**2.2 Genetic diversity of the 12 reference isolates**

The genetic diversity of the 12 reference isolates was evaluated by vegetative compatibility analysis [13] and molecular methods. Vegetative compatibility analysis indicated that three isolates A598, ZN15, and ZN46 belonged to vegetative compatibility group (VCG) US-01; isolates TM2, #24, and A264 belonged to VCG US-02; two isolates 49D and A119 belonged to VCG US-03; and other two isolates IB33 and IB54 belonged to VCG US-04 (**Table 1**). The VCG of isolate ID13 was not determined.

Using Pot 2 primers [23], the repetitive element-based polymerase chain reaction (Rep-PCR) was used to DNA fingerprint the 12 reference isolates. The amplicon patterns of 49D, IB33, and IB54 based on Pot 2 primers were identical; TM2 and ZN7 were identical to each other; isolate 24 and A264 were identical to each other, but they had one extra band compared to that of TM2 and ZN7; ZN15 and ZN46 had similar patterns (**Figure 2**). The mating types of these isolates were determined by using mating-type-specific primers [24]. The results suggested that six isolates, 49D,


### **Table 1.**

*Background information on the 12 US reference isolates of M. oryzae used in this study.*

### **Figure 2.**

A598, IB33, IB54, ZN15, and ZN46, belonged to mating type I, while other six isolates TM2, #24, A119, A264, ID13, and ZN7 belonged to mating type II (**Figure 3**).

Seven avirulence genes were assessed using specific primers to each gene (**Table 2**) [25–28]. The entire *AVR-Pita* fragment could be amplified from nine isolates with primers YL149/YL169, but not from isolates TM2, IB33, and ID1. The coding regions of the avirulence gene *AVR-Pib* was found in all 12 reference isolates (amplified with the *AVR-Pib* F3/R3 primers); however, the promoter region of the *AVR-Pib* gene (amplified with the *AVR-Pib* F2/R2 primers) was not found in isolates 49D, IB33, and IB54. The avirulence gene *AVR-Pikm* was only found in four isolates, 49D, IB33, IB54, and ID13. The other four avirulence genes, *AVR-CO39*, AVR-Pi9, *AVR-Pikz*, and *AVR-Piz-t,* were present in all 12 reference isolates (**Figure 4**).

## **2.3 Testing the US reference isolates on IRRI near-isogenic rice lines**

### *2.3.1 IRRI rice blast near-isogenic lines*

The 12 US reference isolates were tested on 31 LTH NILs (containing 24 blast *R* genes) and 20 CO39 NILs (containing 14 *R* genes) in three independent tests, with two replications in each test. Two cultivars, M204 and Francis, were included as susceptible controls.

### *2.3.2 Inoculation of blast pathogen and disease screening*

Rice seed was planted in plastic trays filled with river sand mixed with potting soil in the greenhouse at the University of Arkansas, Fayetteville, AR, USA. Iron

**57**

**Figure 4.**

**Table 2.**

*Evaluation of Resistance of US Rice Breeding Lines to the Rice Blast Pathogen*

*AVR-Pita* YL149 TGACCGCGATTCCCTCCATT

*AVR-Pi9* AVRPi9F CTG CTC CAT CTT GTT TGG CC

*AVR-Pib* AVR-PibF2 TGGAGAAGACTTTGATGC

*AVR-Pikz* AVR-PikzF TGACGCAGCTTGAGTTGT

*AVR-Pikm* AVR-PikmF TTATCGCCCCTATATTGC

*AVR-Piz-t* AVR-Piz-tF ATGCAGTTCTCAACCATC

*AVR1-CO39* AVR1-CO39F1 GATCTGTAAATTACATA

YL169 CGACCGTTTCCGCC

AVR1-CO39R1 GGATCCGCCGTCGCCTCC

AVR-PibR2 TAGTTGCCATTATGCGTTC AVR-PibF3 ATGCGTTCCTCAACCACTTT AVR-PibR3 TTATTCCACGGTATATTTGCTGCC

AVR-PikzR TCCGAGCAATCAACTCTG

AVR-PikmR TTATCGCCCCAACACGGA

AVR-Piz-tR CTATTGGCGCTGAGCCT

*Primers used to amplify seven avirulence genes from the 12 US reference isolates of M. oryzae.*

AVRPi9R CAC TAG TAC AAG CAC TAA CC

sulfate was applied to the newly emerged seedlings. The plants were fertilized with Miracle-Gro All-Purpose Plant Food 20-20-20 once a week during each test. Plants were inoculated approximately 14–20 days after planting. Each isolate was grown on rice bran agar (RBA) [13] for approximately 7–10 days and then reinoculated on new RBA plates for 7–10 days. Spores were collected in cold water and adjusted to a concentration of 200,000 spores/ml per isolate. Each tray was inoculated with 50 ml of inoculum mixed with 0.02% Tween 20 with an air compressor sprayer. After

*Detection of seven avirulence genes in the 12 US reference isolates of M. oryzae.*

*DOI: http://dx.doi.org/10.5772/intechopen.84980*

**Target gene Primer name Sequences**

**Figure 3.**

*Mating type analysis of the 12 reference blast isolates.*


*Evaluation of Resistance of US Rice Breeding Lines to the Rice Blast Pathogen DOI: http://dx.doi.org/10.5772/intechopen.84980*

### **Table 2.**

*Protecting Rice Grains in the Post-Genomic Era*

A598, IB33, IB54, ZN15, and ZN46, belonged to mating type I, while other six isolates

The 12 US reference isolates were tested on 31 LTH NILs (containing 24 blast *R* genes) and 20 CO39 NILs (containing 14 *R* genes) in three independent tests, with two replications in each test. Two cultivars, M204 and Francis, were included as

Rice seed was planted in plastic trays filled with river sand mixed with potting soil in the greenhouse at the University of Arkansas, Fayetteville, AR, USA. Iron

TM2, #24, A119, A264, ID13, and ZN7 belonged to mating type II (**Figure 3**). Seven avirulence genes were assessed using specific primers to each gene (**Table 2**) [25–28]. The entire *AVR-Pita* fragment could be amplified from nine isolates with primers YL149/YL169, but not from isolates TM2, IB33, and ID1. The coding regions of the avirulence gene *AVR-Pib* was found in all 12 reference isolates (amplified with the *AVR-Pib* F3/R3 primers); however, the promoter region of the *AVR-Pib* gene (amplified with the *AVR-Pib* F2/R2 primers) was not found in isolates 49D, IB33, and IB54. The avirulence gene *AVR-Pikm* was only found in four isolates, 49D, IB33, IB54, and ID13. The other four avirulence genes, *AVR-CO39*, AVR-Pi9, *AVR-Pikz*, and *AVR-Piz-t,* were present in all 12 reference isolates (**Figure 4**).

*Rep-PCR band patterns of 12 reference isolates amplified with Pot 2 primers.*

**2.3 Testing the US reference isolates on IRRI near-isogenic rice lines**

*2.3.1 IRRI rice blast near-isogenic lines*

*Mating type analysis of the 12 reference blast isolates.*

*2.3.2 Inoculation of blast pathogen and disease screening*

susceptible controls.

**Figure 2.**

**56**

**Figure 3.**

*Primers used to amplify seven avirulence genes from the 12 US reference isolates of M. oryzae.*

### **Figure 4.**

*Detection of seven avirulence genes in the 12 US reference isolates of M. oryzae.*

sulfate was applied to the newly emerged seedlings. The plants were fertilized with Miracle-Gro All-Purpose Plant Food 20-20-20 once a week during each test. Plants were inoculated approximately 14–20 days after planting. Each isolate was grown on rice bran agar (RBA) [13] for approximately 7–10 days and then reinoculated on new RBA plates for 7–10 days. Spores were collected in cold water and adjusted to a concentration of 200,000 spores/ml per isolate. Each tray was inoculated with 50 ml of inoculum mixed with 0.02% Tween 20 with an air compressor sprayer. After

inoculation, the plants were incubated at 100% relative humidity in a mist chamber at approximately 22°C for 24 h, allowed to dry for 2–3 h before being moved to the greenhouse. The inoculated plants were incubated in the greenhouse for 6 days. On the 7th day after inoculation, 15–20 plants of each line were scored according to a standard 0–9 disease rating scale developed by IRRI [22]. Lines rated 0 to 3 were considered resistant, whereas those rated 4–9 were considered susceptible.
