**3.2 Improved rice germplasms and genetic stocks**

Germplasms with improved resistance to both blast and sheath blight diseases are helpful for rice breeders to develop new rice cultivars [34]. Four rice germplasms, LJRIL103 (PI 660982), LJRIL158 (PI 660983), LJRIL186 (PI 660984), and LJRIL220 (PI 660985), with resistance to both blast and sheath blight diseases were

### **Figure 3.**

*Photograph showing a view of the rice research plots of USDA ARS DBNRRC and the University of Arkansas Rice Research Center, Stuttgart, Arkansas, USA, 2016. Most rice resources and mapping populations were advanced in similar field plots. The picture was taken with a drone in 2016.*


### **Table 1.**

*List of major genetic resources for blast and sheath blight resistance in the USA. Most of the rice germplasms are available at USDA-GSOR (www.ars.usda.gov/GSOR).*

identified. They were identified from 800 progenies of a cross between US-adapted rice germplasm Lemont with Jasmine 85 [26]. These germplasms contain suitable agronomic traits in addition to the aromatic nature of LJRIL103, LJRIL158, and LJRIL186. Disease resistance and aromatic genes were tagged with DNA makers to ensure their incorporations.

Loss-of-function mutants can help identify the functionality of the corresponding wild-type allele [35]. For example, lesion mimic mutants (LMMs) with a phenotype resembling hypersensitive cell death without pathogen attack are useful for studying the molecular basis of plant innate immunity. A rice LMM was identified from the rice cultivar Katy after treatment with fast neutrons [36]. The severe lesion mimic phenotype of LMM1 can be induced by blast pathogens and water-related stress, respectively (M.S. Jia and Y. Jia, unpublished data). LMM1 has an enhanced resistance to both blast and sheath blight disease [36]. Genetic analysis suggests that a single recessive gene is responsible for the lesion mimic phenotype in LMM1. Further characterization of the underlying gene in LMM1 will help elucidate the mechanisms of plant innate immunity and abiotic stress responses.

The abovementioned mapping populations, characterized rice germplasms and genetic stocks, are now being used to map and clone *R* genes to both rice blast and sheath blight disease and develop DNA markers for marker-assisted breeding [37].

## **4. User-friendly disease evaluation methods**

In the Southern US, genetic resistance to *M. oryzae* was investigated by Drs. Atkins, Johnston, and Marchetti [20, 38, 39]. Analyses of disease reactions to

**41**

**Figure 5.**

*severity of disease reactions.*

**Figure 4.**

*under field conditions.*

*A Toolbox for Managing Blast and Sheath Blight Diseases of Rice in the United States of America*

*Photographic presentation of the massive production of the sheath blight inoculant for field evaluation. Step 1: mixing A (corn chips) and B (rye) in a 2:1 weight ratio, adding water, and autoclaving twice. Step 2: growing mycelia in petri dishes containing PDA media until the appearance of white sclerotia (C). Step 3: mixing mycelia from C with a mixture of A and B from step 1, and incubating in a sterilized plastic or metal container for 3–5 days until the appearance of white sclerotia (D and E). Step 4: air drying mycelia and sclerotia in brown bags at 24°C with a fan (F). Step 5: grinding mycelia with a grinder (G) before inoculating plants* 

*Photographic presentation of two controlled sheath blight evaluation methods. (1) Detached leaf method: mycelia grown on PDA media (A), and PDA plugs removed from a were placed onto detached leaves (6–12 cm in length) (B) at 24°C for 3 days. Symptoms of detached leaves from rice varieties jasmine 85 and M202 after inoculations with three* R solani *isolates versus the control PDA without pathogens (C and D). (2) Soft-drink bottle method: PDA plugs from A were placed onto the bottom of sheaths (E) and covered with 2-L soft-drink bottles (F) for 3–5 days until stable symptoms appeared. Length of lesions was measured for both methods as the* 

*DOI: http://dx.doi.org/10.5772/intechopen.86901*

*A Toolbox for Managing Blast and Sheath Blight Diseases of Rice in the United States of America DOI: http://dx.doi.org/10.5772/intechopen.86901*

### **Figure 4.**

*Protecting Rice Grains in the Post-Genomic Era*

C/M doubled haploid GSOR

USDA core collection GSOR

*available at USDA-GSOR (www.ars.usda.gov/GSOR).*

**Plant identification**

200001–200325

GSOR 100361–100600

GSOR100001– 100355

GSOR 101601–102,174

GSOR 102501–102544

GSOR 303101–303287

GSOR 303301–303536

310001–311795

**Key information**

Sheath blight resistance

> Molecular map/blast resistance

Blast and sheath blight resistance

Blast and sheath blight resistance

**Number Year of** 

Blast resistance 240 2007 [24]

Blast resistance 42 2012 [27]

Blast 187 2015 [28]

Blast resistance 1795 2015 [29–33]

**release**

325 2006 [23]

355 2007 [25]

574 2009 [26]

236 2015 [28]

**Reference**

**Name of genetic sources**

Early/Katy mapping population

K/Z mapping population

SB5 mapping population

Katy//M202 backcrossing lines

Weedy red rice mapping population 1

Weedy red rice mapping population 2

**Table 1.**

ensure their incorporations.

identified. They were identified from 800 progenies of a cross between US-adapted rice germplasm Lemont with Jasmine 85 [26]. These germplasms contain suitable agronomic traits in addition to the aromatic nature of LJRIL103, LJRIL158, and LJRIL186. Disease resistance and aromatic genes were tagged with DNA makers to

*List of major genetic resources for blast and sheath blight resistance in the USA. Most of the rice germplasms are* 

Loss-of-function mutants can help identify the functionality of the corresponding wild-type allele [35]. For example, lesion mimic mutants (LMMs) with a phenotype resembling hypersensitive cell death without pathogen attack are useful for studying the molecular basis of plant innate immunity. A rice LMM was identified from the rice cultivar Katy after treatment with fast neutrons [36]. The severe lesion mimic phenotype of LMM1 can be induced by blast pathogens and water-related stress, respectively (M.S. Jia and Y. Jia, unpublished data). LMM1 has an enhanced resistance to both blast and sheath blight disease [36]. Genetic analysis suggests that a single recessive gene is responsible for the lesion mimic phenotype in LMM1. Further characterization of the underlying gene in LMM1 will help elucidate the mechanisms of plant innate immunity and abiotic stress

The abovementioned mapping populations, characterized rice germplasms and genetic stocks, are now being used to map and clone *R* genes to both rice blast and sheath blight disease and develop DNA markers for marker-assisted

In the Southern US, genetic resistance to *M. oryzae* was investigated by Drs. Atkins, Johnston, and Marchetti [20, 38, 39]. Analyses of disease reactions to

**40**

responses.

breeding [37].

**4. User-friendly disease evaluation methods**

*Photographic presentation of the massive production of the sheath blight inoculant for field evaluation. Step 1: mixing A (corn chips) and B (rye) in a 2:1 weight ratio, adding water, and autoclaving twice. Step 2: growing mycelia in petri dishes containing PDA media until the appearance of white sclerotia (C). Step 3: mixing mycelia from C with a mixture of A and B from step 1, and incubating in a sterilized plastic or metal container for 3–5 days until the appearance of white sclerotia (D and E). Step 4: air drying mycelia and sclerotia in brown bags at 24°C with a fan (F). Step 5: grinding mycelia with a grinder (G) before inoculating plants under field conditions.*

#### **Figure 5.**

*Photographic presentation of two controlled sheath blight evaluation methods. (1) Detached leaf method: mycelia grown on PDA media (A), and PDA plugs removed from a were placed onto detached leaves (6–12 cm in length) (B) at 24°C for 3 days. Symptoms of detached leaves from rice varieties jasmine 85 and M202 after inoculations with three* R solani *isolates versus the control PDA without pathogens (C and D). (2) Soft-drink bottle method: PDA plugs from A were placed onto the bottom of sheaths (E) and covered with 2-L soft-drink bottles (F) for 3–5 days until stable symptoms appeared. Length of lesions was measured for both methods as the severity of disease reactions.*

### **Figure 6.**

*Parafilm method for sheath blight disease evaluation: a PDA containing mycelia (A) in a petri dish containing mycelia after 3 days of culturing at 30°C was removed and covered with parafilm and wrapped onto the second youngest leaf for 3–5 days (B) until stable symptoms appeared. A rating scale based on visual length and area of symptoms was assigned as indicated, with 0 representing immunity and 9 representing extreme susceptibility (C).*

*M. oryzae* have been performed under field conditions where complex biotic and abiotic factors impacting the inheritance of resistance were encountered resulting in inconsistencies of disease reactions. In 1999, Dr. Marchetti and his

**43**

*A Toolbox for Managing Blast and Sheath Blight Diseases of Rice in the United States of America*

colleagues demonstrated that disease reactions under an upland blast nursery were reliable to identify *R* genes among breeding lines [40]. Under greenhouse conditions, the phenotypes of rice to *M. oryzae* are categorized as 0–5 where 0 represents complete immunity, 1 represents hypersensitive cell death showing tiny brown spots, 2 represents infected lesions without mycelia, and, for susceptible reactions, 3–5 exhibit different sizes of lesions with visible mycelia coincident with different levels of resistance [32]. Phenotypes evaluated under the upland rice blast nursery were verified with 200 individuals of a mapping population under greenhouse conditions at DBNRRC [41]. Since then, the greenhouse methods have been used to determine the inheritance and genetic mechanisms of blast resistance [32, 41, 42]. In 2015, several IRRI monogenic lines generously donated by IRRI were added to further identify blast *R* genes

The early evaluation of sheath blight relied on replicated field plot experiments

Disease reactions were scored by visually rating the disease severity on the sheaths

A total of 14 known major blast *R* genes have been used in the USA since 1960s. **Table 2** lists their chromosomal locations, representing germplasms, DNA markers to monitor respective *R* genes, and the avirulent and virulent races of these selected rice germplasms (**Table 2**). Based on field observations, most blast *R* genes are dominant whereas a single haplotype of *R* gene is effective for resistance. Among them, six dominant blast *R* genes *Pia*, *Piks*, *Pi66(t)*, *Pikh*, *Pikm*, and *Pi43(t)/Pi1*, and one recessive *R* gene *pid* were on chromosome 11. Comprehensively, one was found on chromosome 2, two on chromosome 6, one on chromosome 8, one on chromosome 9, and two dominants on chromosome 12. Three of the dominant *R* genes, *Pi9*, *Pi42(t)*, and *Pi43(t)*, provide resistance to all races, while *Pita2/Ptr* is

The genetic markers linked or derived from the cloned *R* genes were developed to predict resistance function and to monitor the existence of each of the *R* genes [31–33, 44–52]. Differential blast races were identified (**Table 2**) and have been

Distinct phenotyping variation of rice after infection via *M. oryzae* in different rice germplasms and in the same germplasm at different growth stages under greenhouse [53] and field conditions are also referred as dilatory, partial, field, and adult resistance interchangeably [54]. A total of 11 blast *R* quantitative trait loci (QTLs) responsible for a phenotypic variation ranging from 5.17 to 26.53% were identified with different blast races under greenhouse conditions [55] (**Table 3**) and verified with different blast isolates/races [56]. Using the same method, four additional blast *R* QTLs were identified from different rice germplasms [57].

and leaves of whole plants. The results of the evaluations are useful for mapping *R* genes. As an alternative, greenhouse methods such as detached leaf, soft-drink bottles, and parafilm methods were developed to validate and verify the function of *R* genes (**Figures 5** and **6**). These greenhouse methods are being used routinely for initial *R* gene discovery because they use less time, labor, land, and fertilizer.

*DOI: http://dx.doi.org/10.5772/intechopen.86901*

under greenhouse conditions [43].

**5. Effective** *R* **genes**

**5.1 Effective major** *R* **genes**

effective to all races except IE1k.

**5.2 Effective minor** *R* **genes**

used to validate their predicted resistance efficacies.

with fungal mycelia grown in corn chips or rye (**Figure 4**).

*A Toolbox for Managing Blast and Sheath Blight Diseases of Rice in the United States of America DOI: http://dx.doi.org/10.5772/intechopen.86901*

colleagues demonstrated that disease reactions under an upland blast nursery were reliable to identify *R* genes among breeding lines [40]. Under greenhouse conditions, the phenotypes of rice to *M. oryzae* are categorized as 0–5 where 0 represents complete immunity, 1 represents hypersensitive cell death showing tiny brown spots, 2 represents infected lesions without mycelia, and, for susceptible reactions, 3–5 exhibit different sizes of lesions with visible mycelia coincident with different levels of resistance [32]. Phenotypes evaluated under the upland rice blast nursery were verified with 200 individuals of a mapping population under greenhouse conditions at DBNRRC [41]. Since then, the greenhouse methods have been used to determine the inheritance and genetic mechanisms of blast resistance [32, 41, 42]. In 2015, several IRRI monogenic lines generously donated by IRRI were added to further identify blast *R* genes under greenhouse conditions [43].

The early evaluation of sheath blight relied on replicated field plot experiments with fungal mycelia grown in corn chips or rye (**Figure 4**).

Disease reactions were scored by visually rating the disease severity on the sheaths and leaves of whole plants. The results of the evaluations are useful for mapping *R* genes. As an alternative, greenhouse methods such as detached leaf, soft-drink bottles, and parafilm methods were developed to validate and verify the function of *R* genes (**Figures 5** and **6**). These greenhouse methods are being used routinely for initial *R* gene discovery because they use less time, labor, land, and fertilizer.
