**Abstract**

Rice false smut (RFS) is the most important grain disease in rice production worldwide. Its epidemics not only lead to yield loss but also reduce grain quality because of multiple mycotoxins generated by the causative pathogen, *Villosiclava virens* (anamorph: *Ustilaginoidea virens*). The pathogen infects developing spikelets and specifically converts individual grain into a RFS ball that is established from mycelia covered with powdery chlamydospores, sometimes generating sclerotia. RFS balls seem to be randomly formed in some grains on a panicle of a plant in the paddy field. However, epidemics differ largely among varieties, fields, and seasons. This chapter introduces current understanding on the disease, mycotoxins, the biology of the pathogen, pathogenesis of RFS, rice resistance, the disease cycle, the disease control, and assay.

**Keywords:** basal defense, biotroph, effector, epiphytic growth, grain filling gene, mycotoxin, sclerotium

### **1. Introduction**

Rice production plays a crucial role in our food security. Rice security is not only an economic issue but also an important parameter to determine social and political stability [1]. Thus, rice research has to be geared up to develop strategies for alleviating losses due to pests and diseases. In the past decades, a number of minor diseases have attained the status of major importance in rice. One such disease is the rice false smut (RFS) disease that is a threat to yield and grain quality.

RFS was previously recorded as a minor disease of rice and considered as a symbol of good harvest in old times. In recent years, increasing occurrence of RFS has been reported in most major rice growing regions throughout the world, such as China, India, and USA [2–5]. The emergence of this disease is believed to be partially due to wide application of hybrid rice varieties, which are mostly susceptible to the RFS. The causative agent of RFS is an ascomycete fungal pathogen *Villosiclava virens* (anamorph: *Ustilaginoidea virens* [Cooke] Takahashi) [6], which specifically infects rice flowers and transforms the latter into RFS balls [3]. RFS balls are small at first growing slowly and enclosing the floral parts. The early balls were found to be slightly flattened and smooth and were covered by a thin membrane. As the pathogen growth intensifies, the RFS ball bursts with chlamydospores and becomes orange then later yellowish-green or greenish-black (**Figure 1A**–**C**). The RFS balls generate sclerotia (**Figure 1D**) when the temperature difference between day and night is large in autumn [3]. RFS ball is the only visible symptom of RFS disease.

#### **Figure 1.**

*Disease symptom of rice false smut: (A)–(C) white, yellow, and dark green false smut balls at early, middle, and late stages, respectively; (D) sclerotia (white arrows) are formed in false smut balls at the late stage; (E) field view of rice false smut disease; and (F) harvested rice grains are contaminated with rice false smut balls. Inset shows that rice grains are covered by chlamydospores from false smut balls.*

The disease induces considerable losses both in yield and quality [7, 8], due to the occurrence of RFS balls and increased sterility of kernels adjacent to the balls [9]. Moreover, RFS balls produce two types of mycotoxins, i.e., ustiloxin and ustilaginoidin, which are poisonous to both humans and animals and impose significant health hazards by contaminating rice grains and straws [10–12]. For example, ustiloxin A causes kidney and liver damage in mice, due to its inhibition activity on microtubule assembly and skeleton formation of the eukaryotic cells [11, 13].

RFS balls seem to be randomly formed in some grains of rice panicles in the paddy field and are inevitably collected during harvest (**Figure 1E, F**). The disease spread varies within a field or between fields and is considered to be more severe in the proximity of drainage channels [14]. Epidemics of RFS disease tend to occur when rice booting and heading stages meet with rainfall periods. However, epidemics differ largely among varieties, fields, and seasons. This chapter describes our current knowledge on the mycotoxins, biology of the pathogen, pathogenesis of RFS, rice resistance, disease cycle, disease control, and disease assay.

### **2. Mycotoxins**

*V. virens* produces two kinds of mycotoxins, ustiloxins and ustilaginoidins. Ustiloxins are water-soluble cyclic peptides, each including a 13-membered ring with an ether linkage [10]. The structures of six ustiloxins, including A–D, F, and G, have been identified so far [10, 15, 16]. A gene cluster has been suggested to be responsible for the ribosomal biosynthesis of ustiloxins [17]. Ustiloxins A and B are

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*Rice False Smut: An Increasing Threat to Grain Yield and Quality*

among the most abundant ustiloxins in RFS ball and are mainly contained in the middle layer of mycelia and immature chlamydospores at early maturity stage [18]. Recently, rapid qualitative or quantitative detection methods, such as monoclonal antibody-based enzyme-linked immunosorbent assay and colloidal gold-based lateral flow immunoassay, have been established for detecting ustiloxins A and B in rice and feed samples [19, 20]. Ustilaginoidins are bis-naphtho-γ-pyrones and can be easily dissolved in organic solvent. Twenty-five ustilaginoidins (A-W) have been isolated from *V. virens* [21–25]. Ustilaginoidins can be isolated from RFS balls and solid rice media culturing *V. virens*. Several tested ustilaginoidins are mainly distributed in the layers embracing chlamydospores of yellowish-green or dark-

Ustiloxins could inhibit polymerization of microtubule proteins and cause abnormal mitosis resembling, which would result in acute necrosis of renal tubular cells and hepatocytes in mice [10]. Ustiloxins also show phytotoxicity, inhibiting elongation of radicle and germ and inducing swelling of seedling root in rice [10, 16]. Cytotoxic activities of ustiloxins have been demonstrated on human tumor cell lines, such as A375, A549, BGC-823, HCT116, and HepG2 [10, 16]. Similar phytotoxic and cytotoxic activities have been detected for ustilaginoidins [25, 27, 28]. In addition, ustilaginoidins show antibacterial activities against several human or plant pathogens, such as *Agrobacterium tumefaciens*, *Bacillus subtilis*, *Pseudomonas lachrymans*, *Ralstonia solanacearum*, *Staphylococcus haemolyticus*, and *Xanthomonas vesicatoria* [24]. Due to their anti-tumor and/or antibacterial abilities, ustiloxins and ustilaginoidins may be used as potential clinical medications. As a result, chemical

synthesis of ustiloxins and their analogs has been carried out [29, 30].

The RFS pathogen belongs to the kingdom: Fungi, phylum: Ascomycota, class: Ascomycetes, subclass: Sordariomycetes, order: Hypocreales, family: Clavicipitaceae, genus: *Villosiclava,* and species: *virens* and its anamorphic stage is *Ustilaginoidea virens* [6]. The colony growth of *V. virens* in culture medium PSA (potato-sucrose-agar) is very slow, with a growth rate of approximately 20 mm in diameter per week [31]. *V. virens* produces pigments during culture in PSA and is prone to generate small colonies and plenty of conidia in PSB (potato-sucrosebroth) (**Figure 2A**–**D**). The conidia are elliptical with diameters ranging from 3 to 5 μm (**Figure 2E**). Upon maturation or under unfavorable conditions, conidia may develop to rounded chlamydospores with prominent spines on the surface (**Figure 2F**) [32–34]. One or two sclerotia, which are the sexual structure of *V. virens*, can be formed in a RFS ball (**Figure 1D**). Sclerotia are horseshoe-shaped and the length ranged from 2 to 20 mm (**Figure 2G**). After several months of dormancy, sclerotia could germinate and produce fruiting bodies with stromata (**Figure 2F**), which ultimately generates ascospores with length reaching 50 μm

Numerous efforts have been undertaken to optimize the culture media and culturing conditions for *V. virens*. PSA and PDA (potato-dextrose-agar) are suitable for culturing *V. virens* in solid media [31]. Moreover, stachyose is a preferential carbon source for *V. virens* and could significantly promote hyphal growth and conidia germination of *V. virens*, much better than other sugars, such as sucrose, glucose, and starch [36]. Stachyose can be also applied in optimization of culture medium for other filamentous fungi. Ammonium chloride, ammonium sulfate, and ammonium nitrate are the suitable nitrogen sources for *V. virens* growth [31]. The

optimal growth of *V. virens* can be achieved at 28°C and pH 6–7 [37].

*DOI: http://dx.doi.org/10.5772/intechopen.84862*

green RFS balls [26].

**3. Biology of the pathogen**

and width 1 μm [35].

*Rice False Smut: An Increasing Threat to Grain Yield and Quality DOI: http://dx.doi.org/10.5772/intechopen.84862*

*Protecting Rice Grains in the Post-Genomic Era*

The disease induces considerable losses both in yield and quality [7, 8], due to the occurrence of RFS balls and increased sterility of kernels adjacent to the balls [9]. Moreover, RFS balls produce two types of mycotoxins, i.e., ustiloxin and ustilaginoidin, which are poisonous to both humans and animals and impose significant health hazards by contaminating rice grains and straws [10–12]. For example, ustiloxin A causes kidney and liver damage in mice, due to its inhibition activity on microtubule assembly and skeleton formation of the eukaryotic cells [11, 13]. RFS balls seem to be randomly formed in some grains of rice panicles in the paddy field and are inevitably collected during harvest (**Figure 1E, F**). The disease spread varies within a field or between fields and is considered to be more severe in the proximity of drainage channels [14]. Epidemics of RFS disease tend to occur when rice booting and heading stages meet with rainfall periods. However, epidemics differ largely among varieties, fields, and seasons. This chapter describes our current knowledge on the mycotoxins, biology of the pathogen, pathogenesis of

*Disease symptom of rice false smut: (A)–(C) white, yellow, and dark green false smut balls at early, middle, and late stages, respectively; (D) sclerotia (white arrows) are formed in false smut balls at the late stage; (E) field view of rice false smut disease; and (F) harvested rice grains are contaminated with rice false smut* 

*balls. Inset shows that rice grains are covered by chlamydospores from false smut balls.*

RFS, rice resistance, disease cycle, disease control, and disease assay.

*V. virens* produces two kinds of mycotoxins, ustiloxins and ustilaginoidins. Ustiloxins are water-soluble cyclic peptides, each including a 13-membered ring with an ether linkage [10]. The structures of six ustiloxins, including A–D, F, and G, have been identified so far [10, 15, 16]. A gene cluster has been suggested to be responsible for the ribosomal biosynthesis of ustiloxins [17]. Ustiloxins A and B are

**90**

**2. Mycotoxins**

**Figure 1.**

among the most abundant ustiloxins in RFS ball and are mainly contained in the middle layer of mycelia and immature chlamydospores at early maturity stage [18]. Recently, rapid qualitative or quantitative detection methods, such as monoclonal antibody-based enzyme-linked immunosorbent assay and colloidal gold-based lateral flow immunoassay, have been established for detecting ustiloxins A and B in rice and feed samples [19, 20]. Ustilaginoidins are bis-naphtho-γ-pyrones and can be easily dissolved in organic solvent. Twenty-five ustilaginoidins (A-W) have been isolated from *V. virens* [21–25]. Ustilaginoidins can be isolated from RFS balls and solid rice media culturing *V. virens*. Several tested ustilaginoidins are mainly distributed in the layers embracing chlamydospores of yellowish-green or darkgreen RFS balls [26].

Ustiloxins could inhibit polymerization of microtubule proteins and cause abnormal mitosis resembling, which would result in acute necrosis of renal tubular cells and hepatocytes in mice [10]. Ustiloxins also show phytotoxicity, inhibiting elongation of radicle and germ and inducing swelling of seedling root in rice [10, 16]. Cytotoxic activities of ustiloxins have been demonstrated on human tumor cell lines, such as A375, A549, BGC-823, HCT116, and HepG2 [10, 16]. Similar phytotoxic and cytotoxic activities have been detected for ustilaginoidins [25, 27, 28]. In addition, ustilaginoidins show antibacterial activities against several human or plant pathogens, such as *Agrobacterium tumefaciens*, *Bacillus subtilis*, *Pseudomonas lachrymans*, *Ralstonia solanacearum*, *Staphylococcus haemolyticus*, and *Xanthomonas vesicatoria* [24]. Due to their anti-tumor and/or antibacterial abilities, ustiloxins and ustilaginoidins may be used as potential clinical medications. As a result, chemical synthesis of ustiloxins and their analogs has been carried out [29, 30].
