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

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.


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][3][4][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.

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

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][33][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].

Infection process of V. virens
The RFS pathogen V. virens specifically attacks rice flowers to form RFS balls, causing economically important disease. The infection process of V. virens in rice flower has been identified cytologically as follows: at late booting stage of rice, spores of V. virens come into contact with developing spikelets and germinate on their surface, or epiphytically grown hyphae reach the surface of developing spikelets. The hyphae could not penetrate the spikelet but extend into the inner space of a spikelet via the gap between the lemma and the palea [38]. After entering, the pathogen primarily infects the stamen filaments intercellularly [39], probably due to loose alignment of cells and flexible cell walls [40]. Lodicules and stigma could also be attacked, although to a lesser extent [39,41]. However, no infection structures, such as appressorium and haustorium, can be detected during infection. Along with time, mycelia grow to enclose all the floral organs, then protrude out of the spikelet, and ultimately form a ball-shape colony covered with chlamydospores. At late stage of infection or in a RFS ball, stamen filaments are replaced by mycelia, but the ovary and lodicules remain intact, suggesting that they may contribute to the formation of RFS ball [42]. In addition, the hyphae of V. virens could not extend into pedicles and stems connecting the spikelets, and no anatomic changes are detected in pedicles [39].
Although RFS disease symptoms are observed at rice grains due to pathogen infections of spikelets, V. virens also grows on other rice organs without obvious symptoms. At the germination stage of rice seed, chlamydospores of V. virens could germinate on coleoptiles and the hyphae are able to extend intercellularly between epidermal cells [43]. At seedling stage, chlamydospores could also germinate on the surface of roots and grow in the intercellular space of root epidermal cells [44,45]. More recently, a detailed observation on V. virens-infected rice roots indicates that the cellulose microfibrils of epidermal cell wall are very loose, similar to those of stamen filaments, and thus are prone to be infected [46]. However, the hyphae of V. virens are stopped by sclerenchyma layer from entering into endodermis and phloem tissues [46]. Again, Rice False Smut: An Increasing Threat to Grain Yield and Quality DOI: http://dx.doi.org /10.5772/intechopen.84862 no appressorium or haustorium can be detected when V. virens infects tender coleoptiles and roots. To date, contribution of the infections of the coleoptiles and roots at the vegetative stage to RFS disease symptoms has not been determined if any, and the evidence of systemic infection of V. virens is currently lacking.

Genetic transformation of V. virens
Genetic manipulation is essential to clarify the pathogenicity of V. virens and its interaction with rice. Several transformation techniques have been successfully applied to V. virens. Electroporation, the process in which a strong electric pulse is applied to an organism in order to transiently increase membrane permeability, has the advantages of being rapid and inexpensive. Through electroporation on conidia, an enhanced green fluorescence protein (eGFP)-expressing V. virens strain was obtained, which was able to infect rice flowers and form RFS balls [47]. Polyethylene glycol (PEG)-mediated approach is typically more efficient than electroporation and generally yields a higher percentage of stable transformants. Ashizawa and colleagues [38] engineered a GFP-tagged V. virens strain via PEGmediated transformation on protoplasts, and identified the infection route of V. virens in rice spikelets with this strain. Agrobacterium tumefaciens-mediated transformation (ATMT) is a fast and easy way to transfer foreign DNA into fungal cells. A few reports have recorded the establishment and optimization of ATMT procedure on V. virens conidia, and construction of T-DNA mutant libraries [48,49], which facilitate characterizing virulence factors in this pathogen. For example, Yu et al. [48] obtained a T-DNA insertion library with 5600 hygromycin-resistant transformants, and identified 37 mutants with impaired pathogenicity. Targeted gene knockout with PEG-or ATMT-mediated transformation has been tried on V. virens; however, the homologous gene replacement frequency was very low [50]. Very recently, Liang and colleagues [51] established a CRISPR-Cas9 system, which remarkably increased the frequency of homologous gene replacement. The knockout efficiency could be as high as 50-90% for some V. virens genes.

Genome and pathogenicity of V. virens
The availability of V. virens genome provides a good basis for charactering its pathogenicity in rice flower. As reported, the genome of UV-8b is approximately 39.4-Mb, encoding 8426 putative proteins [52]. The strain IPU010 possesses a genome of 33.6-Mb, which encodes 6451 predicted proteins [53]. Genome analysis reveals that V. virens is evolutionarily closest to the entomopathogen Metarhizium spp., suggesting host jumping from animal kingdom to plant kingdom [52]. Moreover, genome information provides evidence supporting that V. virens specifically infects rice flower and has a biotrophic lifestyle, since genes responsible for secreted proteins and secondary metabolism are enriched, while genes associated with polysaccharide degradation and nutrient uptake are diminished [52]. A web-based protein-protein interactive database for V. virens-Oryza sativa interaction has been released, greatly facilitating investigation of V. virens pathogenicity [54]. Putatively, 628 secreted proteins are encoded by V. virens genome, 193 of the secreted proteins are predicted to be effectors [52].
Effectors are powerful weapons possessed by pathogens to manipulate host immune system and metabolisms for successful colonization. Characterizing their roles is important for understanding pathogen-host interactions. In V. virens genome, a number of genes encoding effector proteins, such as UV_1261, UV_2508, and UV_2286, have been identified to suppress Burkholderia glumae-induced cell death [52], whilst UV_5823 shows ability to suppress plant RNA silencing [55]. On the contrary, some effectors of V. virens could induce cell death or defense response in rice protoplast. For example, UV_44 induces cell death, and this ability relies on the serine peptidase active sites. UV_1423 could be N-glycosylated, which affects its ability to trigger cell death [56]. However, so far, no effectors have been characterized to function in the flower infection of V. virens.
Nevertheless, several virulence factors have been identified in V. virens. UvSUN2 is a SUN domain protein; loss of function of this factor results in inability of infecting rice flower, as well as abnormal stress responses and mycelium growth [57]. Mutation in UvPRO1 increases sensitivity to abiotic stresses and attenuates virulence, in addition to impaired growth rate and sporulation [49]. In contrast, a low-affinity iron transporter encoded by Uvt3277 negatively regulates virulence in V. virens [58].

Host compatible interaction with V. virens
Monitoring host responses to V. virens infection could help to uncover the pathogenesis of RFS. In an earlier transcriptome study, a series of differentially expressed genes have been identified in a susceptible rice cultivar 93-11 infected with a field V. virens isolate [59]. Among them, genes regulated by Ca 2+ or abscisic acid are down-regulated, while genes regulated by Myb or WRKY transcription factors are up-regulated. OsSWEET11 and OsSWEET14, which may be involved in disease susceptibility [60], are also up-regulated by V. virens infection. Specifically, many pollen development associated genes are down-regulated by V. virens infection, but not responsive to other biotic and abiotic stresses, suggesting that these genes may play unique roles in rice-V. virens interaction [59]. Additionally, several transcriptome analyses on compatible rice-V. virens interactions have been reported. Genes involved in hydrolase, transporter, and flower development tend to be downregulated in susceptible cultivar Huang-Xiu-Zan upon infection [61]. Expression of many defense-related genes such as PAL and PR genes could be suppressed in susceptible rice cultivars infected with V. virens [42,62].
As a successful pathogen, V. virens should have abilities to set up colonization in rice floral organs and acquire abundant nutrients for propagation, in addition to subvert rice immunity. Transcriptome analysis reveals that genes associated with flower opening, such as ARF6 and ARF8 homologs, are down-regulated by V. virens infection [42]. This may contribute to inhibition of flower opening during RFS pathogenesis [42]. Furthermore, V. virens infection causes failure of ovary fertilization. However, a number of grain-filling-specific genes, such as seed-specific starch synthesis related genes and those encoding seed storage proteins, are activated for high expression in V. virens-infected rice spikelets [42]. It is suggested that V. virens may be able to mimic fertilization and hijack rice grain-filling system for nutrient supply to pathogen growth and RFS ball formation. This finding is further supported by an independent study [63]. Although the underlying mechanism needs further investigation, the observation of V. virens activating rice grain filling could provide a promising explanation why mild V. virens infection enhances rice yield traits, including grain weight and filled grain number [9]. Identification and characterization of V. virens factors that manipulate rice grain filling should be an interesting research area in the future.

Sources and inheritance of RFS resistance
Various attempts have been made to screen rice cultivars resistant to RFS. Screening of 186 rice hybrids to RFS resistance was done by Liang and colleagues [64], which identified few hybrids with low disease incidence. They screened the commercial hybrids that had lower rates of diseased panicles and infected florets at Xindu and Qionglai (Sichuan Province, China) in 2011 together with newly registered varieties. Lore et al. [65] evaluated some hybrids and inbred cultivars growing across India for susceptibility/tolerance to RFS. Artificial inoculation of false smut was done by Kaur et al. [66], which identified nine hybrids resistant to RFS among 125 rice genotypes screened. More detailed evaluation of RFS resistance was performed by Huang and colleagues [67]. A total of 843 rice accessions were screened in disease nurseries in 3 years although some of those accessions were planted in different locations and on different dates. Finally, 36 accessions were found to show no disease incidence. A highly susceptible accession Pujiang 6 was identified in this study. Polymorphism analysis determined several resistant accessions which could be used for crossing with Pujiang 6 to construct gene mapping populations [67].
Resistance of genes against V. virens has not been identified yet, but numerous efforts have been undertaken to study the inheritance of the resistance. Earlier, using 266 near-isogenic introgression lines derived from susceptible cultivar Teqing and resistant Lemont and natural infection data in the field, Xu et al. [68] identified two RFS resistance-contributing QTLs, QFsr10 and QFsr12, located on chromosome 10 and 12, respectively. Later, the same group further identified 10 QTLs for RFS resistance [69]. Li et al. [70] developed a population of 157 recombinant inbred lines (RILs) from crossing a susceptible landrace Daguandao (O. sativa subsp. japonica) and a resistant cultivar IR28 (Oryza sativa subsp. indica). Subsequently, different RILs and parents were evaluated following effective artificial inoculation under field conditions. Genetic analysis showed that the RFS resistance was controlled by two major genes with equal effect of 11.41 and polygenes with minor effects. Further work identified seven QTLs for RFS resistance on chromosomes 1, 2, 4, 8, 10, 11, and 12, and the phenotypic variance ranged from 9.8 to 22.5% [71]. The inheritance of RFS resistance in two-line hybrid rice was investigated using natural infection technique. When a moderate susceptible sterile line TGMS 33S was crossed with susceptible restorer lines, the F1 was susceptible to RFS; when TGMS 33S was crossed with resistant restorer lines, 87% of the F1 showed dominant or incomplete dominant inheritance of RFS resistance, the rest showed recessive inheritance [72]. It should be noted that natural infection of RFS varies among fields and seasons, efficient artificial inoculation method is highly recommended to validate the disease phenotype. Although some progresses on screening for resistant materials and determining the resistance inheritance have been achieved, genes responsible for RFS resistance are still unknown.

Molecular basis of RFS resistance
Since many rice cultivars with high RFS resistance or high susceptibility have been identified, comparative transcriptome analysis is a promising method to mine resistance-or susceptibility-related genes in rice. For instance, time-course RNAseq was carried out on susceptible cultivar LYP9 and resistant cultivar IR28 upon V. virens infection [62]. Data analysis revealed that many defense-related genes were only up-regulated in the resistant cultivar IR28, but not in LYP9. Particularly, phytoalexin biosynthetic pathway genes such as OsCPS2, OsMAS, and OsKSL11 were significantly induced in IR28 at early infection stages, indicating that phytoalexins may contribute to rice resistance against RFS. PR family genes, such as β-1,3glucanase and chitinase genes, were specifically up-regulated in IR28, while generally down-regulated in LYP9. Moreover, a chitinase gene cluster was found close to a RFS resistance QTL on chromosome 11 [71], and nine genes in this cluster were activated by V. virens infection. These data suggest that chitinase genes are potential candidates of RFS resistance. In addition, genes encoding receptor-like kinases and WRKY transcription factors may also play roles in RFS resistance. Interestingly, the resistant cultivar IR28 seemed to suppress V. virens genes that are associated with pathogenicity and fungal reproduction [62]. Another comparative transcriptome study demonstrated that peroxidase and flavin-containing monooxygenase genes, and genes involved in hormone metabolism were regulated differently in resistant and susceptible cultivars in response to V. virens infection [61].
Accumulation of H 2 O 2 is a typical plant basal defense fighting against pathogen infections [73]. During a compatible interaction between rice and V. virens, obvious H 2 O 2 accumulation was detected on the lemma and the palea of infected spikelet. H 2 O 2 also enriched in the anthers, stamen filaments, and lodicules of the infected spikelets [74]. However, it needs to be further investigated whether H 2 O 2 accumulation pattern is different between resistant and susceptible rice cultivars upon V. virens infection. A preliminary study described that higher contents of lignin and polyphenolic compounds were detected in spikelets of resistant rice variety Shuijing 3 than in those of susceptible variety 9522 [75], suggesting the role of these secondary metabolites in RFS resistance. To engineer rice resistant to RFS, an elicitor gene hrf1 from Xanthomonas oryzae pv. oryzae was ectopically expressed in R109, which is susceptible to RFS. Artificial inoculation and natural infection both supported that hrf1 conferred high resistance to the RFS pathogen, presumably through enhancing the expression of defense-related genes, including OsPR1a, OsPR1b, and PAL [76].
Based on the current findings that V. virens possesses intercellular infection strategy and biotrophic life style, and that resistant rice cultivars show up-regulation of pathogenesis-related genes and accumulation of secondary metabolites upon infection, basal defense of rice should play a dominant role in resistance against V. virens.

Disease cycle
V. virens attacks rice flowers and forms RFS balls covered with chlamydospores and/or generate sclerotia, which are considered as primary inocula of RFS disease (Figure 3). As to the sexual cycle, a large number of sclerotia can be produced when RFS balls develop in autumn [35]. Sclerotia cannot germinate immediately, requiring a dormancy period of 2-5 months at room temperature or 4°C. They overwinter in the field and could survive up to 10 months with maintaining germination ability to generate ascospores under 25°C and high humidity [77,78]. Even more, sclerotia can survive with high germination rate up to 5 years when stored in a dry environment at 2-4°C. In the next spring, sclerotia start to germinate, and the germination time varies among different sclerotia. Theoretically, a sclerotium could produce up to 21 million ascospores [35]. Although sclerotia are easy to rot in paddy fields under natural conditions, a limited number of sclerotia can still produce plenty of ascospores. This is supported by the fact that ascospores could be trapped 60 cm above ground in paddy fields between May and September, coinciding with rice booting stage and V. virens infection time [35]. Ascospores are able to infect rice flowers to form RFS balls [78,79]. Therefore, it is believed that sclerotia act as primary inocula of RFS and play an important role in the disease cycle.
With regard to the asexual cycle, chlamydospores from RFS balls are easily transmitted by wind and rainfall, and attack developing rice spikelets of late ripening rice cultivars. This is supported by the fact that fresh chlamydospores have high germination rate and could successfully infect rice flowers to form RFS balls [79,80]. Chlamydospores can overwinter in soil and on dead plants, or on harvested RFS balls and rice seeds, and survive up to several months; however, the germination rate decreases rapidly [81]. In the next rice planting season, chlamydospores overwintered in fields and on rice seeds may germinate with hyphae to infect coleoptiles of germinating rice seeds and roots of seedlings [43][44][45]. Since chlamydospores could not be trapped in fields until RFS balls appear [35], it is unclear how chlamydospore germination time couples with rice booting stage for infecting rice flowers. Studies suggest that coleoptile and root infections may lead to asymptomatic colonization of the pathogen in rice plants at subsequent stages. Sensitive PCR methods have been applied to successfully detect V. virens in various tissues of rice before panicle heading [82,83], suggesting the presence of pathogen in rice plants. Furthermore, colorimetric in situ hybridization reveals that V. virens mycelia are present on the surface of tiller buds enclosed by young leaf sheaths at vegetative stage, and also on the surface of elongated stems around leaf axils at the heading stage. As V. virens infection is not systemic, epiphytic growth could explain how the presence of mycelia in rice plants lasts from the germinating stage to the heading stage of rice [84]. Preset of pathogen mycelia in rice plants especially in leaf sheaths should greatly increase chances of attacking flowers.
Epiphytic growth of V. virens is not only found in rice plants, but also detected on leaf surface of various paddy field weeds and on abiotic surfaces (e.g., cellophane and parafilm) [33]. Under wet conditions, V. virens conidia are capable of blastogenesis and could produce a large number of secondary conidia on these surfaces in several days. Chlamydospores and ascospores both germinate to produce conidia [78,81], and the blastogenesis and epiphytic growth greatly increase the amount of inocula under continuing rainy conditions. Therefore, epidemics of RFS disease usually occur when rice booting stage meets with rainy days.
Alternative hosts of a pathogen commonly play an important role in disease cycle. Earlier, paddy field weeds such as Digitaria marginata [85], Panicum trypheron [86], Echinochloa crus-galli, and Imperata cylindrica [87] have been reported as alternative hosts of V. virens. However, a recent survey demonstrated that infection in these potential alternative hosts is very rare in nature [88]. Still, the presence of V. virens in weeds as confirmed by PCR detection [82] and epiphytic colonization on weed leaf surface [33] suggests that paddy field weeds contribute to RFS disease cycle in an unconventional way.

Disease control
In recent years, the RFS disease has become a severe threat to rice production due to its epidemics. In order to minimize direct economic loss, suitable management practices have to be made to manage the disease. Breeding and utilization of resistant cultivar is the most effective and economical way to control RFS disease and ensure the high yield of rice. Attempts have been made to identify sources of resistance against V. virens (see above). As inheritance of RFS resistance is not well understood, breeding for resistant rice is hindered. Late ripening rice cultivars with large panicle and high grain density are prone to RFS and should be carefully chosen for wide application.
Culture managements have been studied to reduce incidence of RFS. Early planted rice has less RFS balls rather than the late planted rice. Excess application of nitrogenous fertilizer should be avoided. Since high rate of nitrogen increases the disease incidence, sensible use of nitrogen is recommended. Fertilizer ratio is often a reasonable parameter for growers to adjust, so as to enhance the stress tolerance of rice plants, and ultimately reduce the RFS incidence. Field ridges and irrigation channels should be kept clean to eliminate alternative hosts. Conservation tillage and furrow irrigation have some effects on suppressing the disease index [2,89]. Using suitable plant spacing and utilizing uncontaminated rice seeds are also recommended.
Chemical control, i.e., fungicide application, can be effective but is often not economical and environment-friendly. Using fungicides with high efficiency, low toxicity, and low residue is currently the best choice to control RFS disease. Fungicides, such as Wenquning (a suspension of Bacillus subtilis in a solution of validamycin), cuproxat SC, simeconazole, tebuconazole, difenoconazole, and hexaconazole, are effective to reduce RFS disease incidence [64,90,91]. It is noteworthy that the timing of spraying fungicides is critical. Application of fungicides after panicle heading should be prevented, as the pathogen infects rice flowers at late booting stage and already successfully colonizes the inner floral organs after heading. As supporting evidence, simeconazole is found to be more effective against RFS when applied 3 weeks before rice heading [92].

Natural infection
To evaluate RFS sensitivity of rice under natural infection, several classification standards of disease incidence have been reported. For example, in 1996, the Rice False Smut: An Increasing Threat to Grain Yield and Quality DOI: http://dx.doi.org /10.5772/intechopen.84862 International Rice Research Institute (IRRI) [93] classified RFS into 6 scales based on incidence of severely infected tillers or infected spikelets, i.e. 0, no incidence; 1, less than 1%; 3, 1-5%; 5, 6-25%; 7, 26-50%; and 9, 51-100%. Later on, Tang and colleagues [94] established a new classification standard, and developed Disease Index to determine RFS incidence. The classification standard was based on aspect ratio and 100-weight of RFS ball, grain weight, seed setting rate, and yield loss of single diseased panicle. Six scales were classified: 0, no RFS ball; 1, one RFS ball; 2, two RFS balls; 3, 3-5 RFS balls; 4, 6-9 RFS balls; and 5, ≥10 RFS balls. Disease index = ∑ (Disease scale value × Diseased plant number)/(Total plant number × Highest disease scale) × 100. Note that only the highest disease scale value is adopted for each plant. This classification standard has been widely applied in recent studies [70][71][72]. When using natural infection method, disease incidence should be evaluated for multiple years at multiple locations, with multiple sowing dates.

Artificial inoculation
Due to uncertainty of environmental conditions under natural infection, a high efficient artificial inoculation method is desired for evaluating V. virens pathogenicity and rice resistance. As the pathogen specifically infects rice stamen filaments at specific rice stage to cause disease, it is difficult and complicated to optimize an efficient inoculation system. Parameters, such as inocula type, inoculation time and method, incubation conditions after inoculation, and so on, should be considered [42,[95][96][97][98]. To date, a number of studies conclude an efficient inoculation method under controlled conditions: first, culture V. virens in PSA at 28°C until white colony grows large enough for inoculating into PSB. Usually, 4-8 plugs of mycelia with around 6 mm diameter each are needed, and incubated in PSB at 28°C in dark, 110-150 rpm for 5-7 days. Second, a mixture of mycelia and conidia is blended as inocula, of which the conidia concentration is adjusted to around 10 6 conidia/mL with 4% potato juice. Third, at late booting stage of rice (5-7 days before heading), inocula are injected into panicles with a syringe until the inocula drip out (Figure 4). Fourth, the inoculated rice plants are kept at 25°C and 95% relative humidity for 5 days, and then moved to 28°C with relative humidity over 75%. Around 4 weeks post inoculation, disease incidence could be recorded. High RFS incidence (90-100%) has been obtained on susceptible rice cultivars such as Pujiang 6, Yueyou 938, and so on [42,97]. It should be noted that the artificial inoculation method needs to be modified when applied to different V. virens isolates and rice cultivars. For example, the highest RFS incidence is achieved when inoculation is carried out 3-5 days before heading for Yueyou 938 [97], and that is 5-7 days before heading for Pujiang 6 [42]. The disease symptom progression also varies among different V. virens-rice combinations under different post-inoculation conditions. As for V. virens PJ52-rice Pujiang 6 interaction under artificial inoculation conditions, no obvious symptom could be found through 1 dpi to 5 dpi. At 9 dpi, white fungal biomass can be seen with the naked eye. The fungal biomass enlarges and protrudes out of rice spikelets as early as 13 dpi, and large RFS balls appear at 17 dpi (Figure 4).
The above-mentioned classification standard and Disease Index [94] can also be applied to evaluation of disease incidence under artificial inoculation. Alternatively, the following method can be adopted when the disease scale is reaching the highest (i.e., scale 5, ≥10 RFS balls) for each plant. This situation is often encountered when using susceptible rice cultivars to evaluate V. virens pathogenicity. Due to high variation of the disease incidence for RFS pathosystem, at least 100 panicles from at least 30 rice plants are recommended to be inoculated. At around 4 weeks post inoculation, each inoculated panicle is collected for counting the number of RFS balls and the number of total spikelets. The number of RFS balls per inoculated panicle is recorded as BPP. Percentage of diseased spikelets (PDS) for each inoculated panicle is calculated as: PDS = 100 × number of diseased spikelets/total number of spikelets. To compare pathogenicity among different V. virens isolates or resistance/ susceptibility among different rice cultivars, BBP and PDS values are recommended to be presented as box-plot. Statistical analysis such as the ANOVA test is required to calculate the significance of difference among BBP or PDS datasets.

Future aspects
RFS is an emerging disease threatening the production safety of rice grains worldwide. Great progresses have been made to understand the RFS pathogen and its interaction with rice. However, many important questions are yet to be addressed. How much the mycotoxins produced by V. virens are contaminated in cooked rice and livestock feed? Whether mycotoxins play a role in pathogenicity? Are there any RFS resistance genes and how do they mediate defense against V. virens? How does V. virens activate rice grain filling system to hijack nutrient supply for the formation of RFS ball? In addition, an efficient inoculation method mimicking natural infection is highly desired for basic research and accurate evaluation of rice resistance.
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