Weedy red rice (Oryza sativa; WRR), a close relative of cultivated rice, is a highly competitive weed that commonly infests rice fields and can also naturally interbreed with rice. Useful genes for biotic stress have been maintained in WRR and can be explored for breeding. Here we describe genetic and physiological traits of WRR that can be beneficial in preventing major rice diseases. Rice blast, caused by the hemibiotrophic fungal pathogen Magnaporthe oryzae, and sheath blight disease, caused by the necrotrophic pathogen Rhizoctonia solani, are the two most damaging biotic stresses of rice. Many major and minor resistance genes and QTL have been identified in cultivated and wild rice relatives. However, novel QTL were recently found in the two major U.S. biotypes of WRR, blackhull-awned (BH) and strawhull-awnless (SH), suggesting that WRR has evolved novel genetic mechanisms to cope with these biotic stresses. Twenty-eight accessions of WRR (PI 653412–PI 653439) from the southern USA were characterized and placed in the National Small Grains Collection, and are available for identification of novel genetic factors to prevent biotic stress.

The fungus Magnaporthe oryzae is the causal agent of a wide range of cereal diseases. For long-term preservation, the fungus is grown and stored desiccated on filter papers at –20 ℃. Inoculated filter papers are cut into pieces of 0.5–1.0 cm diameter prior to storage. In the present study, a fast (11 min) and simple method of preparing DNA suitable for amplifying avirulence genes of M. oryzae by polymerase chain reaction (PCR) was developed. A piece of filter paper containing the fungus was removed from a glass bottle and placed in a 0.2 mL Eppendorf tube containing 100 μL 10 × TE. The suspension was heated for 10 min at 95 ℃ in a PCR machine. The tube was then centrifuged for 1 min at 3000 r min⁻¹. One μL of 10 × TE solution containing DNA was used for PCR. A total of 28 samples were PCR tested. As a positive control, fungal DNA was extracted using a conventional DNA preparation method. DNA samples obtained from both methods were stored at 4 ℃. PCR was performed with DNA on the preparation day and after 4, 8, 10, and 18 days of refrigerated storage. In four samples, samples 12, 13, 14, and 28, AVR-Pi9 failed to be amplified. These four samples were tested with a different set of primers for AVR-Pi9, and for AVR-Pita1, confirming that the quality of the samples was insufficient for PCR. Overall, for nearly 90% (24/28) of the samples, the quality of the DNA prepared directly from the fungus on filter paper appeared suitable for a rapid survey of genetic identity of the rice blast fungus by PCR. This method will be useful and effective for reducing cost and time and could readily be adopted worldwide for analysis of M. oryzae and possibly other fungi.

The AVR-Pita1 gene, from the Chinese isolate O-137 of Magnaporthe oryzae, is an effector that determines the efficacy of the Pi-ta rice blast resistance gene. In the present study, the avirulence function of AVR-Pita1 was induced by transformation of field isolates (TM2, ZN19, B2 and B8) that originally were collected from the U.S. and are virulent on Pi-ta-carrying rice cultivars. The presence of AVR-Pita1 from O-137 in independent transformants was detected by PCR using AVR-Pita1 specific primers and verified by DNA sequencing and Southern blot analysis using the AVR-Pita1 coding region as a probe. The results of pathogenicity assays showed that the AVR-Pita1-transformed isolates were not able to infect rice cultivars Katy and Drew carrying Pi-ta. Control isolates that were transformed with inserts lacking the AVR-Pita1 gene remained virulent. Our findings demonstrate that AVR-Pita1 can be used to induce novel gene-specific blast resistance in nature.