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     

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.     

First Report of the Occurrence of a White Smut Infecting Rice in Arkansas

False smut has recently emerged as an important disease of rice in Arkansas. In 2011, 2012 and 2013, spore balls of a white smut similar to the spore balls of false smut were observed in rice fields in eastern Arkansas. As a white false smut was previously reported in China and Japan, we examined the morphology of chlamydospores and spore balls from some of the infected heads and used selected regions of the rDNA to determine the identity of the causal agent of the disease. We also tested the virulence of an isolate of the white smut to two rice cultivars commonly grown in Arkansas. Our results indicate that the morphology of the spore balls, chlamydospores and conidia is similar to those reported for Ustilaginoidea albicans. However, sequences of ribosomal DNA amplicons indicate a high degree of similarity with both U. virens and U. albicans. The isolate of the white smut was virulent to two rice cultivars, producing spore balls similar to those observed in the field and to those previously described for U. albicans.     

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.     

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。     

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.     

The ‘pears and lemons’ protein UvPal1 regulates development and virulence of Ustilaginoidea virens

Ustilaginoidea virens is an economically important fungus causing a devastating grain disease, rice false smut. An insertional mutagenesis screen was used to explore biological mechanisms underlying infection process of U. virens. T184, a new mutant was identified, with abnormal conidial morphology and deficient virulence. Analysis of the T-DNA inserted gene UvPal1 in the mutant confirmed it as a putative homologue of a cellular morphogenetic protein in yeast, Pal1, whose function has not been well characterized. Deletion of UvPal1 affected hyphal growth, cell morphology, stress adaptation and virulence. UvPal1 could interact with the endocytic proteins, UvEde1 and UvSla2, but was not required for receptor-mediated endocytosis. A yeast two-hybrid (Y2H) analysis was further carried out to screen the UvPal1-interacting proteins, resulting in the identification of 16 putative interacting proteins. Interestingly, UvPal1 interacted with a septin protein, UvCdc11 in vivo and in vitro, and also affected subcellular localization of UvCdc11 protein. Deletion of the four core septins impaired the growth, morphogenesis, stress response and virulence. Collectively, effects on cell morphology, oxidative stress response and virulence are similar to those of UvPal1, suggesting that UvPal1 physically interacts with UvCdc11 to mediate the septin complex to maintain the cellular morphology and virulence of U. virens.     

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.     

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.     

Maize host requirements for Ustilago maydis tumor induction

The biotrophic pathogen Ustilago maydis causes tumors by redirecting vegetative and floral development in maize (Zea mays L.). After fungal injection into immature tassels, tumors were found in all floral organs, with a progression of organ susceptibility that mirrors the sequential location of foci of cell division in developing spikelets. There is sharp demarcation between tumor-forming zones and areas with normal spikelet maturation and pollen shed; within and immediately adjacent to the tumor zone, developing anthers often emerge precociously and exhibit a range of developmental defects suggesting that U. maydis signals and host responses are restricted spatially. Male-sterile maize mutants with defects in anther cell division patterns and cell fate acquisition prior to meiosis formed normal adult leaf tumors, but failed to form anther tumors. Methyl jasmonate and brassinosteroid phenocopied these early-acting anther developmental mutants by generating sterile zones within tassels that never formed tumors. Although auxin, cytokinin, abscisic acid and gibberellin did not impede tassel development, the Dwarf8 mutant defective in gibberellin signaling lacked tassel tumors; the anther ear1 mutant reduced in gibberellin content formed normal tumors; and Knotted1, in which there is excessive growth of leaf tissue, formed much larger vegetative and tassel tumors. We propose the hypothesis that host growth potential and tissue identity modulate the ability of U. maydis to redirect differentiation and induce tumors.     

Mutations associated with floral organ number in rice

How floral organ number is specified is an interesting subject and has been intensively studied in Arabidopsis thaliana. In rice (Oryza sativa L.), mutations associated with floral organ number have been identified. In three mutants of rice, floral organ number 1 (fon1) and the two alleles, floral organ number 2-1 (fon2-1) and floral organ number 2-2 (fon2-2), the floral organs were increased in number centripetally. Lodicules, homologous to petals, were rarely affected, and stamens were frequently increased from six to seven or eight. Of all the floral organs the number of pistils was the most frequently increased. Among the mutants, fon1 showed a different spectrum of organ number from fon2 -1 and fon2 -2. Lodicules were the most frequently affected in fon1, but pistils of more than half of fon1 flowers were unaffected; in contrast, the pistils of most flowers were increased in fon2 -1 and fon2-2. Homeotic conversion of organ identity was also detected at a low frequency in ectopically formed lodicules and stamens. Lodicules and stamens were partially converted into anthers and stigmas, respectively. Concomitant with the increased number of floral organs, each mutant had an enlarged apical meristem. Although meristem size was comparable among the three mutants and wild type in the early phase of flower development, a significant difference became apparent after the lemma primordium had differentiated. In these mutants, the size of the shoot apical meristem in the embryo and in the vegetative phase was not affected, and no phenotypic abnormalities were detected. These results do not coincide with those for Arabidopsis in which clavatal affects the sizes of both shoot and floral meristems, leading to abnormal phyllotaxis, inflorescence fasciation and increased floral organs. Accordingly, it is considered that FON1 and FON2 function exclusively in the regulation of the floral meristem, not of the vegetative meristem.Abbreviation DIC differential interference contrast This work was supported in part by Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science and Culture of Japan.     

Ustilago aldabrensis, a new species from Seychelles, and two other smut fungi on Dactyloctenium

A new smut fungus Ustilago aldabrensis on the grass Dactyloctenium ctenoides is described and illustrated from Aldabra Island in the Seychelles archipelago. It is compared with similar species known on the genus Dactyloctenium. A further two smut fungi infecting this host plant genus, U. dactyloctenii-gigantei and U. idonea are discussed. U. dactyloctenii-gigantei is reported for the first time from Nigeria on a new host plant, D. aegyptium. U. idonea is redescribed, and its nomenclature and geographical distribution are clarified. A key to smut fungi infecting species of Dactyloctenium is provided.     

Specificity and seasonal prevalence of anther smut disease Microbotryum on sympatric Himalayan Silene species

Host sympatry provides opportunities for cross‐species disease transmission and compounded disease effects on host population and community structure. Using the Silene–Microbotryum interaction (the castrating anther smut disease), eleven Himalayan Silene species were assessed in regions of high host diversity to ascertain levels of pathogen specificity. We also investigated disease prevalence, seasonal dynamics of infection and flowering patterns in five co‐blooming Silene species. We identified several new Microbotryum lineages with varying degrees of specialization that is likely influenced by degrees of host divergence and ecological similarities (i.e. shared pollinator guilds). Affected species had 15%–40% of plants infected by anther smut. Flowering was seasonally overlapping among host species (except for the species pair S. asclepiadea and S. atrocastanea), but diseased flowering onset was earlier than healthy plants, leading to dramatic seasonal shifts in observed disease prevalence. Overlapping distributions and flowering provides opportunities for floral pathogen movement between host species, but host specialization may be constrained by the plant phylogenetic relatedness, adaptation to micro‐habitats and difference in pollinator/vector guilds.