带菌土壤和种子是该病害主要侵染源和传播途径,用药荆防治效果可达80％以上;同时建立综合防病体系。     

枯草芽孢杆菌草酸脱羧酶转化拟南芥研究初报

多种真菌在致病过程中会分泌草酸,这些草酸产生菌寄主范围广,可以导致上百种植物病害,造成世界范围农作物的严重减产。草酸可以通过氧化与脱羧两种途径降解。该研究克隆了枯草芽孢杆菌的草酸脱羧酶基因Yvrk,并构建其植物表达载体,通过农杆菌浸花转化拟南芥,收获种子,用除草剂Basta筛选获得15株转基因植株,对其中的11个株系分别离体接种菌核病菌,24h后量病斑,发现有6株转基因植株的病斑显著小于野生型。PCR验证发现大部分转基因植株确有Yvrk的存在,实验结果说明草酸脱羧酶基因确能一定程度上缓解真菌病害。