Current understanding on Villosiclava virens,a unique flower‐infecting fungus causing rice false smut disease

Villosiclava virens (Vv) is an ascomycete fungal pathogen that causes false smut disease in rice. Recent reports have revealed some interesting aspects of the enigmatic pathogen to address the question of why it specifically infects rice flowers and converts a grain into a false smut ball. Comparative and functional genomics have suggested specific adaptation of Vv in the colonization of rice flowers. Anatomical studies have disclosed that Vv specifically infects rice stamen filaments before heading and intercepts seed formation. In addition, Vv can occupy the whole inner space of a spikelet embracing all floral organs and activate the rice grain‐filling network, presumably for nutrient acquisition to support the development of the false smut ball. This profile provides a general overview of the rice false smut pathogen, and summarizes advances in the Vv life cycle, genomics and genetics, and the molecular Vv–rice interaction. Current understandings of the Vv–rice pathosystem indicate that it is a unique and interesting system which can enrich the study of plant–pathogen interactions. Taxonomy: Ustilaginoidea virens is the anamorph form of the pathogen (Kingdom Fungi; Phylum Ascomycota; Class Ascomycetes; Subclass Incertae sedis; Order Incertae sedis; Family Incertae sedis; Genus Ustilaginoidea). The teleomorph form is Villosiclava virens (Kingdom Fungi; Phylum Ascomycota; Class Ascomycetes; Subclass Sordariomycetes; Order Hypocreales; Family Clavicipitaceae; Genus Villosiclava). Disease symptoms: The only visible symptom is the replacement of rice grains by ball‐shaped fungal mycelia, namely false smut balls. When maturing, the false smut ball is covered with powdery chlamydospores, and the colour changes to yellowish, yellowish orange, green, olive green and, finally, to greenish black. Sclerotia are often formed on the false smut balls in autumn. Identification and detection: Vv conidia are round to elliptical, measuring 3–5 μm in diameter. Chlamydospores are ornamented with prominent irregularly curved spines, which are 200–500 nm in length. The sclerotia are black, horseshoe‐shaped and irregular oblong or flat, ranging from 2 to 20 mm. Nested polymerase chain reaction (PCR) and quantitative PCR have been developed to specifically detect Vv presence in rice tissues and other biotic and abiotic samples in fields. Host range: Rice is the primary host for Vv. Natural infection by Vv has been found on several paddy field weeds, including Digitaria marginata, Panicum trypheron, Echinochloa crusgalli and Imperata cylindrica. However, the occurrence of infection in these potential alternative hosts is very rare. Life cycle: Vv infects rice spikelets at the late rice booting stage, and produces false smut balls covered with dark‐green chlamydospores. Occasionally, sclerotia form on the surface of false smut balls in late autumn when the temperature fluctuates greatly between day and night. Both chlamydospores and sclerotia may serve as primary infection sources. Rainfall at the rice booting stage is a major environmental factor resulting in epidemics of rice false smut disease. Disease control: The use of fungicides is the major approach for the control of Vv. Several fungicides, such as cuproxat SC, copper oxychloride, tebuconazole, propiconazole, difenoconazole and validamycin, are often applied. However, the employment of resistant rice cultivars and genes has been limited, because of the poor understanding of rice resistance to Vv. Useful websites: Villosiclava virens genome sequence: http://www.ncbi.nlm.nih.gov/Traces/wgs/?val=JHTR01#contigs     

稻绿核菌(稻曲病菌)分离方法的比较研究

作者对不同情况下稻曲病菌的分离方法进行了比较研究。结果表明成熟早期稻曲球上的绝大多数新鲜的厚垣孢子具有萌发能力,及时进行分离培养是病原菌成功分离的关键。随保存时间的延长,厚垣孢子萌发能力急剧下降;消毒处理可杀死大部分的厚垣孢子。菌核可长期保存并保持极高的萌发生长能力,是稻曲病菌分离最为理想的材料。稻曲球中部的致密菌丝组织分离难度较大,只能作为稻曲病菌分离的一种补救方法。     

稻曲球及稻曲病菌菌落微结构的SEM观察

本文对不同培养条件下稻曲病菌菌落及稻曲球的微结构进行了扫描电镜比较研究。在PS培养液里进行液体培养时,稻曲病菌很少产生分生孢子和厚垣孢子,只有培养后期漂浮在培养液表面的菌落可以产生大量的厚垣孢子。病原菌在进行PSA固体培养时,大部分菌株在培养后期产生大量的成堆分布的厚垣孢子,少部分菌株在菌落上产生散生的厚垣孢子。说明暴露于空气有助于稻曲病菌产生厚垣孢子。在煮熟的带壳谷粒上稻曲病菌的生长明显比在去壳上的要慢得多。微结构分析表明,稻曲球表面是一层密集的厚垣孢子,菌丝与稻粒的胚乳层界限分明,大部分稻曲球中部有大块的发育良好的胚乳,并充满密集的淀粉粒。说明稻曲病菌可能在开花灌浆后开始侵染,而且至少后期是腐生的。     

稻曲球及稻曲病菌菌落微结构的SEM观察

本文对不同培养条件下稻曲病菌菌落及稻曲球的微结构进行了扫描电镜比较研究。在PS培养液里进行液体培养时,稻曲病菌很少产生分生孢子和厚垣孢子,只有培养后期漂浮在培养液表面的菌落可以产生大量的厚垣孢子。病原菌在进行PSA固体培养时,大部分菌株在培养后期产生大量的成堆分布的厚垣孢子,少部分菌株在菌落上产生散生的厚垣孢子。说明暴露于空气有助于稻曲病菌产生厚垣孢子。在煮熟的带壳谷粒上稻曲病菌的生长明显比在去壳上的要慢得多。微结构分析表明,稻曲球表面是一层密集的厚垣孢子,菌丝与稻粒的胚乳层界限分明,大部分稻曲球中部有大块的发育良好的胚乳,并充满密集的淀粉粒。说明稻曲病菌可能在开花灌浆后开始侵染,而且至少后期是腐生的。     

PCR-based Specific Detection of Ustilaginoidea virens and Ephelis japonica

A PCR‐based technique for detection of clavicipitaceous pathogens in rice and related grasses was developed. The target pathogens were Ustilaginoidea virens, which causes rice false smut, and Ephelis japonica, which causes rice udbatta disease and black choke in grasses. To design specific primers, a comparison was made on genetic diversity on the rDNA internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene of U. virens, Ephelis japonica, as well as some other clavicipitaceous fungi. Each fungus was successfully detected by using a specific primer set with high sensitivity. Species‐specific primers designed here were capable of detecting these pathogens in plant tissues. The PCR detection was consistent with conventional histological observation. This nested PCR assay was sensitive and reliable for the detection of U. virens and E. japonica, and thus can be a used to study disease cycles and early prediction of false smut and udbatta‐disease incidence in fields.     

The essential effector SCRE1 in Ustilaginoidea virens suppresses rice immunity via a small peptide region

The biotrophic fungal pathogen Ustilaginoidea virens causes rice false smut, a newly emerging plant disease that has become epidemic worldwide in recent years. The U. virens genome encodes many putative effector proteins that, based on the study of other pathosystems, could play an essential role in fungal virulence. However, few studies have been reported on virulence functions of individual U. virens effectors. Here, we report our identification and characterization of the secreted cysteine-rich protein SCRE1, which is an essential virulence effector in U. virens. When SCRE1 was heterologously expressed in Magnaporthe oryzae, the protein was secreted and translocated into plant cells during infection. SCRE1 suppresses the immunity-associated hypersensitive response in the nonhost plant Nicotiana benthamiana. Induced expression of SCRE1 in rice also inhibits pattern-triggered immunity and enhances disease susceptibility to rice bacterial and fungal pathogens. The immunosuppressive activity is localized to a small peptide region that contains an important ‘cysteine-proline-alanine-arginine-serine’ motif. Furthermore, the scre1 knockout mutant generated using the CRISPR/Cas9 system is attenuated in U. virens virulence to rice, which is greatly complemented by the full-length SCRE1 gene. Collectively, this study indicates that the effector SCRE1 is able to inhibit host immunity and is required for full virulence of U. virens.     

Simple transformation of the rice false smut fungus <Emphasis Type="Italic">Villosiclava virens</Emphasis> by electroporation of intact conidia

A simple procedure is reported for transformation of the rice false smut fungus Villosiclava virens (anamorph: Ustilaginoidea virens) using electroporation of intact conidial cells. The transformation vector pCB1004eGFP was constructed with a green fluorescent protein (eGFP) gene under a constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene of Cochliobolus heterostrophus. When a linearized vector was applied, eGFP-expressing transformants were successfully acquired. An inoculation test in rice plants showed that the eGFP-expressing transformants were able to form rice false smut balls.     

UVI_02019870, a Puptive Effector from <i>Ustilaginoidea virens</i>, Interacts with a Chloroplastic-Like Protein OsCPL1

Ustilaginoidea virens, which causes rice false smut (RFS), is one of the most detrimental rice fungal diseases and poses a severe threat to rice production and quality. Effectors in U. virens often act as a group of essential virulence factors that play crucial roles in the interaction between host and the pathogen. Thus, the functions of individual effectors in U. virens need to be further explored. Here, we found a small secreted hypersensitive response-inducing protein UVI_02019870 was highly conserved in fungi. Furthermore, we performed Y2H and BiFC assay to demonstrated UVI_02019870 interacted with OsCPL1, which was predicted as a chloroplast precursor to regulate chloroplast signaling pathways. Our data provide a theory for gaining an insight into the molecular mechanisms underlying the UVI_02019870 virulence function.     

Identification and evaluation of potential bio-control fungal endophytes against <Emphasis Type="Italic">Ustilagonoidea virens</Emphasis> on rice plants

False smut disease of rice is posing an increasing concern for production, not only because of the hiking epidemic occurrence in rice production, but also because of the challenging specific pathogenesis of the disease. The aim of this work was to evaluate the potential of five fungal endophytes to reduce negative effects of rice false smut fungus (Ustilagonoidea virens) on rice plants, in both the laboratory and greenhouse. Though all the fungal isolates showed the ability to inhibit the growth of U. virens with varying degrees, isolate E337 showed significant antagonistic activity against the pathogenic fungi. The isolate E337 was identified as Antennariella placitae by molecular and morphological data analysis including 18S rDNA sequence analysis. This isolate showed a significant in vitro inhibition of mycelial growth of U. virens by dual culture method and it was subsequently tested for its in vivo biocontrol potential on false smut disease on rice plants. Greenhouse experiments confirmed that applications of conidia of A. placitae protected rice plants by improving rice yield and by decreasing the severity of false smut disease on susceptible rice plants. This is the first report where A. placitae has been identified as a biocontrol organism.     

Teliospore development of Narasimhania alismatis in artificial culture

Development of teliospores interlaced by dermata in clathroid spore balls by the aquatic smut Narasimhania alismatis Pavgi & Thirumalachar was achieved in artificial culture. The spores are morphologically similar to the spore balls in the host (Alisma reniforme D. Don.) leaf. The teliospore development is not conditioned by any nutritrional factor(s) and occurs also in the rich marshy soil,adding another source of primary inoculum     

Development and validation of microsatellite markers for Karnal bunt (Tilletia indica) and loose smut (Ustilago segetum tritici) of wheat from related fungal species

Simple sequence repeats (SSRs) are preferred molecular markers because of their abundance, robustness, high reproducibility, high efficiency in detecting variation and suitability for high‐throughput analysis. In this study, an attempt was made to mine and analyse the SSRs from the genomes of two seed‐borne fungal pathogens, viz Ustilago maydis, which causes common smut of maize, and Tilletia horrida, the cause of rice kernel smut. After elimination of redundant sequences, 2,703 SSR loci of U. maydis were identified. Of the remaining SSRS, 44.5% accounted for di‐nucleotide repeats followed by 29.8% and 2.7% tri‐ and tetranucleotide repeats, respectively. Similarly, 2,638 SSR loci were identified in T. horrida, of which 20.2% were di‐nucleotide, 50.4% tri‐ and 20.5% tetra‐nucleotide repeats. A set of 65 SSRs designed from each fungus were validated, which yielded 23 polymorphic SSRs from Ustilago and 21 from Tilletia. These polymorphic SSR loci were also successfully cross‐amplified with the Ustilago segetum tritici and Tilletia indica. Principal coordinate analysis of SSR data clustered isolates according to their respective species. These newly developed and validated microsatellite markers may have immediate applications for detection of genetic variability and in population studies of bunt and smut of wheat and other related host plants. Moreover, this is first comprehensive report on molecular markers suitable for variability studies in wheat seed‐borne pathogens.     

Microbial secondary metabolite induction of abnormal appressoria formation mediates control of rice blast disease caused by Magnaporthe oryzae

Okinawa, the only subtropical area in Japan with numerous island ecosystems, is expected to have diverse microbial resources. Recently, we reported the construction of a culture filtrate library with microbes originally isolated from soils in Okinawa, including the Yaeyama Archipelago, and validated its phylogenetic diversity. In the present study, we investigated the inhibitory effect of the cell extract (CE) from microbial isolate 3–45 against Magnaporthe oryzae in rice (Oryza sativa). Abnormal appressorium formation by M. oryzae was induced in the presence of the CE from isolate 3–45. Additionally, melanization of appressoria was inhibited in the presence of CE from isolate 3–45. Sequence analysis of the 16S rDNA region of isolate 3–45 indicated that it shared similarities with Streptomyces erythrochromogenes. When rice leaves were inoculated with M. oryzae in the presence of CE from isolate 3–45, blast lesion formation was inhibited compared to leaves treated with M. oryzae in the absence of CE from isolate 3–45. In addition, M. oryzae infective activity was significantly inhibited in rice leaf sheaths treated with CE from isolate 3–45. Furthermore, abnormal appressorium formation was observed in the presence of heat‐treated CE from isolate 3–45. These results suggest that CE from isolate 3–45 can protect rice from blast disease caused by M. oryzae. Further studies are required to identify the active compounds present in 3–45‐CE and to clarify its mechanism of inhibition in full detail. The present study on isolate 3–45 might contribute to the development of a new fungicide for controlling rice blast disease caused by M. oryzae.     

稻曲病两个白化菌株的分离与生物学特性

为了筛选带有自然标记的稻曲病菌菌株,2010年从浙江省象山县和陕西省勉县采集和分离到2个稻曲病白化菌株,ZJa0201和SXa0101。它们在PSA培养基上的生长速度约为其他稻曲病菌株的3倍,未见产生厚垣孢子;在PS培养基上只能产生少量分生孢子。rDNA-ITS和rDNA-IGS序列分析表明,两个白化菌株也与稻曲病菌已知所有菌株的ITS序列同源性高于99.6%;rDNA-IGS序列也属于最为常见的类型,含有2个77bp的重复单元序列。由此推断,这两个白化菌株属于稻曲病菌产孢退化的突变体。白化菌株在PSA上     

2,6‐Dimethoxy‐1,4‐Benzoquinone Enhances Resistance Against the Rice Blast Fungus Magnaporthe oryzae

We investigated the effect of 2,6‐dimethoxy‐1,4‐benzoquinone (DMBQ) on induced resistance to Magnaporthe oryzae in rice. DMBQ concentrations greater than 50 μg/ml inhibited spore germination and appressorium formation in M. oryzae. When rice leaves pretreated with 10 μg/ml DMBQ, which did not show antifungal activity against spore germination and appressorium formation of M. oryzae, were inoculated with M. oryzae spores 5 days after DMBQ pretreatment, blast lesion formation was inhibited compared with control leaves pretreated with distilled water. In addition, infection‐inhibiting activity against M. oryzae was significantly enhanced in rice leaf sheaths pretreated with 10 μg/ml DMBQ. H₂O₂ generation was observed in rice leaves pretreated with DMBQ, and PAL, POX, CHS and PR10a were significantly expressed in these leaves. These results suggested that DMBQ can protect rice from blast disease caused by M. oryzae.     

Spore Liberation and Dispersal in Smut Fungi*

Teliospores are the most important diaspores of smut fungi, albeit not the only ones. The role of basidla, basidiospores, secondary spores, yeast cells, and infected parts of the host for dispersal has often been neglected. Many smut species have soral structures like galls, peridia, and elaters, which cause teliospores to be liberated over prolonged periods. This increases the chance that at least some spores are released under favourable wet climatic conditions and while host plants are susceptible. In this review, the diversity of dispersal units as well as vectors of smut fungi are presented. The importance of timing of diaspore liberation, flexibility in dispersal strategies, and the genetic and evolutionary implications of dispersal strategies of smut fungi are discussed. The general considerations are complemented by examples based on original field and laboratory observations: peridia of Farysia corniculata and certain species of Sporisorium expose the spore mass by hygroscopic movement under wet conditions (hygrochasy) favourable for teliospore germination and infection of a host plant. Basidia with firmly attached basidiospores liberated from spore balls of Doassansiopsis deformans, branched basidiospores of Rhamphospora nymphaeae, needle-shaped basidiospores of species of Entyloma, folded basidiospores of Mycosyrinx cissi, and stellate groups of yeast cells of Trichocintractia utriculicola show enlarged surfaces, which are advantageous for dispersal in water. Galls filled with spore balls of Doassansiopsis limnocharidis and witches' brooms formed by spikelets infected by Cintractia standleyana separate from the host and fall into water where they are dispersed.