False Smut Balls Research Articles

Rice False Smut Fungus, <i>Ustilaginoidea virens</i>, Inhibits Pollen Germination and Degrades the Integuments of Rice Ovule

Ustilaginoidea virens is a flower-infecting fungus that forms false smut balls in rice panicle. Rice false smut has long been considered a minor disease, but recently it occurred frequently and emerged as a major disease in rice production. In vitro co-cultivation of U. virens strain with young rice panicles showed that U. virens enters inside of spikelets from the apex and then grows downward to infect floral organs. In response to U. virens infection, rice host exhibits elevated ROS accumulation and enhanced callose deposition. The secreted compounds of U. virens can suppress rice pollen germination. Examination of sectioning slides of freshly collected smut balls demonstrated that both pistil and stamens of rice flower are infected by U. virens, hyphae degraded the contents of the pollen cells, and also invaded the filaments. In addition, U. virens entered rice ovary through the thin-walled papillary cells of the stigma, then decomposed the integuments and infected the ovary. The invaded pathogen could not penetrate the epidermis and other layers of the ovary. Transverse section of the pedicel just below the smut balls showed that there were no fungal hyphae observed in the vascular bundles of the pedicel, implicating that U. virens is not a systemic flower-infecting fungus.

Determination and Analysis of Ustiloxins A and B by LC-ESI-MS and HPLC in False Smut Balls of Rice

Ustiloxins are cyclopeptide mycotoxins produced by the pathogenic fungus Villosiclava virens of rice false smut. Ustiloxins A and B as two main mycotoxins were determined conveniently by LC-ESI-MS in the water extract from rice false smut balls which were mostly composed of the chlamydospores and mycelia of the pathogen. Both ustiloxins A and B in the water extract were also quantitatively analyzed by HPLC. This is the first report on the determination and analysis of ustiloxins A and B simultaneously by LC-ESI-MS and HPLC in false smut balls of rice.

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.

Morphological structure of propagules and electrophoretic karyotype analysis of false smut Villosiclava virens in rice

The target pathogen Villosiclava virens (teleomorph: claviceps oryzae-sativae) was isolated from the infected rice, where it caused false smut. In our study, the forming processes of the chlamydospores, chlamydospore balls, conidiospores, and secondary conidiospores during the asexual reproduction were observed more precisely and in greater detail than previous descriptions. The microstructure of the infected rice kernel showed that the outer dense chlamydospores piled around the false smut balls grown on XBZ medium; moreover the sclerotia consisting of dense mycelium were found. The different morphology was observed across the different growing conditions. In addition, we observed the nuclear numbers of both the conidiospores and hyphae using 4',6-diamidino-2-phenylindole (DAPI) staining. Because the fungus has small chromosomes and the numbers were not previously known, we analyzed the electrophoretic karyotype using a pulsed field gel electrophoresis (PFGE) technique. The results showed that V. virens has at least 10 chromosomes ranging in size from 0.6 kb to 6 Mb. The V. virens genome size is estimated to be 23 Mb. Here, we report the morphological characteristics of the fungus and the process of asexual spores forming asexual propagules, along with the first analyze the molecular karyotype of V. virens. These results supply a foundation for further study of the pathogenicity and biology of this devastating pathogen.

イネ稲こうじ病粒のウスチロキシンAの含有量における菌株間の差異

A protocol to quantify ustiloxin A (ustA) in a bulk sample of false smut balls of rice (J. Vet. Med. Sci. 71: 239-241) was modified to quantify ustA in single balls collected from a paddy field after natural infection and in balls formed after rice plants were inoculated with either of two isolates of Ustilaginoidea virens isolated from different commercial fields in Fukui Prefecture. To test whether these isolates differed in their capacity to produce ustA, we compared the mean quantity of ustA that each produced in individual balls. The two isolates varied greatly in ustA production; the differences in ustA in balls between the two ranged from 42-fold to 115-fold. Such massive differences in ustA production indicate that the pathogenic isolate is an important contributing factor to the quantity of ustA produced in false smut balls.

Simple transformation of the rice false smut fungus Villosiclava virens 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 vectorwas 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.

Simple transformation of the rice false smut fungus <i>Villosiclava virens</i> 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 vectorwas 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.

Quantification of the rice false smut pathogen Ustilaginoidea virens 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 (CT) 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.

Phytoalexins and endophyte related mycotoxins as hazards to the animal industry

Phytoalexins, compounds produced by plants in response to pathogens, and biologically active metabolites produced by endophyte are beneficial to plant growth. There are many trials to utilize these biologically active substances for plant disease control. On the other hand, some of these substances are toxic to mammals. We experienced ryegrass staggers of cattle caused by endophyte infected ryegrass straw. In this review, toxicities of phytoalexins and endophyte related substances to animals are overviewed. Furthermore, toxic substances found in false smut balls in rice plant are cited.

A Convergent Total Synthesis of Ustiloxin D via an Unprecedented Copper-Catalyzed Ethynyl Aziridine Ring-Opening by Phenol Derivatives

[reaction: see text] The ustiloxins are a family of heterodetic cyclopeptides that have been isolated from the water extracts of false smut balls on the panicles of rice plants caused by the fungus Ustilaginoidea virens. A concise total synthesis of ustiloxin D has been achieved via an unprecedented ethynyl aziridine ring-opening of phenol derivatives. The longest linear sequence of the synthesis is 15 steps from commercially available compounds.

Interaction of ustiloxin a with bovine brain tubulin

Ustiloxin A is a modified peptide derived from false smut balls on rice panicles, caused by the fungus Ustilaginoidea virens; structurally, it resembles phomopsin A. Ustiloxin A is cytotoxic and is an inhibitor of microtubule assembly in vitro. Because of its resemblance to phomopsin A, we examined its interaction with tubulin and compared the results with those obtained with phomopsin A and dolastatin 10, both of which were found previously to have very similar effects. We determined that ustiloxin A inhibited the formation of a particular intra-chain cross-link in β-tubulin, as do vinblastine, maytansine, rhizoxin, phomopsin A, dolastatin 10, halichondrin B and homohalichondrin B; this is in contrast to colchicine and podophyllotoxin which do not inhibit formation of this cross-link. Ustiloxin A also inhibited the alkylation of tubulin by iodo[ 14C]acetamide, as do phomopsin A and dolastatin 10; vinblastine was almost as potent an inhibitor of alkylation as ustiloxin A, whereas maytansine, halichondrin B and homohalichondrin B have little or no effect. In addition, ustiloxin A inhibited exposure of hydrophobic areas on the surface of the tubulin molecule. In this respect, ustiloxin A was indistinguishable from phomopsin A but slightly more effective than dolastatin 10 and considerably more effective than vinblastine; this provides a strong contrast to maytansine, rhizoxin, and homohalichondrin B which have no effect on exposure of hydrophobic areas and to halichondrin B which enhances exposure. Lastly, ustiloxin A strongly stabilized the binding of [ 3H]colchicine to tubulin. The combination of ustiloxin A with colchicine stabilized tubulin with a half-life of over 8 days, comparable with results obtained with phomopsin A and colchicine. A comparison of the structures of ustiloxin A, phomopsin A and dolastatin 10 raised the possibility that the strong stabilization of the tubulin structure may require a short segment of hydrophobic amino acids such as the modified valine-isoleucine sequence present in all three compounds. The rest of the structure, specifically the large ring of ustiloxin A and phomopsin A, may serve to place this sequence in an appropriate conformation to interact with tubulin.

Ustiloxins, antimitotic cyclic peptides from false smut balls on rice panicles caused by Ustilaginoidea virens.

Ustiloxins A (1a), B (1b), C (1c), D (1d) and E (1e), antimitotic peptides, have been isolated from the water extract of false smut balls caused on the panicles of rice plant by a fungus Ustilaginoidea virens. The structure of 1b was assigned from its spectral property and its amino acid analysis in relation to 1a whose structure was determined previously by a combination of X-ray crystallographic and amino acid analyses. Structures of 1c and 1d were elucidated by their spectroscopic data, specially based on their 1H and 13C NMR spectra. Bioactivities of these compounds against microtubule assembly as well as mammal, plant and fungal cells have been studied.

"Lupinosis"-like lesions in mice caused by ustiloxin, produced by Ustilaginoieda virens: a morphological study.

A crude toxin, obtained by methanol-extraction of a water extract of false smut balls (INA-KOUJI in Japanese) caused by Ustilaginoidea virens on rice panicles, was injected into mice intraperitoneally. Single injection of 100 or 200 mg/kg body weight of the extract was not lethal but caused acute, occasional necrosis of hepatocytes and renal tubular cells, followed by increased number of mitotic figures with occasional multinuclear giant cells. Erosions and ulceration of the forestomach and atrophy of the thymus were observed a week later. Repeated intraperitoneal injection of the crude toxin at the levels of 12 and 25 mg/kg body weight induced more severe necrosis of the liver and kidneys, with delayed occurrence of mitosis. Forestomach erosion also occurred. Serial injection for 10-12 days of either 3 or 6 mg/kg of the crude extract or 400 micrograms/kg of ustiloxin A, using the purified crystals, caused relatively mild but definite liver and kidney lesions similar to those described above. The lesions in the liver and kidney were quite similar to those observed in lupinosis caused by phomopsin A, a mycotoxin produced by Phomopsis leptostromiformis. Isolation of the toxic substance indicates that the contaminated rice panicles may cause toxicosis of cattle, although no field outbreaks have occurred yet.

Ustiloxin: a phytotoxin and a mycotoxin from false smuth balls on rice panicles

Ustiloxin( 1), an antimitotic tetrapeptide containing a 13-membered ring including an ether linkage, was isolated from the water extract of false smut balls caused by Ustilaginoidea virens on the panicles of rice plant. The absolute structure was determined by a combination of X-ray crystallographic and amino acid analyses. Its structure and biological activity are closely related to those of phomopsin A, a mycotoxin to cause the lupinosis.