PEST MANAGEMENT: WEEDS

PEST MANAGEMENT: WEEDS Outcrossing Frequency and Phenotypes of Outcrosses Based on Flowering of Red Rice Accessions and Clearfield Cultivars in the Grand Prairie V.K. Shivrain, N.R. Burgos, J.A. Bullington, D.R. Gealy, and H.L. Black ABSTRACT Outcrossing between Clearfield (CL) cultivars and red rice (RR) has been documented in experimental plots as well as in fields. The authors hypothesize that CL cultivars, red rice type, planting time, and flowering time of CL and RR influence the transfer of imazethapyr-resistance genes from CL rice to RR. Our objectives were to 1) evaluate the flowering behavior of RR accessions and CL rice cultivars with respect to planting dates, 2) determine outcrossing rates between CL cultivars and RR accessions, and 3) determine the phenotypes of outcrosses. The experiments were conducted at the Rice Research and Extension Center (RREC), Stuttgart, and Southeast Research and Extension Center (SEREC), Rohwer, Ark., in the summers of 2005 and 2006. The experimental design was a split-split plot with three replications. Planting time (4), CL cultivar (2), and RR accessions (12) were main-, sub-, and sub-sub-plot, respectively. Each RR accession was planted in the middle of 9-row, 10-ft long plots with four rows of CL rice on both sides. Red rice seeds were collected in 2005 from individual plots for outcrossing rate determination. Seeds collected in 2005 were planted in the summer of 2006. Red rice seedlings at the 2-lf stage were sprayed with imazethapyr. Survivors of imazethapyr applications were characterized morphologically at maturity. Leaf tissues were also collected from survivors for confirmatory test of outcrossing by DNA analysis. Earlier-planted RR accessions and CL rice took longer to flower than later-planted ones. Flowering period of RR accessions ranged from 83 to 114, 83 to 110, 70 to 100, and 70 to 94 days after planting (DAP), in the first, second, third, and fourth plant- 237

AAES Research Series 550 ings, respectively. On average, CL XL-8 flowered 3 to 5 days earlier than CL 161, although flowering was completed within a week in all plantings in both cultivars. CL XL-8 had a higher outcrossing rate in all planting dates compared with CL 161. Outcrosses between CL 161 and red rice accessions were phenotypically uniform. In contrast, outcrosses between CL XL-8 and red rice accessions segregated in terms of flowering time, height, and various other plant characteristics. INTRODUCTION Red rice control in the drill-seeded delayed-flood cultural system using traditional rice herbicides is challenging due to the genetic similarity between cultivated rice and RR. Selective control of RR can be achieved in a CL rice production system. However, the transfer of the acetolactate synthase (ALS)-resistant gene from CL rice to RR is a valid concern. Imazethapyr-resistant RR plants were found in farmers fields in Arkansas and Louisiana within 2 years of the introduction of CL rice (Scott and Burgos, 2004; Zhang et al., 2004). Synchronization in the flowering time of red rice and rice is an important factor in gene transfer (Gealy et al., 2003). Red rice accessions in Arkansas have been documented as highly diverse in terms of morphology, especially in their flowering time (Shivrain, 2004). Outcrossing rate varies within CL cultivars, planting date, and synchrony in flowering time (Shivrain et al., 2007). Disparity in CL cultivars, planting time, and flowering time of RR accessions have significant impacts on gene transfer. As more CL cultivars and hybrids are released, gene transfer will become more complex due to the diversity in RR populations. Understanding the flowering pattern of different types of RR and CL cultivars will help us develop better strategies in mitigating outcrossing and managing RR. PROCEDURES Experiments were conducted at the Rice Research and Extension Center (RREC) at Stuttgart, and the Southeast Research and Extension Center (SEREC), Rohwer, Ark., in the summers of 2005 and 2006, respectively. The soil at the RREC experimental site is a DeWitt silt loam (fine, smectitic, thermic Typic Albaqualfs) with 1.2% organic matter and a ph of 5.8; the soil at the SEREC experimental site was a Sharkey clay with <1% organic matter and a ph of 7.2. In 2005, the experimental design was split-split plot with three replications. Planting time, CL cultivar, and RR accessions were main-, sub-, and sub-sub-plot, respectively. Planting dates were selected to cover early to late rice-planting time in Arkansas. There were four plantings: 16 April (PD 1), 27 April (PD 2), 13 May (PD 3), and 26 May (PD 4). Two CL cultivars, CL 161 and hybrid CL XL-8, were planted at 90 and 30 lb/acre, respectively, with 12 RR accessions representing RR from four rice-growing zones in Arkansas: White River, Grand Prairie, Delta 1, and Delta 2 (the Delta region is divided into two due to differences in flowering time of RR accessions from these regions) in Arkansas. The accessions represent an assortment 238

B.R. Wells Rice Research Studies 2006 of characteristics: strawhull, blackhull, and brownhull color; short and tall; awned and awnless; and early and late to flower. Each RR accession was planted in the middle of 9-row, 10-ft long plots with four rows of rice on both sides. Both CL cultivars were planted at a distance of 30-ft to prevent pollen movement from one cultivar to the other. Propanil and Facet were applied at 3 qt. and 0.35 lb/acre, respectively, at 5 weeks after planting to control other weeds. Volunteer RR plants were rouged by hand. Standard agronomic and pest-management practices were implemented during the growing season. Data on flowering were collected three times a week. When flowering of red rice was over, panicles were enclosed in Delnet bags to collect seeds. At maturity, RR seeds collected in the Delnet bags and those remaining on the panicles were harvested, cleaned, and stored until screened for imazethapyr-resistant outcrosses. In 2006, a sub-sample of approximately 3,000 seeds from each plot was planted at SEREC. Red rice seedlings were treated with three applications of Newpath at 8 oz/acre starting at 2-lf stage. Leaf tissues were collected from survivors for DNA analysis; SSR primers RM 253 and 234 were used to confirm the outcrosses. Outcrossing rate was calculated based on the number of confirmed hybrids. Outcrosses were grown until the end of the season and characterized morphologically. RESULTS AND DISCUSSION Results on four RR accessions (2 strawhull, 1 blackhull, 1 brownhull) that originated from the White River Zone are presented in this report. CL rice and RR accessions planted earlier took relatively more days to flower than those planted later. Flowering period of RR accessions ranged from 83 to 114, 83 to 110, 70 to 100, and 70 to 94 DAP, in the first, second, third, and fourth plantings, respectively (Fig. 1). Early flowering at later planting date is common among strawhull RR and CL cultivars (Burgos et al., 2004). On average, CL XL-8 flowered at 95 to 98 DAP and CL 161 flowered 3 to 5 days later. The outcrossing rate with RR accessions ranged from 0 to 1.55% (Fig. 2). Interactions between planting date by CL cultivars (Table 1) and planting date by RR accessions (Fig. 2) were significant (p<0.05) for outcrossing rate. However, no interaction was detected between CL cultivars and RR accessions for outcrossing rate. In general, higher outcrossing was observed with brownhull type compared with blackhull and strawhull types. The outcrossing rate differed among accessions in the same planting date due to various degrees of overlap in flowering. CL XL-8 had higher outcrossing rate in all planting dates compared with CL 161 (Table 1). Most of the red rice in the southern U.S. is an indica type, whereas cultivated rice, including CL 161, is a japonica type (Vaughan et al., 2001). The reason for higher outcrossing rate between the CL XL-8 hybrid and RR accessions is not clear. The outcrosses between CL 161 and RR accessions were phenotypically uniform (Fig. 3), and were significantly taller than both parents, which is consistent with observations in other studies (Shivrain et al., 2007). All the outcrosses had pale-colored, rough-textured leaves, which are traits similar to those of the RR parent. Sixty-five 239

AAES Research Series 550 percent of outcrosses were late in flowering and did not mature in the field. Increased height, flag-leaf length, and general plant size indicate that the outcrosses would be more competitive in terms of occupying space, intercepting light, and acquiring nutrients in the field compared with RR and CL rice cultivars. The outcrosses between CL XL-8 and RR accessions segregated in terms of flowering time, height (Fig. 4), and leaf color and texture. Nearly 50% of the outcrosses flowered during the season. The outcrosses started flowering 5 weeks earlier than any of the RR accessions or CL XL-8 hybrid and continued flowering until the onset of cold weather. A wide range in height of outcrosses was observed (70 to 165 cm). Therefore, some outcrosses were shorter than the shortest parent and others were taller than the tallest parent (data not shown). The outcrosses that are smaller than the cultivated rice will be difficult to detect in the fields. The outcrosses that produce seeds and shatter before rice harvest will increase the ALS-resistant RR seed bank. SIGNIFICANCE OF FINDINGS Results of this study suggest that the flowering pattern of RR accessions varies and affects outcrossing rate. CL XL-8 has higher outcrossing rate with RR accessions than CL 161. Brownhull RR has higher outcrossing rate than strawhull and blackhull RR with both CL cultivars. Outcrosses between CL 161 and RR accessions were phenotypically uniform, whereas outcrosses between CL XL-8 and RR accessions segregated. This experiment demonstrates that outcrossing rate is influenced by CL cultivars, RR type, and planting time. Hence, outcrossing mitigation and RR management strategies need to consider these factors. ACKNOWLEDGMENTS This study was supported by the rice growers checkoff funds through the Arkansas Rice Research and Promotion Board. We would like to thank Mellisa Jia at USDA-ARS, Stuttgart, Ark., for her help in DNA analysis. The authors would also like to extend their gratitude to Larry Earnest and his crew for their help in this research. LITERATURE CITED Burgos, N.R., V.K. Shivrain, D.R. Gealy, and H.R. Black. 2004. Planting time matters in Clearfield rice outcrossing. Proc. Ark. Crop Prot. Assoc. Res. Conf. 8:7. Gealy, D.R., W. Yan, and J.N. Rutger. 2003. Characterization of hybrid populations from rice crossed with awned and awnless red rice. Proc. 3rd Temperate Rice Conference. Paper #140, 7 pp. (Available On CD Rom Only) Scott, R.C. and N.R. Burgos. 2004. Clearfield/red rice out-cross confirmed in Arkansas field. Delta Farm Press Nov:18. Shivrain, V.K., N.R. Burgos, S.N. Rajguru, M.M. Anders, J. Moore, and M.A. Sales. 2007. Gene flow between Clearfield TM rice and red rice. Crop Protection 26:349-356. 240

B.R. Wells Rice Research Studies 2006 Shivrain, V.K. 2004. Molecular characterization of acetolactate synthase (ALS) gene and phenotypic diversity in red rice (Oryza sativa L.). M.S. Thesis. University of Arkansas, Fayetteville, Ark., U.S.A. 180 pp. Vaughan, L.K., B.V. Ottis, A.M. Prazak-Havey, C.A. Bormans, C. Sneller, J. M. Chandler, and W.D. Park. 2001. Is all red rice found in commercial rice really Oryza sativa? Weed Sci. 49:468-476. Zhang, W., S. Linscombe, E. Webster, and J. Oard. 2004. Risk assessment and genetic analysis of natural outcrossing in Louisiana commercial fields between Clearfield rice and the weed, red rice. 30 th Rice Technical Working Group Meeting, New Orleans, La. p. 125. Table 1. Estimated outcrossing rates as affected by cultivar and planting date. Red rice seedlings screened with imazethapyr at the Southeast Research and Extension Center, Rohwer, Ark., in 2006. Outcrossing rate Planting date (2005) CL 161 CL XL-8 ----------------------------- (%)------------------------------- 16 April (PD 1) 0.06 0.40 27 April (PD 2) 0.05 0.13 13 May (PD 3) 0.07 0.19 26 May (PD 4) 0.05 0.48 LSD z (0.05) ---------------------------(0.11)------------------------- z LSD values for comparing cultivar within planting dates or comparing planting dates within cultivar are the same. 241

AAES Research Series 550 Fig. 1. Flowering of blackhull (BH), brownhull( BRH), strawhull1 (SH 1), and strawhull 2 (SH 2) accessions; CL 161; and CL XL-8 at four planting dates (PD) at the Rice Research and Extension Center, Stuttgart, Ark., 2005. Shaded areas represent the period of time when at least one CL cultivar overlapped 50% in flowering with any red rice accession. 242

B.R. Wells Rice Research Studies 2006 Fig. 2. Outcrossing rate (%) between strawhull (SH1 and SH 2), blackhull (BLH), brownhull( BRH) and CL 161 and/or CL XL-8 at four planting dates (PD) at the Southeast Research and Extension Center, Rohwer, Ark., 2006. LSD (0.05) values for comparing outcrossing rates of red rice accessions within and between planting dates are 0.3 and 1.02, respectively. 243

AAES Research Series 550 Fig. 3. Two outcrosses between CL161 x Red Rice. Note uniformity in height and flowering of both plants. Fig. 4. Three outcrosses between CLXL-8 x Red Rice. Note segregation of height and flowering time. 244