**6. 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

**97**

**Figure 3.**

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

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 [43–45]. 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

*spikelet; le, lemma; pa, palea; sf, stamen filament; lo, lodicule; ov, ovary; and fsb, false smut ball.*

*Disease cycle of rice false smut. Rice false smut balls with chlamydospores and sclerotia are formed in rice spikelets (*①*), and overwinter in field (*②*). Next spring, spores in soil (*③*) and on contaminated rice grains (*④*) germinate and attack rice roots and coleoptiles when rice seeds are germinating. Hyphae grow intercellularly in roots and coleoptiles, but could not infect seedlings systemically. Instead, hyphae may grow epiphytically on leaf surface or leaf sheath, and reach the external surface between tiller buds at the late vegetative stage (*⑤*) or even the surface of elongated stems at the heading stage. It is possible that the pathogen hyphae reach the inner space of rice panicles and initiate infection at the late booting stage (*⑥*). Meanwhile, conidia produced by chlamydospores and/or ascospores from sclerotia (*⑦*) also initiate attack on rice spikelets in developing panicles (*⑧*). Spores could firstly germinate on the surface of a spikelet (*⑨*), and the hyphae extend into the inner space of the spikelet via the gap between the lemma and the palea (*⑩*). Stamen filaments are the major infection sites for the pathogen (*⑪*). After successful colonization in floral organs, a large amount of fungal mass are formed and eventually grow into a false smut ball (*⑫*). The route of infection is indicated by arrows and numbers. Arrows with dotted lines are the steps needing further exploration. Red arrows indicate the main infection sites of the pathogen. Red curve lines represent pathogen hyphae. co, coleoptile; p, panicle; sp,* 

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

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

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

### **Figure 3.**

*Protecting Rice Grains in the Post-Genomic Era*

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

Accumulation of H2O2 is a typical plant basal defense fighting against pathogen infections [73]. During a compatible interaction between rice and *V. virens*, obvious H2O2 accumulation was detected on the lemma and the palea of infected spikelet. H2O2 also enriched in the anthers, stamen filaments, and lodicules of the infected spikelets [74]. However, it needs to be further investigated whether H2O2 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*.

*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

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 germina-

tion 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

of RFS and play an important role in the disease cycle.

and susceptible cultivars in response to *V. virens* infection [61].

**96**

**6. Disease cycle**

*Disease cycle of rice false smut. Rice false smut balls with chlamydospores and sclerotia are formed in rice spikelets (*①*), and overwinter in field (*②*). Next spring, spores in soil (*③*) and on contaminated rice grains (*④*) germinate and attack rice roots and coleoptiles when rice seeds are germinating. Hyphae grow intercellularly in roots and coleoptiles, but could not infect seedlings systemically. Instead, hyphae may grow epiphytically on leaf surface or leaf sheath, and reach the external surface between tiller buds at the late vegetative stage (*⑤*) or even the surface of elongated stems at the heading stage. It is possible that the pathogen hyphae reach the inner space of rice panicles and initiate infection at the late booting stage (*⑥*). Meanwhile, conidia produced by chlamydospores and/or ascospores from sclerotia (*⑦*) also initiate attack on rice spikelets in developing panicles (*⑧*). Spores could firstly germinate on the surface of a spikelet (*⑨*), and the hyphae extend into the inner space of the spikelet via the gap between the lemma and the palea (*⑩*). Stamen filaments are the major infection sites for the pathogen (*⑪*). After successful colonization in floral organs, a large amount of fungal mass are formed and eventually grow into a false smut ball (*⑫*). The route of infection is indicated by arrows and numbers. Arrows with dotted lines are the steps needing further exploration. Red arrows indicate the main infection sites of the pathogen. Red curve lines represent pathogen hyphae. co, coleoptile; p, panicle; sp, spikelet; le, lemma; pa, palea; sf, stamen filament; lo, lodicule; ov, ovary; and fsb, false smut ball.*

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 [43–45]. 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.

### **7. 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].

### **8. Disease assay**

### **8.1 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

**99**

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

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 [70–72]. When using natural infection method, disease incidence should be evalu-

ated for multiple years at multiple locations, with multiple sowing dates.

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, 95–98]. 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

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

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 *b*alls *p*er inoculated *p*anicle

conidia/mL with 4% potato juice.

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

**8.2 Artificial inoculation**

the conidia concentration is adjusted to around 106

balls appear at 17 dpi (**Figure 4**).

*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 [70–72]. When using natural infection method, disease incidence should be evaluated for multiple years at multiple locations, with multiple sowing dates.

### **8.2 Artificial inoculation**

*Protecting Rice Grains in the Post-Genomic Era*

cycle in an unconventional way.

**7. Disease control**

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

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

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

To evaluate RFS sensitivity of rice under natural infection, several classification standards of disease incidence have been reported. For example, in 1996, the

when applied 3 weeks before rice heading [92].

disease usually occur when rice booting stage meets with rainy days.

**98**

**8. Disease assay**

**8.1 Natural infection**

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, 95–98]. 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 106 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 *b*alls *p*er inoculated *p*anicle

### **Figure 4.**

*Artificial inoculation of rice false smut pathogen. (A) Inocula of V. virens are artificially injected with a syringe into a rice panicle at the late booting stage (5–7 days before heading). (B)–(H) Symptom development on rice spikelets after artificial inoculation. No obvious symptom is seen at 1 dpi (day post inoculation) and 5 dpi. White fungal mass could be detected in inner space of the infected spikelets at 9 dpi (E). The fungal mass grows larger at 13 dpi (G), and at 17 dpi eventually forms a ball-shape colony, called false smut ball (H). Scale bar, 0.5 cm. Image (A) is courtesy of Dr. Junjie Yu from Jiangsu Academy of Agricultural Sciences, China.*

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.

### **9. 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.

### **Acknowledgements**

We thank Drs Dongwei Hu (Zhejiang University) and Junjie Yu (Jiangsu Academy of Agricultural Sciences) for kindly providing images as indicated in figure legends. We apologize to the colleagues whose work could not be included in this book chapter due to space limitation. This work was supported by grants from the National Natural Science Foundation of China (grant no. 31501598 and 31772241) and Key Projects of Sichuan Provincial Education Department.

**101**

**Author details**

University, Chengdu, China

provided the original work is properly cited.

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

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

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Rice Research Institute and Key Lab for Major Crop Diseases, Sichuan Agricultural

Wen-Ming Wang\*, Jing Fan and John Martin Jerome Jeyakumar

\*Address all correspondence to: j316wenmingwang@sicau.edu.cn

### **Conflict of interest**

The authors declare no conflict of interest.

*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*

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

*Artificial inoculation of rice false smut pathogen. (A) Inocula of V. virens are artificially injected with a syringe into a rice panicle at the late booting stage (5–7 days before heading). (B)–(H) Symptom development on rice spikelets after artificial inoculation. No obvious symptom is seen at 1 dpi (day post inoculation) and 5 dpi. White fungal mass could be detected in inner space of the infected spikelets at 9 dpi (E). The fungal mass grows larger at 13 dpi (G), and at 17 dpi eventually forms a ball-shape colony, called false smut ball (H). Scale bar, 0.5 cm. Image (A) is courtesy of Dr. Junjie Yu from Jiangsu Academy of Agricultural Sciences, China.*

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.

We thank Drs Dongwei Hu (Zhejiang University) and Junjie Yu (Jiangsu Academy of Agricultural Sciences) for kindly providing images as indicated in figure legends. We apologize to the colleagues whose work could not be included in this book chapter due to space limitation. This work was supported by grants from the National Natural Science Foundation of China (grant no. 31501598 and 31772241) and Key Projects of Sichuan Provincial Education Department.

calculate the significance of difference among BBP or PDS datasets.

**100**

**9. Future aspects**

**Figure 4.**

**Acknowledgements**

**Conflict of interest**

The authors declare no conflict of interest.
