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.     

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.     

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。     

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.     

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。     

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.     

Intercellular invasion of rice roots at the seedling stage by the rice false smut pathogen, <Emphasis Type="Italic">Villosiclava virens</Emphasis>

False smut is a serious disease affecting rice production worldwide. Initial infection of rice seedlings by Villosiclava virens was clarified using axenically cultured chlamydospores to inoculate rice roots. Chlamydospores were found on rice roots at 1 day post inoculation (dpi), and were germinating at 1–4 dpi. At 4 dpi, the infection germ tube had invaded the intercellular space between epidermal rice root cells. Between 5 and 11 dpi, branching and fusion-like structures were observed that may contribute to the establishment of the hyphal network on the root surface.     

Infection process of <Emphasis Type="Italic">Pseudocercospora musae</Emphasis> on banana leaf

Yellow Sigatoka that is caused by Pseudocercospora musae is an important banana disease. The aim of this study was to elucidate the infection process of P. musae in banana leaves by scanning electron microscopy. Leaf samples were inoculated on the abaxial surface with P. musae and then analysed at 12, 24, 36, 48, 72, 96, 120, 144, and 168 h post inoculation (hpi) and at 36 and 50 days post inoculation (dpi). The conidia were found to be germinated between 24 and 36 hpi and penetrated through the stomata between 96 and 120 hpi, or more generally from 144 hpi. P. musae colonized the spongy parenchyma at 36 dpi and the palisade parenchyma at 50 dpi. Sporulation occurred at 50 dpi on the adaxial surface of leaves through the emergence of conidia on conidiophores through the stomata. Considering the importance of yellow Sigatoka in banana production, our results provide a better understanding of the life cycle of the fungus for treatment processes.     

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.     

Effects of panicle development stage and temperature on rice panicle blast infection by Magnaporthe oryzae and visualization of its infection process

Panicle blast, caused by the fungus Magnaporthe oryzae (syn. Pyricularia oryzae), directly contributes to yield loss in the field. The effects of panicle development stage and temperature on panicle blast were studied and the infection process of M. oryzae in panicles was visualized. Rice panicles at different development stages from three rice cultivars were inoculated with a conidial suspension in vitro. The rice cultivar Lijiangxintuanheigu was highly susceptible to panicle blast at 5 days postinoculation (dpi) when the pulvinus distance was 15–20 cm. Nanjing 9108 was moderately susceptible to panicle blast when the pulvinus distance was 8–10 cm, but Yliangyou 800 was resistant. The effect of temperature on panicle blast was determined under 22–35 °C temperature treatments. Inoculated panicles placed at temperatures of 28 and 30 °C showed the highest lesion grade based on lesion length at 5 dpi. The infection process of M. oryzae in rice panicles was observed by confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM). M. oryzae initially formed the appressorium to invade through the epidermis of rice panicles at 24 hours postinoculation (hpi). As the disease progressed, the invasive hyphae formed dense mycelial networks in the inner parenchyma cells at 60 hpi. Our results will contribute to the understanding of panicle development stage and temperature effects on panicle blast and improve resistance evaluation methods. Additionally, visualization of the infection process by CLSM and TEM are valuable methods to observe M. oryzae invasive hyphae inside rice panicle cells.     

Histology of waxflower (Chamelaucium spp.) flower infection by Botrytis cinerea

Botrytis cinerea infects waxflower (Chamelaucium spp.) flowers and can induce them to abscise from their petioles before disease becomes evident. Botrytis cinerea infection of flowers was studied on two waxflower cultivars by light and electron microscopy. Pot‐grown waxflower flowers were harvested, inoculated with aqueous suspensions of B. cinerea conidia, incubated at 20–22°C and >95% RH and examined within 96 h post‐inoculation (hpi). Conidial germination on petals started 4 hpi, penetration via germ tube tips was 6 hpi and protoappressoria were formed 8 hpi. Germination on petals approximately doubled every 4–6 h to 18 hpi. Conidial germination was ca. 50% at 22–24 hpi. Botrytis cinerea infected most waxflower flower organs, including petals, anthers and filaments, stigma and hypanthium, within 24 hpi. Hyaline and lobate appressoria were observed 36 hpi. Infection cushions on stamen bases were formed 36 hpi by saprophytic hyphae that originated from anthers. This infection process can give rise to tan‐coloured symptoms typical of botrytis disease that radiate from this part of the flower. Subcuticular hyphae were present at high density near stamen bases and evidently resulted from multiple penetrations from single infection cushions. The subcuticular hyphae grew within the hypanthium and towards the centre of the floral tube. When flower abscission occurred, floral tube tissues close to the abscission zone remained uninfected. This observation infers possible transmission of a signal (e.g. ethylene) upon B. cinerea infection. Thus, B. cinerea causes flower abscission apparently as a defence response.     

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.     

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.     

Infection process of <Emphasis Type="Italic">Gilbertella persicaria</Emphasis> in papaya (<Emphasis Type="Italic">Carica papaya</Emphasis> L.) fruits

Gilbertella persicaria is a pathogenic fungus recently reported as a causative agent of soft rot in papaya fruits. Here the interactions between G. persicaria and papaya fruits was analyzed under laboratory conditions using histological techniques and optical microscopy to elucidate the process of pathogenesis. Healthy and disinfested fruits of papaya cv. Maradol were also inoculated with a suspension of sporangiospores of G. persicaria. Tissue sections were cut, which were subjected to differential staining with safranin-fast green for different times. Sporangiospores presumably adhered to the cuticle of the fruit by 3 h post inoculation (hpi) and germinated by 6 hpi; invasive intracellular hyphae were growing in host cells by 9 hpi. By 15 hpi, fruit epidermis was macerated, presumably by enzymatic activity reported for mucoral fungal species and appeared as a wet-looking lesion on the cuticle. Fruit mesocarp was colonized by 30 hpi, and asexual reproduction structures had formed by 48 hpi. This process of infection and disease development of G. persicaria in papaya fruits corresponds to that used by pathogens with a necrotrophic lifestyle.     

Comparison between the resistance to blast in panicles exserted from the main culm and primary tillers as measured in six rice varieties

Three rice japonica varieties Jiajing3768, Xiushui09, and Xiushui63 and three rice indica varieties Qianyou930, Liangyoupeijiu, and Liangyou6326 were used to analyze the resistance to neck blast, comparing panicles excised from the main culm and from the first primary tiller. Disease incidence in the necks, disease incidence in the rachis nodes, lesion length in the necks, and number of conidia in the necks were measured after artificial inoculation of excised neck pieces. Resistance to neck blast was higher in the panicles excised from the main culm than from the first primary tiller for the varieties Jiajing3768, Xiushui63, and Liangyou6326. However, the opposite was true for Xiushui09 and Liangyoupeijiu. In Qianyou930, the type of panicles had no significant influence on the resistance to neck blast.     

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.     

Geographic distribution of false smut disease of rice in India and efficacy of selected fungicides for its management

False smut disease of rice caused by the fungus Ustilaginoidea virens, has become a serious production constraint in various rice-growing regions of India. Analysis of rice false smut disease survey data of last 24 years revealed significant changes in the intensity of the disease in various rice-growing regions of India over the years. We evaluated three fungicide formulations viz., trifloxystrobin-tebuconazole (TT) combination (0.04%), kresoxim methyl (KM) (0.1%) and propiconazole (PC) (0.1%) for their effectiveness against false smut disease of rice. Under in vitro condition, TT and PC completely inhibited the fungal growth while KM inhibited the growth by 80%. We evaluated the efficacy of these chemicals in a multi-location trial in 3 hot spot locations, consecutively for three years. A single fungicide spray was given in 3 different crop stages (booting, 50% panicle emergence and 100% panicle emergence). The results indicated that all the treatments significantly reduced the percentage of both infected panicles and spikelets in all the three locations. Both TT and PC were most effective in reducing the disease intensity and increasing the yield when applied either in booting or in 50% panicle emergence stage.     

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.