Screening the Presidio/Oryza rufipogon rice advanced backcross population for reaction to leaf blast disease using two techniques

Authors: Grunden, Quynh; Eizenga, Georgia; Jia, Yulin

Submitted to: Rice Technical Working Group Meeting Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 1/31/2025
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: The ancestral species of cultivated rice (Oryza sativa), O. rufipogon and O. nivara, are collectively referred to as the Oryza rufipogon Species Complex (ORSC) and are a potential source of novel alleles for rice improvement, especially tolerance to biotic and abiotic stresses. Unfortunately, many ORSC accessions shatter seed, exhibit sterility when crossed with O. sativa and several produce limited seed. Blast disease, a major fungal disease of rice worldwide, is caused by Magnaporthe oryzae. To determine if any of the 94 accessions included in an ORSC collection carried a novel blast resistance gene/allele, the collection was screened with three of the most virulent US blast races (IA1, IB49 and IB33) using a modified seedling inoculation technique to accommodate accessions with few seed and have more uniform seedling development for inoculation. Modifications to the standard procedure were dehulling and sterilizing the ORSC seeds, placing the seed on an Orchid tissue culture media in a Magenta box, growing the plants under 12-hr light/12-hr dark for about 10 days (3-4 leaf stage) in an incubator, transplanting 3-4 seedlings into 6 x 6 cm pots placed in trays for inoculation, and rating on a 0 (resistant) to 9 (susceptible) scale. From these screenings two accessions were resistant to IB49 and IB33, and one O. rufipogon accession (IRGC103404) from Bangladesh was resistant to all three races. An advanced backcross (ABC) mapping population was developed using this O. rufipogon as the donor parent and Presidio, a US long grain cultivar, as recurrent parent. The 244 BC2F3:4 backcross inbred lines (BILs) comprising the population were screened with blast races IA1, IB49 and IB33 by inoculating seedlings, grown in soil, without the tissue culture step. Due to limited BIL seed, the Spot Inoculation Method was used to evaluate this population for reaction to blast races IB54 and IE1K. Briefly, steps in this method include placing filter papers containing fungal mycelia on oatmeal agar plates and incubating under a 28-hr light/8-hr dark cycle at 24oC for seven days; transferring hyphal tips to oatmeal agar and incubating for 12-hr to produce conidia; harvesting conidia in a sterile 0.25% gelatin solution and determining the concentration of conidia using a hemacytometer. Lastly, Tween 20 was added to the conidial suspension to promote attachment to the leaf surface. Filter paper was placed into a 23.5 x 23.5 cm square petri dish and four 5-cm young leaf segments were cut and placed into the petri dish. Sterile water was used to saturate the filter paper. Using a multichannel pipet, four equal sized 5-µl droplets of the conidial suspension were placed on each leaf segment. The petri dish was maintained at 21o to 24oC under continuous fluorescent light for 24-hr after which the droplets were removed by blotting with a laboratory tissue. Disease reactions were rated based on the development of the necrotic lesions (7 to 10 days) using a 0 (uniform dark brown spot) to 4 (large spot lesions with sporulated mycelia and conidia) scale. To date, three BILs were rated as resistant to IB49, IA1 and IB33. Currently the BILs are being screened for reaction to races IB45 and IB54 using the seedling method. Once the ratings for the six races are complete, comparisons will be made between the ratings using these two methods.