稻曲病菌成灾机制与防控技术研究进展

过去数十年来我国稻曲病的发生一直呈不断加重趋势,并逐渐由一个水稻次要病害发展成为一个水稻主要病害。最近不断强化的针对稻曲病成灾机制和防控技术的研究有了新发现和新观点。稻曲病菌主要侵染水稻花期的花丝等细胞壁相对疏松的组织,细胞外扩展。我国长江中下游地区稻曲病的加重与稻曲病菌菌核生成密切相关,菌核在其生活史中发挥了重要作用,可能是其主要的初侵染源。目前水稻孕穗初期使用杀菌剂是最为有效的稻曲病防控措施。未来对其生活史深入持续的研究是稻曲病发生流行中长期预测预警的重要前提。     

来源于同一穗不同稻曲球的稻曲病菌的致病性及遗传多样性

 本研究从源于6穗稻曲病穗的48个稻曲球中分离获得稻曲病菌（Ustilaginoidea virens）48株,从3个稻曲球的不同部位分离获得稻曲病菌23株。用注射接种法将菌株分别接种到水稻品种两优培九（感病品种）、淮稻5号（中抗品种）和武育粳3号（抗病品种）上,结果显示分离的菌株致病力分化较大,而菌株在水稻品种上的致病力强弱与已知水稻品种对稻曲病菌的感、抗性趋势基本一致。相同孢子量接种水稻,不同分离菌株之间仍有致病力分化,生长速率测定也发现菌株之间可能存在差异。利用REP PCR (repetitive extragenic palindromic sequence PCR)技术进行菌株遗传多样性分析表明,同穗不同稻曲球分离的菌株中,1号穗分离的4个菌株聚在同一簇群,其余5穗的菌株分别聚在3～5个簇群;同一稻曲球不同部位分离的菌株中,一个稻曲球分离的8个病菌聚在同一簇群,而其余2个稻曲球分离的病菌则分别聚在2～3个簇群。由此推测同一稻穗上不同稻曲球可能是由来源不同的稻曲病菌侵染所形成;而一个稻曲球可以由同一稻曲病菌引起,也存在多个侵染源共同侵染的可能。     

水稻稻曲病室内人工接种技术

为提高稻曲病人工接种的发病效果和稳定性,在温棚条件下采用水稻孕穗期注射接种法分别研究了稻曲病菌不同接种体、培养时间、接种浓度和接种时期的接种效果。采用病菌马铃薯蔗糖液体培养基（potato sucrose broth,PSB）作为接种体,其穗发病率为100%,明显好于病菌米糠培养液（23.33%）。病菌在PSB中培养5～7天接种效果较好,随病菌培养天数的延长,接种效果明显下降。接种的分生孢子浓度越低水稻病穗率和病粒数也越低。在水稻品种两优培九孕穗中后期,采用含分生孢子浓度为4×106个/mL的病菌PSB培养液注射接种,穗发病率达100%,平均病粒数35.1粒,最高达87粒。研究表明,温棚条件下建立的稻曲病人工接种技术能获得稳定的发病效果,并可区别水稻品种间的抗性差异。     

稻曲病菌致病机制研究进展

 稻曲病是一种水稻穗部病害,在世界各水稻主产区均有发生,已成为世界性病害,在我国也属于水稻主要病害之一。稻曲病的发生严重影响水稻产量和稻米品质。研究其病原菌致病机制,对找寻防治药物的特异性靶标具有重要作用,可为抗病分子育种提供新的思路,为稻曲病菌防治药剂研发提供新的靶标。本文综述了稻曲病菌侵染过程、致病相关基因功能研究、水稻响应稻曲病菌侵染等方面的研究进展,提出进一步研究的策略。现有研究表明,稻曲病菌能成功侵染水稻孕穗期雄蕊的花丝引起发病。病原菌可通过抑制多个水稻免疫途径实现成功侵染;还可模仿胚珠受精激活水稻灌浆、糖代谢等途径为稻曲球的形成提供营养物质;全基因组测序完成和基因编辑技术在基因敲除中的成功应用,为稻曲病菌功能基因研究提供了很好的基础。稻曲病菌独特的侵染过程和营养利用机制是未来研究的重要方向。     

稻曲病菌致病机制研究进展

 稻曲病是一种水稻穗部病害,在世界各水稻主产区均有发生,已成为世界性病害,在我国也属于水稻主要病害之一。稻曲病的发生严重影响水稻产量和稻米品质。研究其病原菌致病机制,对找寻防治药物的特异性靶标具有重要作用,可为抗病分子育种提供新的思路,为稻曲病菌防治药剂研发提供新的靶标。本文综述了稻曲病菌侵染过程、致病相关基因功能研究、水稻响应稻曲病菌侵染等方面的研究进展,提出进一步研究的策略。现有研究表明,稻曲病菌能成功侵染水稻孕穗期雄蕊的花丝引起发病。病原菌可通过抑制多个水稻免疫途径实现成功侵染;还可模仿胚珠受精激活水稻灌浆、糖代谢等途径为稻曲球的形成提供营养物质;全基因组测序完成和基因编辑技术在基因敲除中的成功应用,为稻曲病菌功能基因研究提供了很好的基础。稻曲病菌独特的侵染过程和营养利用机制是未来研究的重要方向。     

稻曲病菌离体培养体系的构建与优化

稻曲病是水稻穗期的一种重要病害,严重影响稻米的产量和品质。稻曲病菌Ustilaginoidea virens 在离体培养条件下生长缓慢,严重制约了杀菌剂室内生测试验的观察和高效筛选。本研究以稻曲病菌培养基为研究对象,通过筛选、组合和优化固体培养体系的氮源、碳源、凝固剂以及阳离子浓度,以期构建适合稻曲病菌生长的室内培养体系。结果发现:在5种常见的商品化培养基中,稻曲病菌的生长速率仅为0.7~2.3 mm/d;而在18种配制培养基中,有77%的培养基对稻曲病菌表现出不同程度的生长促进效应,其中蛋白胨蔗糖结冷胶 (PSGG) 培养基的促进效果最显著 (P < 0.05),生长速率达3.4 mm/d,且其对不同地理分布的稻曲病菌菌株均有生长促进效应。此外,在以结冷胶作为凝固剂的培养基中添加质量分数为0.02%的硫酸镁 (Mg2+),可有效提高培养基的凝固程度,且对菌丝体的生长速率无影响。在适合稻曲病菌生长的PSGG培养基中,蛋白胨和蔗糖是合适的氮源和碳源组合,结冷胶是合适的凝固剂。此研究结果可提高室内培养稻曲病菌的生长速率,为进一步理解稻曲病菌在离体条件下的生长机制及提高室内杀菌剂生测试验的效率提供了重要参考。     

稻曲病菌有性生殖中光诱导基因的转录组分析

 稻曲病菌(Villosiclava virens)能够以菌核的形式安全越冬并在来年萌发产生子囊孢子,成为稻曲病菌的重要初侵染源;而菌核产生子实体的过程需要光的诱导。为了探索光诱导的分子机制,本文对不同光照处理条件下稻曲病菌菌核萌发过程的转录组学进行了比较分析。结果发现,与黑暗条件下菌核的萌发相比,光照处理后542个基因显著差异表达,其中上调359个,下调183个。GO富集分析发现,DEGs显著富集于糖基水解酶活性、细胞壁生物合成、氧化还原酶活性等。KEGG分析发现,DEGs显著富集于脂质代谢、氨基酸代谢、次生代谢产物合成等代谢通路。DEGs中子实体独有基因组氨酸激酶为光感受器,其响应光信号引起下游基因的表达,同时,与有性生殖过程相关的水通道蛋白和甲基转移酶蛋白编码基因表达活跃,共同调控了菌核萌发产生子实体过程。     

寒地水稻秆腐菌核病菌的生物学研究

研究结果表明,水稻秆腐菌核病菌可利用多种碳源和氮源,碳源以可溶性淀粉最好,氮源以酒石酸铵最好,混合碳源(蔗糖+鼠李糖)对病菌菌丝生长有促进作用,比单独蔗糖和鼠李糖生长的好;病菌能在多种天然培养基上生长并形成菌核;菌丝在碳氮比为60∶1的培养基上生长最好;生长物质VB₆+肌醇、VB₆+谷氨酰胺等11个组合对菌丝生长有促进作用;微量元素中亚铁、锰和硼对菌丝生长有抑制作用;液体培养中以Czapek培养基生长最好,但不利于菌核的形成;菌核在15～35℃均可萌发,最适温度为30℃;菌核在0.05%葡萄糖溶液、蒸馏水、水琼脂上均可萌发,以0.05%葡萄糖溶液萌发最好。     

瓶式平板培养制备稻曲病菌薄壁分生孢子

利用三角瓶装纳马铃薯琼脂培养基制备稻曲病菌薄壁分生孢子。结果发现,瓶装培养基的体积与产孢量无关。各菌株在瓶式培养平板上均能正常形成后代孢子。瓶式培养比常规培养皿平板培养具有更高的产孢效率。产孢平板上产生的孢子收取后,菌落继续培养,新形成的孢子数量大幅下降。由于三角瓶能高效避免污染,因而认为瓶式培养是制备稻曲病菌薄壁分生孢子的理想技术。     

稻曲病菌厚垣孢子侵染时期和侵染途径的研究

用田间自然发病病粒上新形成的稻曲病菌厚垣孢子,喷雾或注射接种不同生育期的稻穗获得成功。水稻孕穗末期至破口期是稻曲病菌厚垣孢子侵染的主要时期。在孕穗末期喷雾接种2次萌发孢子的发病率高达50.8%。解剖736个病粒内部都含有花药,镜检119条花药和35个柱头发现,病粒内大多数花粉粒的内容物已基本充实,柱头大多处于分枝状突起阶段。解剖刚发病的病粒柱头上有多根菌丝,并向花药等处扩展。萌发的厚垣孢子可以侵染处于乳头状突起期的柱头,并形成菌丝及产生小孢子。稻曲病菌厚垣孢子主要在水稻破口前1—4天至破口时从水稻的柱头侵入引起发病。     

水稻穗部农艺性状与稻曲病抗性的相关性分析

 人工接种试验和田间多年自然发病调查发现,目前生产上应用的水稻品种均感稻曲病;但在自然条件下,不同水稻品种稻曲病的发生程度存在较大差异,可以人为的分为多病粒高感品种和寡病粒相对抗病的品种。为了探究水稻穗部性状与其病害抗性间的关系,本文对孕穗期不同阶段的不同水稻品种的穗部性状进行了比较分析。结果发现表型为多病粒的高感多病粒品种与表型为寡病粒的相对抗病的品种之间在穗子大小、小花密度、穗鞘闭合程度和密封性、旗叶面积等方面均存在明显差异,寡病粒相对抗病的水稻品种穗鞘闭合程度优于高感多病粒品种。     

The role of <Emphasis Type="Italic">Ustilaginoidea virens</Emphasis> sclerotia in increasing incidence of rice false smut disease in the subtropical zone in China

Rice false smut is heavily and increasingly occurring in subtropical zones in China in the past decades. The pathogen of the disease, Ustilaginoidea virens, can produce both chlamydospores and sclerotia, and the sclerotia seem to form frequently in temperate or high-altitude regions in China. Which of these structures play a dominant role in the pathogen’s life cycle in subtropical zones remains unclear. Here we found that Ustilaginoidea virens could produce a great number of sclerotia in subtropical zones and the maximal number of sclerotia could reach to 2.25 million per hectare. In the year with relatively low autumn temperatures, the disease severity and sclerotia numbers of U. virens increased significantly. Although there was a few sclerotia in subtropical zones capable of overwintering successfully, one individual sclerotium could produce large numbers of ascospores. In the rice-growing paddy field, the ascospores could be trapped in both temperate and subtropical zones in May–September, when rice was at the booting stage, the critical infection period of rice false smut. This suggested that the sclerotia of rice false smut in subtropical zone played an important role in the life cycle of Ustilaginoidea virens and acted as the primary inoculum. Experiments in the laboratory showed that mature sclerotia of rice false smut remained dormant for about 2–5 months, and that light was essential for fruiting body differentiation. As with ergot, the fruiting bodies of Ustilaginoidea virens secreted sticky droplets on the stromata that prevented the ascospores from dispersing into the air, implying that the transfer of ascospores of Ustilaginoidea virens to rice plants in paddy field needed an intermediary vector.     

稻曲病菌菌核降解微生物的筛选与作用机制分析

菌核是稻曲病菌天然的越冬菌源和来年病害发生的重要初侵染源,控制田间菌核越冬数量能够从源头上遏制和减轻稻曲病的发生。为此,本研究对来自稻田土壤、菌核表面、以及青海高原的能够降解菌核的真菌进行了筛选,得到了6个有较好降解作用的生防菌株。在实验室条件下它们能够在30~70 d内彻底降解稻曲病菌菌核。真菌形态特征和rDNA-ITS序列分析表明,这些菌株分别为淡色生赤壳菌Bionectria ochroleuca、粘鞭霉Gliomastix polychroma、烟曲霉Aspergillus fumigatus、草酸青霉Penicillium oxalicum、粉红粘帚霉Gliocladium spp.和疣孢漆斑菌Myrothecium verrucaria。进一步超微结构观察发现,不同生防菌对菌核的降解机制可分为以重寄生作用和直接降解为主2种基本模式。春季田间撒施试验表明,其中2种生防菌的防治效果可达33.9%和46.8%。     

Rice false smut pathogen, Ustilaginoidea virens, invades through small gap at the apex of a rice spikelet before heading

Ustilaginoidea virens, the false smut pathogen of rice, produces false smut balls on spikelets after heading. To clarify how the fungus invades spikelets during the booting stage, we developed a fungal strain that expresses a green fluorescent protein gene and injected conidia from this strain into rice sheaths. Observations at 48?h post-inoculation showed many conidia were present on spikelet surfaces, and the conidia had germinated and the hyphae have gradually grown by 120?h post-inoculation. By 144?h, hyphae had invaded spikelets through their apices, via the small gap between the lemma and palea and had already reached all floral organs.     

稻曲病的接种技术研究

 探索在田间条件下进行稻曲病的人工接种技术。通过比较3种接种体发现,上年保存于-20℃的厚垣孢子不能引起发病;用PS液体培养获得的薄壁分生孢子可引起发病,孢子浓度越高引起病穗率越高;菌丝片段-分生孢子混合液接种发病最严重。病原菌的培养菌龄对接种效果有影响。在水稻的3个时期接种,以抽穗破口前6~9d的效果最好。在一天的3个时段接种,以下午4~6时的病情最重。在接种液里添加马铃薯煮汁可显著提高接种发病率。本文的接种技术能高效引发稻曲病,最高穗发病率100%,最大病情指数93.96,最多穗病粒数110粒。该接种技术可鉴别出品种抗病性的差异。     

Epiphytic colonization of Ustilaginoidea virens on biotic and abiotic surfaces implies the widespread presence of primary inoculum for rice false smut disease

Ustilaginoidea virens (Uv), the causative agent of rice false smut disease, infects developing rice spikelets at the booting stage, and transforms individual grains of the panicle into smut balls. Epidemics of the disease occur when the rice booting and heading stages coincide with rainy days. Using a green fluorescent protein (GFP)‐labelled Uv isolate that can form false smut balls on rice panicles, it was found that under high humidity and free water conditions the Uv isolate could colonize leaves of plants belonging to various families including the Poaceae (Oryza sativa, Echinochloa crusgalli, Digitaria sanguinalis and Leptochloa chinensis), the Brassicaceae (Arabidopsis thaliana) and the Solanaceae (Nicotiana benthamiana) without symptoms. Over several days, some conidia could germinate on the leaves of these plants and in water on the surface of Parafilm and cellophane, form hyphae and differentiate conidiophores to generate a large number of secondary conidia, while other conidia were able to directly produce secondary conidia. Conversely, in the absence of water some conidia could either bud to form new conidia or were converted into chlamydospores. These data indicate that Uv is one of a few fungal pathogens reported to have epiphytic characteristics. The rapid generation of a large number of spores on biotic and abiotic surfaces greatly increases the inoculum that can infect rice spikelets, resulting in the occurrence of rice false smut disease epidemics. These findings are important in the development of disease control strategies.     

Colonization of the vegetative stage of rice plants by the false smut fungus Villosiclava virens,as revealed by a combination of species‐specific detection methods

Rice false smut disease caused by the ascomycete fungus Villosiclava virens (Clavicipitaceae) reduces rice yield worldwide. It invades rice panicles and forms dark‐green false smut balls composed of thick‐walled conidia. Although the infection process during the booting stage is well studied, its infection route before this is unclear. It was hypothesized that the thick‐walled conidia in soil penetrate rice roots, and the fungus latently colonizes roots and tiller buds at the vegetative stage. This hypothesis was tested using species‐specific detection methods. First, real‐time PCR with species‐specific primers and probe was used to estimate thick‐walled conidial number in the paddy field soil. Secondly, nested PCR with species‐specific primers showed that fungal DNA was detected in roots and shoot apices of rice plants in the vegetative stage. Thirdly, colourimetric in situ hybridization with a species‐specific oligonucleotide probe targeting 18S rRNA suggested that sparse mycelia or tightly condensed mycelia were present on the external surface of tiller buds enveloped by juvenile leaf sheaths at the vegetative stage. Thin hyphae were found around leaf axils at the surface of elongated stems at the heading stage, and the fungal hyphae grew in the rice root tissues. In addition, it was demonstrated that eGFP‐tagged transformants of the fungus invaded rice roots and colonized the surface of roots and leaf sheaths under artificial conditions.