Research progress on the resistance of Brassica napus to Sclerotinia sclerotiorum

Ka ZHANG, Hao-jie LI, Jin-fang ZHANG, Cheng CUI, Liang CHAI, Ben-chuan ZHENG, Jun JIANG, Cheng DAI, Jin-xing TU, Liang-cai JIANG

CHINESE JOURNAL OF OIL CROP SCIENCES ›› 2023, Vol. 45 ›› Issue (6) : 1095-1102.

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CHINESE JOURNAL OF OIL CROP SCIENCES ›› 2023, Vol. 45 ›› Issue (6) : 1095-1102. DOI: 10.19802/j.issn.1007-9084.2023212

Research progress on the resistance of Brassica napus to Sclerotinia sclerotiorum

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Abstract

Brassica napus (oilseed rape) is the primary source of vegetable edible oil in China, playing an important role in ensuring and stabitity the safety of national edible oil supply. The growth and development of oilseed rape are frequently attacked by various pathogens, among which Sclerotinia sclerotiorum is generally considered one of the most economically damaging, widely studied and highly concerned diseases. This article refers to relevant research results, elaborates on the pathological cycle and pathogenic mechanism of S. sclerotiorum, summarizes the progress of resistant germplasms, defense-ralated genes and mechanisms in oilseed rape. Additionary, this review introduces the newly reporeted pathogenicity factors such as SsCP1; it contains breakthroughs in the creation of resistant germplasm inherited from Brassica species; multiple studies that found the co-localization of genetic loci controlling flowering time and resistance to S. sclerotiorum in oilseed rape; and the research reveals the molecular mechanism mediated by the WRKY28-WRKY33 module finely regulates defense strength of oilseed rape after being infected with S. sclerotiorum. This review also looks forward to future research on oilseed rape resistance against S. Sclerotiorum, with a focus on exploring resistant germplasm. It aims to provide a reference for the comprehensive control of S. sclerotiorum in China.

Key words

Brassica napus / Sclerotinia sclerotiorum / resistance related loci / resistance mechanism

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Ka ZHANG , Hao-jie LI , Jin-fang ZHANG , Cheng CUI , Liang CHAI , Ben-chuan ZHENG , Jun JIANG , Cheng DAI , Jin-xing TU , Liang-cai JIANG. Research progress on the resistance of Brassica napus to Sclerotinia sclerotiorum[J]. CHINESE JOURNAL OF OIL CROP SCIENCES, 2023, 45(6): 1095-1102 https://doi.org/10.19802/j.issn.1007-9084.2023212
油菜是我国第一大油料作物,常年种植面积超过730万公顷(1.1亿亩)。世界上油菜主要有白菜型(Brassica rapa)、芥菜型(Brassica juncea)和甘蓝型(Brassica napus)三种栽培类型[1]。我国是白菜型和芥菜型油菜的起源中心之一,白菜型和芥菜型油菜在我国已有数千年栽培历史[2]。甘蓝型油菜起源于地中海沿岸,最初主要种植在德国、法国、瑞典等国家,因此也被称为欧洲油菜,大约在20世纪30年代初期引入国内。甘蓝型油菜株型高大、产量高、抗病性较好,因此20世纪50年代后半期到20世纪70年代在我国长江流域油菜主产区大面积推广,自此甘蓝型油菜成为我国最主要的栽培类型[3]。甘蓝型油菜是在约7500年前由白菜和甘蓝通过自然杂交形成的异源四倍体物种[4]。菌核病是由核盘菌(Sclerotinia sclerotiorum)侵染引起的一种世界性病害,是长期以来制约油菜提质增效的关键因素之一;在我国油菜主产区年年发病,造成油菜减产10%~20%,重病年高达70%[5]

1 核盘菌的病害循环

核盘菌寄生范围广,可侵染全球600多种植物,包括油菜、大豆、花生、向日葵等双子叶植物,以及郁金香、洋葱等单子叶植物[6,7]。核盘菌一直被认为是一种典型的死体营养型真菌病原菌;但近几年有研究提出,其实际上可能是一种半活体营养型病原菌。在核盘菌侵染的早期,菌丝扩展过程中并未引起宿主细胞坏死,在定殖点的外围仍保持着短暂的生物营养化行为,病原菌成功定殖后,宿主产生大量活性氧、组织逐渐坏死,病原菌进入死体营养阶段[5]。以甘蓝型油菜茎秆为例,在感染核盘菌后,发病部位初期呈现浅褐色,随后变成白土色、组织腐烂、茎秆内部中空,在高湿度条件下,茎秆表面产生白色菌丝体和黑色菌核[8]。在油菜开花期,土壤中残留的菌核萌发形成菌丝或子囊孢子,借气流传播附着在油菜的花瓣或者叶片;染菌的花瓣是核盘菌生长繁殖的营养载体,也成为了传播病原菌的媒介,飘落在健康的茎秆、花瓣、叶片上形成再侵染;到了油菜生育期后期,随着气温升高、田间气候干燥等环境条件的变化,核盘菌致病性受到限制,形成菌核残留在土壤中或者被带入种子中,进入下一个病害循环[9]。菌核的萌发和致病过程对环境湿度有较高的要求,相对湿度一般需要达到85%以上;对环境温度有一定的要求,一般在0~30℃均可,20℃是最有利的温度条件[8,10]。因此,当油菜越冬时遭遇气候湿冷、早春温湿多雨,一般会导致菌核病的爆发。

2 核盘菌的致病机理

目前的研究表明,核盘菌的致病因子主要包括两类分泌物:一类是降解植物细胞壁的酶类,另一类是草酸[8]。核盘菌分泌的植物细胞壁降解酶有果胶酶、纤维素酶、半纤维素酶、木质素降解酶等[11],其中以果胶酶为主[12],果胶酶中尤以多聚半乳糖醛酸酶(polygalacturonase, PG)为主。核盘菌基因SsPG1在侵染油菜早期即诱导表达,并帮助核盘菌在寄主体内扩展[13]。在拟南芥叶片中异源表达核盘菌的SsPG3SsPG6基因,能引起植物叶片浸水性细胞死亡[14]。病原菌分泌的草酸可导致寄主细胞坏死,诱发侵染点产生病斑;不能产生草酸的突变株丧失致病力[15]。有研究表明草酸的致病原因主要有4个方面:(1)草酸可使寄主侵染细胞周围的pH降低至5.0左右,与许多植物细胞壁降解酶最适催化pH相当,促进寄主细胞壁的降解;此外,低pH可降低寄主产生的多聚半乳糖醛酶抑制蛋白(PGIP)的活性,从而抑制了寄主的防御作用[16]。(2)草酸可螯合细胞壁中的Ca2+,既抑制了Ca2+作为第二信使的信号传递作用,又帮助PG降解植物细胞壁中失去了Ca2+的果胶酸酯[17]。(3)草酸可抑制保卫细胞中脱落酸诱导的气孔关闭作用,引起叶片萎蔫[18]。(4)草酸可诱导寄主细胞程序性死亡,寄主死亡为营腐生的病原菌提供营养来源[19]
除了上述两类主要的致病因子,近年来也发现了核盘菌还存在其他致病因子。SsSOD1突变病原株可正常产生菌丝及草酸,但致病力显著下降,SsSOD1为一个超氧化物歧化酶,可能与清除寄主产生的活性氧有关[20]。分泌蛋白Ss-Caf1起到与寄主相互识别的作用,诱导寄主细胞程序性死亡,也与菌核的形成相关[21]。核盘菌分泌的Ss-Bi1蛋白与菌株毒性密切相关,突变后导致病原菌致病力减弱、全毒性丧失[22]。核盘菌侵染过程中可通过菌丝分泌SsCP1,进而与寄主防御蛋白PR1互作,降低PR1对病原菌的抑制作用,促进侵染。此外,高浓度的SsCP1可引起寄主植物细胞坏死,从而有利于核盘菌从寄主中获取营养物质[23]

3 油菜菌核病抗性鉴定方法

建立准确可靠的油菜菌核病抗性鉴定方法,是判断种质资源的抗性强弱、筛选优良抗性材料、开展抗病育种的重要环节。目前,国内外报道了多种用来评估油菜菌核病抗性强弱的鉴定方法,按照生育期可分为苗期鉴定和花期鉴定,按照接种部位可分为叶片接种和茎秆接种,按照取样方式可分为离体鉴定和活体鉴定。其中,苗期离体叶片接种、活体叶片接种,以及盛花期离体茎秆菌丝块接种、活体茎秆菌丝块接种、活体茎秆牙签接种等是常用的几种抗病表型筛选方法[24]。油菜苗期离体叶片接种法操作简单、易于控制,是苗期抗病性鉴定、抗性相关基因功能验证最常用的方法;育种家在育种实践中通常通过多年多点自然条件下的发病率及发病指数来判断育种材料的菌核病抗性;品种/系的植保鉴定一般采用田间病圃抗性鉴定方法,病圃每年于苗期每平方米均匀增施2~3粒菌核并定期检测土层中菌核的丰度和均匀度,于油菜花期通过人工喷雾系统保证周边环境湿度,以诱导病害的发生;设置统一的抗病性较好的对照材料,并将受外部条件影响较大的病害值转换成相对抗性指数来评判每份材料的抗性水平[25]。但由于受环境因素影响及病原菌致病力差异等,不同方法鉴定结果相关性不显著,且室内抗性鉴定结果较自然发病存在一定差异[26]。为了科学准确地评估不同品种/系的抗病性,应结合多年度、多种鉴定方法的结果进行综合判断[27]

4 抗性资源的创制策略

4.1 杂交聚合抗性位点

为了降低菌核病对油菜生产的影响,最经济、环保、有效的方式是培育和推广抗病品种。在我国油菜抗病遗传改良的历程中,通过传统的杂交育种策略选育出了秦油2号、中油821、中双9号、宁RS-1等在室内接种鉴定和田间自然发病条件下均表现出良好菌核病抗性的品种,并得到了广泛的推广应用[28-31]。然而,这些品种也仅达到中抗菌核病水平,其中未发现高抗品种,至今在油菜乃至整个十字花科物种中也还未找到对菌核病完全免疫的种质资源。有研究表明芸薹属物种中对菌核病抗性最强的是甘蓝,最弱的是白菜型油菜,芥菜型油菜、甘蓝型油菜和黑芥的抗性居中[32]。利用野生甘蓝优良的菌核病抗性特征,通过远缘杂交将野生甘蓝中的抗性位点导入甘蓝型油菜中双9号,创制出了抗病性优于中双9号、具有38条染色体和育性正常的甘蓝型油菜新材料[33],并通过杂交聚合C亚基因组上的3个抗性位点,选育出了抗病品系M726[34]

4.2 阻遏病原菌致病因子的作用

前文提到核盘菌主要分泌多聚半乳糖醛酸酶(PG)和草酸(OA)两种致病因子,有研究就提出了通过抑制分泌蛋白的作用来降低病原菌的致病性的方法。在甘蓝型油菜中过表达来源于水稻的编码多聚半乳糖醛酶抑制蛋白(OsPGIP)的基因,能有效提高转基因油菜对菌核病的抗性,过表达OSPGIPs可能通过抑制核盘菌侵染过程中对寄主细胞壁的降解作用,增加油菜茎秆细胞壁的纤维素和半纤维素含量从而加强了防御屏障[35,36]。核盘菌在侵染油菜过程中通过分泌草酸产生致病作用,油菜自身不能分解草酸,在油菜中过表达来源于大麦的编码草酸氧化酶的基因OXO,转基因株系的草酸盐含量降低、过氧化氢介导的防御反应增强,从而部分增强了油菜的菌核病抗性[37]。此外,有研究者发现,油菜中细胞色素b-c1复合物的一个亚基是核盘菌致病因子的识别因子,利用基因编辑技术在油菜中敲除BnQCR8基因,突变体在遭受核盘菌侵染时与致病分泌蛋白的相互作用受到影响,降低了病原菌的侵染效率[38]

4.3 限制病原菌的传播

前文提到,油菜花瓣可为核盘菌提供营养,也是病害进一步传播和扩散的媒介。基于此,有研究者在较早的时候就提出了油菜的无花瓣育种理念,并利用种间杂交获得了无花瓣新材料[39,40]。田间发病率调查发现,无花瓣材料仅为1.75%,比正常的四瓣材料发病率降低85.93%~94.76%[39]。除了利用无花瓣育种技术,有研究者提出了创制花瓣不掉落或少掉落的新材料,切断病原菌传播的温床,减少发病率。在这方面有两项研究都表明,拟南芥中IDA基因编码一个具有N末端信号肽的小分泌蛋白,与花器官的脱落密切相关,该基因在不同作物中的功能比较保守,在油菜中敲除该基因的两个同源拷贝,突变体的花瓣不脱落或少脱落,并且不容易感染菌核病,此外花期延长也增加了旅游收入[41,42]。一种真菌病毒SsHADV-1可以将其宿主核盘菌从一种典型的坏死性病原体转化为有益的内生真菌,利用该真菌病毒制作成生物防治制剂,发现喷施这种药剂后,油菜接种病原菌后病斑变小了,产量还得到了提升[43]

5 油菜中抗性相关基因挖掘和抗性机理解析

5.1 油菜菌核病抗性相关QTL分析

研究表明油菜对菌核病的抗性是由多基因控制的数量性状,呈现加性效应和显性效应相叠加的遗传模式,并具有中等遗传力[44]。Zhao等利用中抗菌核病材料宁RS-1与一个菌核病敏感材料杂交构建分离群体,利用F2:3家系的128个单株构建油菜菌核病抗性的遗传连锁图谱,检测到6个与菌核病抗性相关的QTL,其中3个QTL与苗期叶片抗性相关,解释了40.7%的表型变异,另外3个QTL与成株期的抗病性相关,解释了49.0%的表型变异[45]。之后利用两个独立的DH(双单倍体)系构建连锁分析群体,共鉴定到5个茎秆抗性QTL,并进一步提出了油菜菌核病的显性效应和加性效应[46]。Wu等对一个DH系群体进行了多年份多环境条件下的抗性鉴定,共定位到3个苗期叶片抗性QTL和10个成株期茎秆抗性QTL,并发现位于C06连锁群上的一个主效QTL在所有环境中稳定存在,可解释表型变异29.01%~32.61%[47]。Wei等利用油菜60 K芯片对347份种质进行了全基因组关联分析,在A08和C06两个连锁群上发现了多个茎秆抗性关联位点[48]。Wu等也是利用60 K芯片进行了全基因组关联分析,通过调查448份材料的菌核病抗性,鉴定到了位于C04、C06和C08上的3个抗性位点,并在该染色体区间预测了39个候选基因[49]。Roy等利用187个品系,在4个环境条件下,在田间活体植株上接种带菌琼脂块考察抗病性,利用全基因组关联分析在两个或两个以上的环境中检测到19个显著的SNPs,鉴定出69个与抗性相关的候选基因[50]。Roy等采用叶柄接种法对337份材料进行苗期抗病性调查,通过全基因组关联分析,使用单基因座和多基因座模型分别鉴定到41个和208个显著相关的SNP,这些SNP解释了1.25%~12.22%的表型变异[51]
一般来说,早花的油菜品种菌核病发病较重,早花与抗菌核病几乎是矛盾的。有研究对包含521份自交系的自然群体进行表型调查,发现油菜菌核病抗性与开花时间呈现显著的负相关[52],并在A02、A06、C02、C08等染色体上鉴定到多个菌核病抗性与开花时间共定位的QTL[52,53]。有研究利用一个抗性较好但相对晚花材料与一个抗性较差但相对早花的材料构建的重组自交系群体,利用油菜60 K芯片对群体进行基因分型,并在6个开花时间和9个菌核病抗性环境下进行表型分型,共检测到30个菌核病抗性相关的QTL以及22个控制开花期的QTL,并发现这两个性状相关的一些主要QTL是共定位的,这表明它们之间存在遗传连锁[54]。有研究利用一个杂交育种骨干亲本和另一个常规种构建的151个单株组成的DH系群体,在多个环境下鉴定群体抗病性,并扫到13个茎秆抗性相关的QTL、9个病情指数相关的QTL,发现多个位点与以往鉴定到的开花时间相关QTL置信区间相重合[55]。这些研究从遗传上揭示了难以选育出既早花又抗菌核病品种的原因,同时多个研究均表明A02和C02染色体上可能存在比较保守的控制这两个性状的位点,通过规避这些紧密连锁的基因座,可以为育种亲本的抗性改良提供参考。

5.2 油菜菌核病抗性机理解析

虽然油菜中有部分品种或材料达到中抗菌核病水平,并利用QTL定位鉴定到了许多与抗性相关的位点,但到目前为止,还未检测到稳定存在、效应值大的QTL,还未见将鉴定到的QTL转化为有效的分子标记,并应用于育种实践的报道,这也使得油菜对菌核病抗性机理的解析变得困难。近年来,关于油菜响应核盘菌侵染后防御机制的研究取得了一些进展,所报道的基因主要集中在植物先天免疫系统组分、转录因子、激素信号转导分子等几个方面。
MAPK(mitogen-activated protein kinase,丝裂原活化蛋白激酶)级联磷酸化是植物应答病原菌侵染的重要环节,一个完整的MAPK级联由MAPKKK、MAPKK和MAPK三类蛋白激酶组成,通过逐级磷酸化发挥信号传递与放大的作用[56]。茉莉酸、水杨酸和乙烯是三种被报道与菌核病抗性相关的植物激素。PDF1.2是茉莉酸介导的抗病信号通路中的重要基因,在甘蓝型油菜中过量表达MAPK4增强了转基因株系的抗菌核病能力,进一步分析发现,MAPK4通过促进PDF1.2的表达增强了防御方应[57]。此外,核盘菌侵染、茉莉酸或乙烯处理能诱导油菜中MAPK3基因的表达,核盘菌侵染、茉莉酸或水杨酸处理能诱导油菜中MAPK6基因的表达,MAPK3MAPK6过表达油菜株系的菌核病抗性显著增强,而RNAi株系抗病性均减弱;并认为这两个基因均通过影响茉莉酸、水杨酸和乙烯介导的信号传导相关基因的表达来参与抗病反应[58,59]NPR1在水杨酸介导的抗病信号通路中发挥核心作用,通过RNAi技术在油菜中降低NPR1基因的表达,显著减弱了转基因植株对菌核病的抗性[60]。有研究证明了BnaA03.MKK5与BnaA06.MPK3/BnaC03.MPK3相互作用,以及BnaA03.MKK5对BnaA06.MPK3/BnaC03.MPK3的磷酸化作用,在油菜中构建了MAPKK5-MAPK3分子模块,并验证其在菌核病抗性中的作用[61]
甘蓝型油菜中WRKY33基因受到核盘菌侵染诱导表达[62],在油菜中过表达WRKY33,接种核盘菌后转基因株系相比野生型植株菌斑面积小,WRKY33是油菜菌核病响应过程中的一个正向调控因子[63,64]。围绕该转录因子进一步研究,构建了一条完整的油菜响应核盘菌侵染的信号通路:发病早期,MAPK信号级联被激活并磷酸化激活WRKY33,WRKY33与自身启动子结合帮助植物体抵御病原菌入侵;发病后期,WRKY28与VQ12被诱导并形成复合体,使WRKY28优先于WRKY33与WRKY33的启动子结合。由于WRKY28的转录活性低于WRKY33的转录活性,因此WRKY28优先结合WRKY33启动子后导致WRKY33的转录低于防御早期时的水平。WRKY33调控的抗病基因的表达量也会降低,这相当于抗病反应的“刹车”作用。同时,在叶腋处表达的WRKY28通过与BRC1启动子结合,降低其表达,从而刺激腋生分生组织的起始和腋芽的发育,并促进形成完整的分枝。在这一阶段,植物体的抗性强度降低,生长恢复活跃。这种防御“刹车”有助于油菜实现防御和生长之间的平衡,增加病害条件下生存繁衍的机会[65]。有研究利用RNAi技术在油菜中降低转录因子BnMYB43的表达,转基因株系茎秆中纤维素和木质素含量分别下降了17.49%和16.21%,果胶含量增加了71.92%。与野生型相比,接种核盘菌后,转基因植株茎秆的病变长度显著减少了52.10%,表明抗病性显著提升;BnMYB43通过调控防御相关基因的表达负向作用于油菜菌核病抗性[66]。此外,有研究发现油菜中编码β-1,3-葡聚糖酶的基因BnaC4.PR2受到核盘菌侵染诱导表达,BnaC4.PR2过表达可诱导寄主防御过程中活性氧爆发,从而触发部分细胞死亡和增强系统获得性抗性[67]

6 展望

一直以来,油菜菌核病受到广泛关注,虽然在油菜中鉴定到了一些菌核病抗性相关的QTL,挖掘了一些抗病相关的基因,解析了部分抗性相关的分子机理,但由于缺乏对菌核病免疫的种质资源,使得抗菌核病品种选育工作未取得突破性的进展,仍未培育出高抗菌核病的品种/系,达中抗水平的品种也相对较少。对于未来油菜菌核病相关研究,重点还是要进一步挖掘和鉴定抗性种质;油菜是一个形成不足万年的异源四倍体,是一个相对年轻的物种,目前未找到对菌核病免疫的种质不代表一定不存在;特别是近年来有研究者在甘蓝中找到了抗性较好的资源,通过远缘杂交将抗性位点转到甘蓝型油菜中,再通过不断转育筛选结实正常、抗病性好的油菜材料[33,34],为未来的研究方向提供了重要参考。抗性种质筛选和鉴定的范围可以扩展到油菜的近缘种,甚至整个十字花科物种。例如目前在油菜中广泛应用的抗根肿病位点,最初来源就是同属于十字花科的芜菁[68]。近期,有研究发现一种野生羽衣甘蓝(B. villosa,BRA1896)经接种鉴定达到高抗菌核病水平,并在该材料C07染色体上定位到与抗病性紧密连锁的区段,为改良甘蓝型油菜菌核病抗性提供了一个新的、独特的遗传来源[69]。培育抗病、优质、高产的品种是油菜育种的目标,但产量与抗病往往是两个相互关联的性状,也涉及大量基因的参与及调控,所以要选育出综合性状好的品种,要注重抗性、品质与产量之间平衡。

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