Quantification of the rice false smut pathogen <Emphasis Type="Italic">Ustilaginoidea virens</Emphasis> from soil in Japan using real-time PCR

Ustilaginoidea virens is the causal agent of false smut disease of rice. In this study, we developed a real-time polymerase chain reaction (PCR) assay to clarify the relationship between false smut occurrence on rice and quantification of U. virens from soil in Japan. The method here described is sensitive, detecting less than 50 fg of pathogen DNA, and specific to the nuclear ribosomal DNA for U. virens when tested across 27 rice-pathogenic fungi and bacteria, 26 other fungi and bacteria and four plant species. As few as eight chlamydospores of U. virens per gram soil were detected when added to sterilized Gley and Ando soils. The real-time PCR assay for the soil samples was at least 100-fold more sensitive than the conventional and nested-PCR assays tested. By quantification of U. virens with real-time PCR using DNA extracted from naturally contaminated Gley soils and visual assessment of the disease in agricultural fields, a linear correlation between cycle threshold (C_T) values and the number of false smut balls was revealed. Therefore, this specific quantitative assay could be a useful tool for optimization of disease control strategies, and for studying the ecology of U. virens.     

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.     

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.     

Effect of calcined lime (CaO) on chlamydospores of the rice false smut pathogen,Villosiclava virens,and their root attachment and infection

Rice false smut caused by Villosiclava virens is a devastating disease. The smut balls contain chlamydospores, which fall onto paddy soils to become the primary inoculum. After transplanting, the chlamydospores subsequently germinate and infect rice roots. The application of a CaO-containing fertilizer to paddy soils can inhibit the development of rice false smut disease; however, the underlying mechanism is unknown. In this study, we evaluated the suppression of chlamydospore behaviour due to CaO. Specifically, we clarified the effects of the following on chlamydospore morphology and germination: (a) pH, (b) calcium (Ca) concentration, (c) Ca concentration (pH adjusted to 6.5), and (d) CaO-added soil solution. Germination was suppressed at pH 4 and 10, in contrast with the normal germination at pH 6, 7, and 8. Treatments with more than 10 mg/L Ca melted the outer layer of chlamydospores and suppressed germination regardless of whether the pH was adjusted to 6.5. A saturated CaO solution induced bursting of chlamydospores. Suppressed germination and a melted outer chlamydospore layer were also observed, even though the soil buffering effect initially prevented the CaO-mediated pH increase. Furthermore, the chlamydospores within 15 mm from the CaO small lump exhibited suppressed germination in soil. In addition, there was no effect of CaO treatment on chlamydospore attachment to rice roots and hyphal invasion of rice roots in in vitro inoculation tests. These results suggest that Ca concentration is an important factor for inhibiting the occurrence of rice false smut disease.     

Frequency distribution of sensitivity of Ustilaginoidea virens to four EBI fungicides, prochloraz, difenoconazole, propiconazole and tebuconazole, and their efficacy in controlling rice false smut in Anhui Province of China

False smut, caused by Ustilaginoidea virens, is an important emerging disease of rice (Oryza sativa L.) in China. Up to now, as most varieties with high yielding and good quality are susceptible or even highly susceptible to false smut in most rice-growing ecological regions, especially in Anhui Province, chemical control with fungicides would be an important measure for the control of this disease. The ergosterol biosynthesis inhibitor (EBI) fungicides, such as prochloraz, difenoconazole, propiconazole and tebuconazole, are extensively used in China for the control of rice diseases, such as rice sheath blight and rice blast. In this study, a total of 102 U. virens isolates (from Anhui Province of China) were tested for their sensitivity to these four EBI fungicides during the stage of mycelial growth. The EC₅₀ ranges of values for prochloraz, difenoconazole, propiconazole and tebuconazole inhibiting mycelial growth of the 102 U. virens isolates were 0.04–0.75, 0.04–1.08, 0.04–0.38 and 0.03–0.57 μg?ml^?1, with the average EC₅₀ values of 0.32?±?0.08, 0.45?±?0.08, 0.19?±?0.03 and 0.21?±?0.06 μg?ml^?1, respectively. These values suggested that the tested U. virens isolates were very sensitive to these four EBI fungicides. Results of field trials showed that two sprays of three of the fungicides exhibited greater control efficacy than a single spray for the control of rice false smut. Two sprays of each was better than a single spray for the control of rice sheath blight. Two sprays of 50% propiconazole EC at 300 g a.i. ha^?1 gave the best control of rice false smut at both two sites during the two consecutive years, 2010 and 2011, with the control efficacy ranging from 71.5 to 74.3%. Sensitivity of the field U. virens isolates to EBI fungicides should be monitored. Mixtures, as well as alternation with other fungicides with different modes of action, should be tested.     

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.     

A refined inoculation method to evaluate false smut resistance in rice

False smut, caused by Ustilaginoidea virens, is a serious disease of rice worldwide. To evaluate false smut resistance in rice, we developed a method combining the cultivation of the main culm of rice plants in the greenhouse and rapid preparation of a conidial suspension to inject into the leaf sheath. The method was used to evaluate false smut resistance in 18 varieties/lines of rice. For comparison, field trials were also carried out in 2007 and 2008. The results indicated that the greenhouse method was more reproducible than field trials: commercial varieties tested were resistant; almost all the forage varieties were highly susceptible; and blast-resistant varieties/lines were mostly resistant to false smut. Thus, this inoculation method will be useful for determining the level of false smut resistance in rice and for breeding resistant varieties.     

Simple and rapid detection of rice false smut pathogen Ustilaginoidea virens in rice seeds

A method for simple and rapid detection of Ustilaginoidea virens in rice seeds was developed based on specific polymerase chain reaction (PCR). To design the specific primers for detection of U. virens, the comparison was made on 5.8S rDNA intra- and inter-specific variations in nucleotide sequences of U. virens and other pathogens: Fusarium verticillioides, Sclerotinia sclerotiorum, Tilletia barclayana, Fusarium graminearum and Magnaporthe oryzae. A 346-bp fragment could be amplified by using the specific primers uvr-F and uvr-R in U. virens, but not in other test fungi, indicating that the designed primers were specific for the detection of U. virens. U. virens was detected in nine of the 24 tested rice samples, indicating that 37.5% of the rice samples were contaminated with the false smut pathogen. The development of the simple and rapid detection technique using specific primers will be applicable for direct identification of U. virens in rice seeds to screen large numbers of samples.     

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.     

Infection processes of Ustilaginoidea virens during artificial inoculation of rice panicles

Ustilaginoidea virens is a ubiquitous plant pathogen that causes rice false smut disease, one of the most destructive diseases of rice (Oryza sativa L.) production. However, data concerning the effect of inoculation on disease development and the infection process of this pathogen are not comprehensive. In this study, the developmental processes of U. virens in rice panicles were characterized using an enhanced green fluorescent protein (EGFP) labelled strain. A mixture of hyphae and conidia of U. virens was used to inoculate rice panicles by leaf sheath injection during the booting stage of rice plants grown in a greenhouse. The panicles were assessed to determine the relationship between artificial inoculation and disease occurrence. Increasing volumes of inocula (0.2, 0.5, 1, and 2 ml of a mixture of hyphae-fragment and 2?×?10⁶ conidia/ml suspension) caused more severe infections, and small differences were also observed for the different inoculation sites at the base, apex and mid-point of rice panicles. The optimum inoculation condition was 1–2 ml inoculum injected into the mid-point of rice panicles. Spikelet samples were collected as the disease progressed and observed by confocal laser scanning microscopy and scanning electron microscopy. The images collected showed that the primary site of U. virens colonization was at the base of the filaments with the inner spikelets becoming infected by hyphae at 24 h post inoculation (hpi). The accumulation of hyphae reached its highest level at 168 hpi, before the rice heading stage, as the infection extended upward from basal filaments to the anther apex, and then enclosed all the floral organs to produce a velvety smut ball.     

Isolation and characterization of <Emphasis Type="Italic">Ustilaginoidea virens</Emphasis> and survey of false smut disease of rice in India

The intensity of rice false smut disease in selected states of northwest and south India was studied. In northern Indian states as a whole, disease incidence (percentage of false smut-infected tillers) varied from 2% to 75%. In the state of Haryana, maximum infection was recorded on hybrids like PA 6444 and PA 6129 while in Punjab state, 10–20% disease incidence was recorded in popular inbred rice varieties like PR 114, PA 116 and PAU 201. In the southern state of Tamil Nadu, the disease incidence varied from 5% to 85%. A heavy incidence of the disease was noticed in variety BPT 5204 and due to this, the air above the infected field gave a black smoky appearance from a distance as a result of release of spore mass in the atmosphere. In severe cases the number of infected grains reached even more than 100 per panicle. The pathogen Ustilaginoidea virens was isolated in potato dextrose agar medium and was characterized by both pathogenicity test and molecular analysis. Under glasshouse conditions, when a conidial suspension of the pathogen was injected during boot leaf stage of the rice variety TN1, typical smut balls were observed. The identity of the pathogen was further confirmed through polymerase chain reaction (PCR) analysis using U. virens-specific internal transcribed spacer (ITS) primers. The primer pair US 1-5/US3-3 and US2-5/US4-3 amplified 380 bp and 232 bp product, respectively, which are typical for the U. virens fungus.     

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。     

籼型杂交水稻对稻曲病的田间抗性差异

为了解不同水稻品种在田间的抗性差异及其与农艺性状的相互关系,在相同的土壤环境和栽培条件下,利用稻曲病菌诱发接种鉴定,对56个籼型杂交水稻进行田间抗性差异比较,初步探讨了抽穗期和农艺性状与品种田间抗性的关系.结果表明,不同水稻品种对稻曲病田间抗性存在显著差异,病穗率为0.43%～33.04%,病情指数为0.05～16.14;不同抽穗期的品种稻曲病发病率不同,同一抽穗期的品种对稻曲病的抗性亦表现出明显差异;水稻株高与品种抗性之间呈正相关,相关系数为026(P<0.05);水稻分蘖率、有效穗数、剑叶性状等与品种的田间抗性有一定相关性,但差异不显著(P>0.05);水稻株叶形态和叶色与品种田间抗性没有直接相关性.     

人工培养基上稻曲病菌拟菌核诱导的初步研究

 稻曲病菌在水稻孕穗期侵染水稻小花,在水稻灌浆后期只在个别稻曲球上形成菌核。因此,稻曲病菌菌核分化与发育基因功能研究的相关试验难度较高。为了探究稻曲病菌在人工培养基上能否形成菌核,本文利用低温或杀菌剂环境对稻曲病菌进行了筛选。结果发现,在81个菌株中有13个在人工培养基上形成拟菌核组织,且它们可在低温处理时稳定地产生拟菌核组织。这些拟菌核组织在结构上与菌核类似,但体积较小且组织较为疏松,无法像菌核一样越冬和完成有性生殖。     

Elucidation of the infection process of Ustilaginoidea virens (teleomorph: Villosiclava virens) in rice spikelets

This study reports an efficient inoculation protocol that allowed cytological analysis of the infection process of the rice false smut pathogen Ustilaginoidea virens. Examination of serial semithin and ultrathin sections of infected spikelets showed that the primary infection sites for the pathogen were the upper parts of the three stamen filaments located between the ovary and the lodicules. The stigma and lodicules were also occasionally infected to a limited extent. The pathogen infected the filaments intercellularly and extended intercellularly along the filament base. The host cells were degraded gradually. The pathogen did not penetrate host cell walls directly and did not form haustoria. In the balls the ovary remained alive and was never infected. This suggests that the pathogen is a biotrophic parasite that grows intercellularly in vivo.     

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。     

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。     

Detection of quantitative resistance loci associated with resistance to rice false smut (Ustilaginoidea virens) using introgression lines

False smut, caused by Ustilaginoidea virens, is a rice disease of increasing importance worldwide, with no source of high‐level resistance in the existing rice germplasm. To facilitate breeding varieties with good levels of field resistance to false smut, quantitative resistance loci (QRL) were identified using 213 introgression lines (ILs) from a cross between Teqing (recipient) and Lemont (donor) evaluated using natural infection at two hotspots of false smut in northeast China. Ten QRL affecting percentages of diseased hills, diseased panicles and diseased spikelets were detected and mapped to rice chromosomes 2, 3, 4, 6, 8, 10, 11 and 12. The Lemont alleles at all QRL increased false smut resistance. Four QRL (qFSR‐6‐7, qFSR‐10‐5, qFSR‐10‐2 and qFSR‐11‐2) had relatively larger and consistent effects across the two testing sites. Promising resistant ILs were identified, most of which had multiple QRL, suggesting that pyramiding multiple QRL by marker‐assisted selection would be an effective strategy for improving rice resistance to false smut. The identified QRL and their linked DNA markers will facilitate this breeding effort in the future.     

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。     

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。