**3. Biology of the pathogen**

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 and width 1 μm [35].

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].

### **Figure 2.**

*Morphology of the rice false smut pathogen. Colony of Ustilaginoidea virens (anamorph) in PSA medium from top view (A) and back view (B). (C) Colonies of U. virens in PSB. (D) A single colony of U. virens in PSB. (E) Conidia of U. virens in PSB. (F) Chlamydospores of U. virens from false smut balls. (G) Sclerotia of Villosiclava virens (telemorph). (H) Sclerotia of V. virens germinate to produce fruit bodies. Images (G) and (H) are courtesy of Prof. Dongwei Hu from Zhejiang University, China.*
